Leverage kizumab composition

Abstract

The present disclosure relates, in part, to a lived chizumab composition having a reduced level of companion protein PLA2, poloxamer 188, and / or a reduced immunogenicity.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 352,459 filed on June 15, 2022, U.S. Provisional Application No. 63 / 455,495 filed on March 29, 2023, U.S. Provisional Application No. 63 / 444,178 filed on February 8, 2023, and U.S. Provisional Application No. 63 / 444,182 filed on February 8, 2023, the contents of each of which are incorporated herein by reference in their entirety.

[0003] References to Sequence Listing XML

[0004] This application contains a sequence listing that has been submitted electronically in XML format. The sequence listing is incorporated herein by reference. The XML file created on June 13, 2023 is named AVR-05525_SL.xml and has a size of 16,072 bytes. Background Art

[0005] Risankizumab (approved by the U.S. Food and Drug Administration (FDA) as risankizumab-RZAa and marketed under the trade name (sold) is a humanized immunoglobulin G1 (IgG1) monoclonal antibody against the p19 subunit of IL-23. The binding of lishengqizhumab to IL-23p19 inhibits the effects of IL-23 inducing and maintaining T helper (Th) 17 cells, innate lymphoid cells, γδT cells, and natural killer (NK) cells that cause tissue inflammation, destruction, and abnormal tissue repair. Lishengqizhumab is particularly effective in treating autoimmune diseases and inflammatory diseases such as psoriasis. Clinical studies have revealed the excellent safety and efficacy of lishengqizhumab in the treatment of, for example, plaque psoriasis and psoriatic arthritis.

[0006] Lisenqizumab can be formulated for subcutaneous injection at different concentrations. For example, 60 mg / mL, 90 mg / mL, and 150 mg / mL concentrations of Lisenqizumab formulations have been approved by the FDA. Various Lisenqizumab formulations are described in international applications PCT / US2013 / 038109 and PCT / IB2020 / 058347, the contents of which are incorporated herein by reference in their entirety.

[0007] The commercial formulations described above contain the surfactant polysorbate 20 (PS20). It has been suggested that trace amounts of hitchhiker protein contaminants in the formulation of certain recombinantly produced biopharmaceutical products can cause hydrolysis of polysorbate 20, leading to particle formation (aggregates of therapeutic protein and / or free fatty acids), and thus reducing shelf life (Khan et al. (2015) European Journal of Pharmaceutics and Biopharmaceutics 97: 60-67).

[0008] An important factor contributing to the presence of concomitant protein impurities in recombinant therapeutic monoclonal antibody formulations is their association with the product monoclonal antibody (mAb) (Nogal et al. (2012) Biotechnol. Prog. 28: 454-458). It has been reported that mAbs can preferentially bind to select concomitant proteins (HPs), and the degree of interaction and / or identity of the associated HPs can vary depending on the mAb (Nogal et al. (2012) Biotechnol. Prog. 28: 454-458). It has also been demonstrated that the mAb concomitant protein content in protein A (PrA) eluates is specific to a particular antibody (Zhang et al., (2016) Biotechnol. Prog. 32: 708-717), and that the primary sequence of the mAb can cause the binding of specific concomitant proteins and thus co-purification (Bee et al. (2016) Biotechnol. Prog. 00: 1-6). Another factor is that some companion proteins have similar physicochemical characteristics to the specific mAb to be purified, which causes them to copurify with the mAb. Because different companion proteins copurify with different recombinant antibodies, it is impossible to predict before the experiment whether you will encounter companion protein problems during the generation of a new antibody, let alone which companion protein will be problematic.

[0009] Furthermore, a major problem with protein-based therapeutics is their immunogenicity, i.e. their tendency to trigger unwanted immune responses against themselves, producing so-called "anti-drug antibodies" or "ADAs."

[0010] To date, there remains a need to develop risankizumab compositions with reduced levels of accompanying protein impurities and with improved properties such as reduced immunogenicity. Summary of the invention

[0011] The present disclosure is based in part on the discovery of a specific companion protein phospholipase A2 (PLA2) that co-purifies with levitra, the presence of which adversely affects the stability of polysorbates (e.g., polysorbate 20 and / or polysorbate 80) in levitra liquid drug formulations, and reducing the concentration of PLA2 in the formulation beneficially increases the long-term stability of the formulation (e.g., reduces particle formation, increases the shelf life of levitra drug products, etc.). Increased stability of levitra formulations can also be achieved by using poloxamer 188 (P188) instead of PS20 or PS80. The present disclosure also describes new levitra compositions with reduced levels of levitra material modified with high mannose N-glycans (e.g., M5, M6, and / or M7) and increased purity. These levitra compositions exhibit reduced immunogenicity in human subjects.

[0012] Thus, in one aspect, the present disclosure relates to a liquid composition comprising: (1) lisenqizumab; and (2) PLA2 in an amount less than about 250 pg / mg lisenqizumab.

[0013] In another aspect, the present disclosure relates to a composition comprising levitra, wherein the composition has one or more of the following characteristics: (a) less than about 5.4% of the total levitra substance having N-glycosylation has high mannose N-glycans; and / or (b) the incidence of anti-drug antibodies (ADA) that emerge during treatment in humans is less than about 4.7% after a single subcutaneous 150 mg dose of the pharmaceutical composition is administered to the humans.

[0014] In yet another aspect, the present disclosure relates to a composition comprising: (1) risankizumab; and (2) poloxamer 188 (P188), wherein the composition does not comprise polysorbate 20 (PS20) and / or polysorbate 80 (PS80). BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Shown are the amounts of low molecular weight (LMW) chaperones (HPs), total HPs, and LMW HPs and total HPs in DP1 and DP2 obtained by affinity purification.

[0016] Figure 2 Western blots probed with anti-PLA2G15 antibody are shown: lane 1, MW standard; lane 2, 1 ng PLA2G15 (MW: 47 kDa); lane 3, 0.1 ng PLA2G15; lane 6, DP1; lane 7, DP2_#1; lane 8, DP2_#2; lane 9, DP3; and lane 10, DP4.

[0017] Figure 3A Shown are PS20 stability at 5°C of samples made from various control and knockout cell lines as measured by the CAD assay.

[0018] Figure 3B Shown are PS20 stability at 25°C for samples made from various control and knockout cell lines as measured by the CAD assay. #5 Placebo: PS20 control. #6: BDS control: DP4 BDS.

[0019] Figure 3C Shown are PS20 stability at 5°C measured by FFA assay for samples made from various control and knockout cell lines. #5 Placebo: PS20 control. #6: BDS control: DP4 BDS.

[0020] Figure 3D Shown are PS20 stability at 25°C for samples made from various control and knockout cell lines as measured by FFA assay. #5 Placebo: PS20 control. #6: BDS control: DP4 BDS.

[0021] Figure 4A Shown are overlays of the PS20 subspecies chromatograms for sample C1 injection 1 (DP2 after 30 months at 2-8°C), sample A1 injection 2 (DP3 spiked with 1 μg / mL PLA2G15 after approximately 9 hours of incubation at room temperature and 25°C), and sample D1 injection 1 (no spiked DP3 material, no meaningful PS20 degradation).

[0022] Figure 4B Shown is an overlay of PS20 subspecies chromatograms of PS20 degradation in DP4 solutions at different PLA2G15 doping levels after 4 days of incubation at 25°C (more sample information can be found in Table 20). Arm 8 is a DP2 control sample. Since this peak was not observed in historical data, the small difference of nearly 42 minutes in arm 8 may be caused by leaching from the syringe filter used in this laboratory filling of the arm.

[0023] Figure 5 Shown are overlays of PS20 subspecies chromatograms for sample E3 injection 2 (DP3; no meaningful PS20 degradation), sample A3 injection 6 (DP3 spiked with 5 μg / mL PLBL2 after approximately 30 hours of incubation at 25°C; total incubation time after spiked was approximately 20 hours at 2-8°C plus 26 hours at 25°C), and sample D3 injection 2 (PS20 in DP2 after storage at 2-8°C for approximately 30 months).

[0024] Fig. 6AShown are overlays of the PS20 subspecies chromatograms of sample E3 injection 2 (DP3; no significant PS20 degradation) and sample B3 injection 6 (DP3 spiked with 5 μg / mL CES 1 after approximately 30 hours of incubation at 25°C; total incubation time after spiked was approximately 20 hours at 2-8°C plus 27 hours at 25°C).

[0025] Figure 6B The PS20 subspecies chromatogram overlays of sample D3 injection 2 (PS20 degradation in DP2 after storage at 2-8°C for about 30 months) and sample B3 injection 6 (DP3 spiked with 5 μg / mL CES1 after incubation at 25°C for about 30 hours; the total incubation time after spiked was about 20 hours at 2-8°C plus 27 hours at 25°C) are shown.

[0026] Figure 7 Shown are the overlays of the PS20 subspecies chromatograms of sample E3 injection 2 (DP3, no meaningful PS20 degradation) and sample C3 injection 6 (DP3 spiked with 5 μg / mL SIAE after approximately 30 hours of incubation at 25°C; the total incubation time after spike was approximately 20 hours at 2-8°C plus 28 hours at 25°C).

[0027] Figure 8 Shown are overlays of PS20 subspecies chromatograms for sample D4 injection 2 (DP3, no meaningful PS20 degradation), sample A4 injection 7 (DP3 spiked with 5 μg / mL PRDX6 after approximately 30 hours of incubation at 25°C; total incubation time after spiked was approximately 27 hours at 25°C), and sample C4 injection 2 (PS20 in DP2 after storage at 2-8°C for approximately 30 months).

[0028] Fig. 9 Shown are overlays of PS20 subspecies chromatograms for sample D4 injection 2 (DP3, no meaningful PS20 degradation), sample B4 injection 7 (DP3 spiked with 5 μg / mL PLA2G7 after incubation at 25°C for about 30 hours; total incubation time after spiked was about 28 hours at 25°C), and sample C4 injection 2 (PS20 in DP2 after storage at 2-8°C for about 30 months).

[0029] Fig.10The PS20 subspecies chromatograms of samples H7-9, H7-8, and H7-7 are shown as overlays. H7-9: DP2 (kept at -80°C); H7-8: DP2 (kept at room temperature for two weeks); H7-7: DP2 doped with 0.9ug / mL fosinopril (kept at room temperature for two weeks). The signals were normalized to correct for concentration changes due to doping (normalized with the peak at 37 minutes, which is stable in DP2 material based on historical data).

[0030] Fig.11 The PS20 subspecies chromatograms of samples H7-9, H7-8, and H7-6 are shown as overlays. H7-9: DP2 (kept at -80°C); H7-8: DP2 (kept at room temperature for two weeks); H7-6: DP2 spiked with 3.8ug / mL fosinopril (kept at room temperature for two weeks). The signals were normalized to correct for concentration changes due to doping (normalized with the peak at 37 minutes, which is stable in DP2 material based on historical data).

[0031] Fig.12 The PS20 subspecies chromatogram overlay of samples H7-9, H7-8, and H7-3 is shown. H7-9: DP2 (kept at -80°C); H7-8: DP2 (kept at room temperature for two weeks); H7-3: DP2 spiked with 27.8ug / mL fosinopril (kept at room temperature for two weeks). The signal was normalized to correct for concentration changes due to doping (normalized with the peak at 37 minutes, which is stable in DP2 material based on historical data). The higher background in the spiked samples at about 39 to 42 minutes should come from the co-elution of fosinopril.

[0032] Fig.13 The PS20 subspecies chromatograms of samples A6 and C6 are shown as an overlay. A6 DP2 material (kept at -80°C, diluted 1:1 with water before testing); C6: DP2 material (spiked with 930ug / mL Fosinopril; kept at room temperature for two weeks; diluted 1:1 with water before testing). The signal from C6 was normalized to better compare with A5.

[0033] Fig.14 A general overview of the newly developed purification process for the risankizumab drug substance (referred to herein as Process 4 Development) is shown.

[0034] Fig.15A Shown are the PS20 stability in DP2 (PS20) and DP3 (PS20) measured by the CAD assay at 5°C.

[0035] Fig. 15BShown are the PS20 stability in DP2 (PS20) and DP3 (PS20) measured by the CAD assay at 25°C.

[0036] Fig. 15C Shown are the PS20 stability in DP2 (PS20) and DP3 (PS20) measured by the CAD assay at 40°C.

[0037] Fig.16A Shown is the stability of PS20 in DP2 (PS20) and DP3 (PS20) measured by FFA assay at 5°C.

[0038] Fig. 16B Shown is the stability of PS20 in DP2 (PS20) and DP3 (PS20) measured by FFA assay at 25°C.

[0039] Fig.17A Shown are the PS20 stability in DP2 (PS20) and DP4 (PS20) measured by the CAD assay at 5°C.

[0040] Fig. 17B Shown are the PS20 stability in DP2 (PS20) and DP4 (PS20) measured by the CAD assay at 25°C.

[0041] Fig. 17C Shown are the PS20 stability in DP2 (PS20) and DP4 (PS20) measured by the CAD assay at 40°C.

[0042] Fig.18A and Fig.18B Shown is the stability of PS20 in DP2 (PS20) and DP4 (PS20) measured by FFA assay at 5°C.

[0043] Fig.18C and Fig.18D Shown is the stability of PS20 in DP2 (PS20) and DP4 (PS20) measured by FFA assay at 25°C.

[0044] Fig.19A Shown are the PS80 stability in DP2 (PS80), DP3 (PS80), and DP4 (PS80) measured by the CAD assay at 5°C.

[0045] Fig.19B Shown are the PS80 stability in DP2 (PS80), DP3 (PS80), and DP4 (PS80) measured by the CAD assay at 25°C.

[0046] Fig.19CShown are the PS80 stability in DP2 (PS80), DP3 (PS80), and DP4 (PS80) measured by the CAD assay at 40°C.

[0047] Fig. 20A and Fig. 20B Shown are the PS80 stability in DP2 (PS80), DP3 (PS80), and DP4 (PS80) measured by FFA assay at 5°C.

[0048] Fig. 20C and Fig.20D Shown are the PS80 stability in DP2 (PS80), DP3 (PS80), and DP4 (PS80) measured by FFA assay at 25°C.

[0049] Fig.20E and Fig.20F Shown are the PS80 stability in DP2 (PS80), DP3 (PS80), and DP4 (PS80) measured by FFA assay at 40°C.

[0050] Fig.21A and Fig. 21B 2-AB and HILIC-FL chromatograms of process 4 bulk drug substance (DS) batches and process 1 reference standard DS1-RS2 are shown. Process 4 batch DS4-001 and process 1 reference standard DS1-RS2 were analyzed side by side. Results accompanying the reference standards of the other three process 4 DS batches are not shown. As expected, slight differences in retention times from different runs were observed. Assay performance (relative peak quantitation) was not affected. Fig. 21B yes Fig.21A The expanded diagram of .

[0051] Fig. 22 RapiFluor and HILIC-FL chromatograms for Process 1, 2, and 4DS batches are shown.

[0052] Fig.23A The relative distribution of UP-SEC monomer results for lisdexizumab process 1, 2 and 4DS batches is shown.

[0053] Fig. 23B The relative distribution of UP-SEC HMW results for Risenqizumab Process 1, 2 and 4DS batches are shown.

[0054] Fig.23C UP-SEC results of lisdexizumab process 4 DS batch DS4-005 and process 1 reference standard DS1-RS2 are shown.

[0055] Fig.23D yes Fig.23C The expanded diagram of .

[0056] Fig.24A The relative distribution of CGE-NR main peak results for lisdexizumab process 1, 2 and 4DS batches is shown.

[0057] Fig. 24B The relative distribution of CGE-NR LMW results for lisdexizumab process 1, 2 and 4DS batches is shown.

[0058] Fig.24C CGE-NR results for risankizumab Method 4 DS batch DS4-005 and Method 1 reference standard DS1-RS2 are shown.

[0059] Fig.24D Shows Fig.24C The expanded diagram of .

[0060] Fig.25 P188 and PS20 levels in DP2 DS are shown.

[0061] Fig.26 P188 and PS20 levels in DP3 DS are shown. DETAILED DESCRIPTION

[0062] In some aspects, the present disclosure is based in part on the discovery of a specific companion protein, PLA2, the presence of which adversely affects the stability of polysorbates (e.g., PS20 and / or PS80) in a levitra liquid drug formulation, and that reducing PLA2 in the formulation beneficially increases the stability of the formulation (e.g., reduces particle formation, increases the shelf life of the levitra drug product, etc.). It was also discovered that increased stability of the levitra formulation can also be achieved by using poloxamer 188 instead of P20 or P80.

[0063] The initial drug formulation developed for risankizumab had a concentration of 90 mg / ml. The 150 mg / ml formulation was subsequently approved by the U.S. FDA to enable a single subcutaneous injection of the entire 150 mg therapeutic dose. The commercially available 75 mg / 0.83 ml (90 mg / mL) and 150 mg / ml risankizumab formulations are disclosed in the FDA-approved drug label and the December 2022 revised (Risenqizumab-RZA), the contents of each of which are incorporated herein by reference in their entirety. Both FDA-approved risanqizumab formulations contain a highly purified, recombinantly produced risanqizumab active pharmaceutical ingredient (API). However, when the 150 mg / ml risanqizumab formulation was diluted to explore the feasibility of developing specific product displays (such as those used with on-body devices), unacceptable levels of particles composed of risanqizumab and / or free fatty acid aggregates were formed under certain storage conditions.

[0064] In one embodiment of the present invention, this unexpected problem is believed to be caused by residual trace levels of companion proteins that are co-purified with the other high purity Risenqizumab API purified using the prior art orthogonal column chromatography process. Because the identity of the companion proteins that co-purify with the monoclonal antibody (mAb) varies depending on the mAb, it is impossible to predict before the experiment whether a companion protein problem will be encountered during the generation of a new antibody, let alone which companion protein will be problematic.

[0065] In some aspects, the present disclosure identifies PLA2 as a specific problematic companion protein that co-purifies with Risenqizhumab. It is demonstrated herein that PLA2 co-purified with Risenqizhumab causes degradation of the surfactant polysorbate 20 (PS20), leading to particle formation in the Risenqizhumab product. An optimized purification process has been developed that is specifically directed to reducing the level of PLA2 co-purified with Risenqizhumab. In some aspects, the present disclosure therefore provides a Risenqizhumab liquid composition with reduced levels of PLA2 and improved stability and shelf life.

[0066] In some aspects, the present disclosure relates to new levitra compositions having reduced levels of levitra substances modified with high mannose N-glycans (e.g., M5, M6, and / or M7) with reduced immunogenicity.

[0067] In some aspects, the present disclosure relates to levofloxacin compositions having reduced levels of levofloxacin substances having high mannose N-glycans (M5, M6 and / or M7) and reduced immunogenicity. Reduced immunogenicity (e.g., a lower incidence of anti-drug antibodies arising during treatment after administration of a single 150 mg subcutaneous dose of a liquid composition to a human) also indicates an improved product quality of the levofloxacin compositions described herein.

[0068] As disclosed herein, the present disclosure relates to the following embodiments.

[0069] Embodiment 1. A liquid composition comprising: (1) levitra or an anti-IL23 monoclonal antibody comprising two light chains having an amino acid sequence of SEQ ID NO: 9 and two heavy chains having an amino acid sequence of SEQ ID NO: 10; and (2) phospholipase A2 (PLA2) in an amount less than about 250 pg / mg of levitra.

[0070] Embodiment 2. The liquid composition of embodiment 1, comprising about 60 mg / ml to about 150 mg / ml of lisenqizumab.

[0071] Embodiment 3. The liquid composition of embodiment 1 or 2, wherein the PLA2 is PLA2G15.

[0072] Embodiment 4. A liquid composition as described in any of embodiments 1-3, wherein the level of PLA2 is less than about 240 pg, less than about 220 pg, less than about 200 pg, less than about 180 pg, less than about 160 pg, less than about 140 pg, less than about 120 pg, less than about 100 pg, less than about 90 pg, less than about 80 pg, less than about 70 pg, less than about 60 pg, less than about 50 pg, less than about 40 pg, less than about 30pg, less than about 25pg, less than about 20pg, less than about 15pg, less than about 10pg, less than about 9pg, less than about 8pg, less than about 7pg, less than about 6pg, less than about 5pg, less than about 4.4pg, less than about 3pg, less than about 2pg, less than about 1pg, less than about 0.5pg, less than about 0.1pg, less than about 0.05pg or less than about 0.01pg / mg of lisenqizumab.

[0073] Embodiment 5. A liquid composition as described in any of embodiments 1-3, wherein the level of PLA2 is greater than about 240 pg, greater than about 220 pg, greater than about 200 pg, greater than about 180 pg, greater than about 160 pg, greater than about 140 pg, greater than about 120 pg, greater than about 100 pg, greater than about 90 pg, greater than about 80 pg, greater than about 70 pg, greater than about 60 pg, greater than about 50 pg, greater than about 4 0pg, greater than about 30pg, greater than about 25pg, greater than about 20pg, greater than about 15pg, greater than about 10pg, greater than about 9pg, greater than about 8pg, greater than about 7pg, greater than about 6pg, greater than about 5pg, greater than about 4pg, greater than about 3pg, greater than about 2pg, greater than about 1pg, greater than about 0.5pg, greater than about 0.1pg, greater than about 0.05pg or greater than about 0.01pg / mg of lisenqizumab.

[0074] Embodiment 6. A liquid composition as described in any of embodiments 1-3, wherein the level of PLA2 is about 200 to about 249, about 160 to about 200, about 120 to about 160, about 100 to about 120, about 80 to about 100, about 60 to about 80, about 40 to about 60, about 25 to about 40, about 10 to about 25, about 5 to about 10, about 4 to about 10, about 1 to about 5, about 1 to about 4, about 1 to about 3, about 1 to about 2, about 0.5 to about 1, about 0.1 to about 0.5, about 0.05 to about 0.1, about 0.01 to about 0.5, or about 70 to about 240 pg / mg of lisenqizumab.

[0075] Embodiment 7. A liquid composition as described in any of embodiments 1-3, wherein the level of PLA2 is about 240, about 220, about 200, about 180, about 160, about 140, about 120, about 100, about 90, about 80, about 70, about 60, about 50, about 40, about 30, about 25, about 20, about 15, about 10, about 9, about 8, about 7, about 6, about 5, about 4.4, about 3, about 2, about 1, about 0.5, about 0.1, about 0.05 or about 0.01 pg / mg of lisenqizumab.

[0076] Embodiment 8. The liquid composition of any one of embodiments 1-7, wherein the level of PLA2 is determined by ELISA.

[0077] Embodiment 9. The liquid composition of any one of embodiments 1-8, wherein the risenizumab is produced in a CHO cell line.

[0078] Embodiment 10. The liquid composition of any one of embodiments 1-9, further comprising one or more of a surfactant, a polyol, and a buffer.

[0079] Embodiment 11. The liquid composition of embodiment 10, wherein the polyol is selected from the group consisting of trehalose, mannitol, sucrose and sorbitol.

[0080] Embodiment 12. The liquid composition of embodiment 11, wherein the polyol is trehalose.

[0081] Embodiment 13. The liquid composition of embodiment 12, wherein the amount of trehalose is about 150 to about 220 mM.

[0082] Embodiment 14. The liquid composition of embodiment 13, wherein the amount of trehalose is about 185 mM.

[0083] Embodiment 15. A liquid composition as described in any of embodiments 10-14, wherein the buffer is selected from the group consisting of acetate buffer, histidine buffer, citrate buffer, phosphate buffer, glycine buffer and arginine buffer.

[0084] Embodiment 16. The liquid composition of embodiment 15, wherein the buffer is acetate buffer.

[0085] Embodiment 17. The liquid composition of embodiment 16, wherein the amount of acetate buffer is about 5 to about 50 mM.

[0086] Embodiment 18. The liquid composition of embodiment 17, wherein the amount of acetate buffer is about 10 mM.

[0087] Embodiment 19. A liquid composition as described in any of embodiments 10-18, wherein the surfactant is selected from the group consisting of polysorbate 20 (PS20), polysorbate 80 (PS80), polysorbate 40 (PS40), polysorbate 60 (PS60), polysorbate 65 (PS65) and poloxamer 188.

[0088] Embodiment 20. A liquid composition as described in Embodiment 19, wherein the surfactant is PS20.

[0089] Embodiment 21. A liquid composition as described in Embodiment 20, wherein the amount of PS20 is about 0.2 mg / mL.

[0090] Embodiment 22. A liquid composition as described in embodiment 21, comprising: 150 mg / ml risankizumab; 185 mM trehalose; 10 mM acetate; and 0.20 mg / mL polysorbate 20, wherein the liquid composition has a pH of about 5.7.

[0091] Embodiment 23. A liquid composition as described in Embodiment 21, comprising: 150 mg / ml risankizumab; 0.054 mg / mL acetic acid; 1.24 mg / mL sodium acetate trihydrate; 70 mg / mL trehalose dihydrate; 0.20 mg / mL polysorbate 20; and water for injection, wherein the liquid composition has a pH of about 5.7.

[0092] Embodiment 24. The liquid composition of any one of embodiments 20-23, wherein after storage at 5°C for 6 months, at least 80% of the initial concentration of PS20 is present in the composition.

[0093] Embodiment 25. The liquid composition of any one of embodiments 20-23, wherein after storage at 5°C for 24 months, at least 70% of the initial concentration of PS20 is present in the composition.

[0094] Embodiment 26. The liquid composition of any one of embodiments 20-23, wherein after storage at 25°C for 6 months, at least 60% of the initial concentration of PS20 is present in the composition.

[0095] Embodiment 27. The liquid composition of any one of embodiments 20-23, wherein after storage at 40°C for 6 months, at least 40% of the initial concentration of PS20 is present in the composition.

[0096] Embodiment 28. The liquid composition of any one of embodiments 20-23, wherein the total concentration of free fatty acids (FFA) present in the composition does not increase by more than 1.5 times after storage at 5°C for 6 months.

[0097] Embodiment 29. The liquid composition of any one of embodiments 20-23, wherein the total concentration of FFA present in the composition does not exceed 20 nmol / ml after storage at 5°C for 6 months.

[0098] Embodiment 30. The liquid composition of any one of embodiments 20-23, wherein the total concentration of FFAs present in the composition does not exceed 3.2 times after storage at 25°C for 6 months.

[0099] Embodiment 31. The liquid composition of any one of embodiments 20-23, wherein the total concentration of FFA present in the composition does not exceed 25 nmol / ml after storage at 25°C for 6 months.

[0100] Embodiment 32. The liquid composition of any one of embodiments 20-23, wherein the total concentration of FFAs present in the composition does not exceed 3 times after storage at 40°C for 6 months.

[0101] Embodiment 33. The liquid composition of any one of embodiments 20-23, wherein the total concentration of FFA present in the composition does not exceed 35 nmol / ml after storage at 40°C for 6 months.

[0102] Embodiment 34. A liquid composition as described in Embodiment 19, wherein the surfactant is PS80.

[0103] Embodiment 35. The liquid composition of embodiment 34, wherein after storage at 5°C for 6 months, at least 80% of the initial concentration of PS80 is present in the composition.

[0104] Embodiment 36. The liquid composition of embodiment 34, wherein after storage at 25°C for 6 months, at least 60% of the initial concentration of PS80 is present in the composition.

[0105] Embodiment 37. The liquid composition of embodiment 34, wherein after storage at 40°C for 6 months, at least 60% of the initial concentration of PS80 is present in the composition.

[0106] Embodiment 38. The liquid composition of embodiment 34, wherein the total concentration of FFAs present in the composition does not increase by more than 8-fold after storage at 5°C for 6 months.

[0107] Embodiment 39. The liquid composition of embodiment 34, wherein the total concentration of FFA present in the composition does not exceed 40 nmol / ml after storage at 5°C for 6 months.

[0108] Embodiment 40. The liquid composition of embodiment 34, wherein the total concentration of FFAs present in the composition does not increase by more than 12-fold after storage at 25°C for 6 months.

[0109] Embodiment 41. The liquid composition of embodiment 34, wherein the total concentration of FFA present in the composition does not exceed 60 nmol / ml after storage at 25°C for 6 months.

[0110] Embodiment 42. The liquid composition of embodiment 34, wherein the total concentration of FFAs present in the composition does not increase by more than 2.5 times after storage at 40°C for 6 months.

[0111] Embodiment 43. The liquid composition of embodiment 34, wherein the total concentration of FFA present in the composition does not exceed 15 nmol / ml after storage at 40°C for 6 months.

[0112] Embodiment 44. A liquid composition as described in any of Embodiments 24-43, wherein the PS20 or PS80 is measured using HPLC-CAD.

[0113] Embodiment 45. The liquid composition of any one of Embodiments 24-43, wherein the FFA is measured using an LC-FFA assay.

[0114] Embodiment 46. The liquid composition of any one of embodiments 1-45, wherein no visible or sparkling particles are observed within 24 months at 4°C.

[0115] Embodiment 47. A liquid composition as described in any of Embodiments 1-46, wherein the liquid composition is packaged in a vial, a pre-filled syringe, or an on-body device.

[0116] Embodiment 48. The liquid composition of any one of Embodiments 1-47, wherein the liquid composition is a pharmaceutical composition and is suitable for subcutaneous injection.

[0117] Embodiment 49. The liquid composition of any one of Embodiments 1-47, wherein the liquid composition is a pharmaceutical composition and is suitable for intravenous injection.

[0118] Embodiment 50. A method of treating an immune disease using the composition of any one of embodiments 1-49.

[0119] Embodiment 51. A composition comprising lisenqizumab, wherein the composition has one or more of the following characteristics: (a) less than about 5.4% of the total lisenqizumab species with N-glycosylation have high mannose N-glycans; (b) at least about 99.1% of the lisenqizumab is present as monomers and / or no more than about 0.4% of the lisenqizumab is present as high molecular weight (HMW) species as measured by ultra-high performance size exclusion chromatography (UP-SEC); (c) greater than about 97.5% of the lisenqizumab is present as a major peak and / or less than about 2.2% of the lisenqizumab is present as low molecular weight (LMW) species as measured by capillary gel electrophoresis under non-reducing conditions (CGE-NR); and / or (d) after administration of a single subcutaneous dose of the composition to humans, the incidence of anti-drug antibodies (ADA) that arise during treatment is less than about 4.7%.

[0120] Embodiment 52. The composition of embodiment 51, comprising about 60 mg / ml to about 150 mg / ml of lisenqizumab.

[0121] Embodiment 53. The composition of embodiment 51 or 52, wherein the pharmaceutical composition has at least the following characteristic (a): less than about 5.4% of the total lisenqizumab species with N-glycosylation have high mannose N-glycans.

[0122] Embodiment 54. The composition of embodiment 53, wherein the high mannose N-glycan comprises one or more high mannose N-glycans selected from mannose 5 N-glycan (M5), mannose 6 N-glycan (M6), and mannose 7 N-glycan (M7).

[0123] Embodiment 55. The composition of embodiment 54, wherein the high mannose N-glycans are M5, M6, and M7.

[0124] Embodiment 56. A composition as described in any of embodiments 51-55, wherein the level of lisenqizumab with high mannose N-glycans is less than 5.3%, less than about 5.2%, less than about 5.1%, less than about 5.0%, less than about 4.9%, less than about 4.8%, less than about 4.7%, less than about 4.6%, less than about 4.5%, less than about 4.4%, less than about 4.3%, less than about 4.2%, less than about 4.1%, less than about 4.0%, less than about 3.9%, less than about 3.8% or less than about 3.7% of the total lisenqizumab material with N-glycosylation.

[0125] Embodiment 57. A composition as described in any of embodiments 51-56, wherein the level of lisenqizumab with high mannose N-glycans is greater than about 5.3%, greater than about 5.2%, greater than about 5.1%, greater than about 5.0%, greater than about 4.9%, greater than about 4.8%, greater than about 4.7%, greater than about 4.6%, greater than about 4.5%, greater than about 4.4%, greater than about 4.3%, greater than about 4.2%, greater than about 4.1%, greater than about 4.0%, greater than about 3.9%, greater than about 3.8%, greater than about 3.7% or greater than about 3.6% of the total lisenqizumab material with N-glycosylation.

[0126] Embodiment 58. A composition as described in any of embodiments 51-57, wherein the level of lisenqizumab with high mannose N-glycans is about 3.6% to about 5.3%, about 3.6% to about 5.0%, about 3.6% to about 4.8%, about 3.6% to about 4.5%, about 3.6% to about 4.1%, about 3.6% to about 3.8%, about 3.8% to about 5.3%, about 4.1% to about 5.3%, about 4.5% to about 5.3%, about 4.8% to about 5.3%, about 5.0% to about 5.3%, about 4.3% to about 4.9% or about 3.6% to about 4.9% of the total lisenqizumab material with N-glycosylation.

[0127] Embodiment 59. A composition as described in any of embodiments 51-58, wherein the level of lisenqizumab with high mannose N-glycans is about 5.3%, about 5.2%, about 5.1%, about 5.0%, about 4.9%, about 4.8%, about 4.7%, about 4.6%, about 4.5%, about 4.4%, about 4.3%, about 4.2%, about 4.1%, about 4.0%, about 3.9%, about 3.8%, about 3.7% or about 3.6% of the total lisenqizumab material with N-glycosylation.

[0128] Embodiment 60. The composition of embodiment 54, wherein the high mannose N-glycan is M5.

[0129] Embodiment 61. A composition as described in embodiment 60, wherein the level of lisenqizumab with M5 is less than 5.3%, less than about 5.2%, less than about 5.1%, less than about 5.0%, less than about 4.9%, less than about 4.8%, less than about 4.7%, less than about 4.6%, less than about 4.5%, less than about 4.4%, less than about 4.3%, less than about 4.2%, less than about 4.1%, less than about 4.0%, less than about 3.9%, less than about 3.8%, less than about 3.7%, less than about 3.6%, less than about 3.5%, less than about 3.4%, less than about 3.3%, less than about 3.2%, less than about 3.1%, less than about 3.0%, less than about 2.9% or less than about 2.8% of the total lisenqizumab material with N-glycosylation.

[0130] Embodiment 62. A composition as described in embodiment 60 or 61, wherein the level of lisenqizumab with M5 is greater than about 5.2%, greater than about 5.1%, greater than about 5.0%, greater than about 4.9%, greater than about 4.8%, greater than about 4.7%, greater than about 4.6%, greater than about 4.5%, greater than about 4.4%, greater than about 4.3%, greater than about 4.2%, greater than about 4.1%, greater than about 4.0%, greater than about 3.9%, greater than about 3.8%, greater than about 3.7%, greater than about 3.6%, greater than about 3.5%, greater than about 3.4%, greater than about 3.3%, greater than about 3.2%, greater than about 3.1%, greater than about 3.0%, greater than about 2.9% or greater than about 2.8% or greater than about 2.7% of the total lisenqizumab material with N-glycosylation.

[0131] Embodiment 63. A composition as described in any of embodiments 60-62, wherein the level of lisenqizumab with M5 is about 2.7% to about 5.2%, about 3.1% to about 5.2%, about 3.5% to about 5.2%, about 4.0% to about 5.2%, about 4.5% to about 5.2%, about 5% to about 5.2%, about 2.7% to about 5.0%, about 2.7% to about 4.5%, about 2.7% to about 4.0%, about 2.7% to about 3.5%, about 2.7% to about 3.1%, about 3.2% to about 3.7% or about 2.7% to about 3.7% of the total lisenqizumab material with N-glycosylation.

[0132] Embodiment 64. A composition as described in any of embodiments 60-63, wherein the level of lisenqizumab with M5 is about 2.7%, about 2.8%, about 2.9%, about 3.0%, about 3.1%, about 3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4.0%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, about 5.0%, about 5.1% or about 5.2% of the total lisenqizumab material with N-glycosylation.

[0133] Embodiment 65. The composition of embodiment 54, wherein the high mannose glycan is M6.

[0134] Embodiment 66. A composition as described in embodiment 65, wherein the level of lisenqizumab with M6 is less than about 2.6%, less than about 2.5%, less than about 2.4%, less than about 2.3%, less than about 2.2%, less than about 2.1%, less than about 2.0%, less than about 1.9%, less than about 1.8%, less than about 1.7%, less than about 1.6%, less than about 1.5%, less than about 1.4%, less than about 1.3%, less than about 1.2%, less than about 1.1%, less than about 1.0%, less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6% or less than about 0.5% of the total lisenqizumab material with N-glycosylation.

[0135] Embodiment 67. A composition as described in embodiment 65 or 66, wherein the level of lisenqizumab with M6 is greater than about 2.5%, greater than about 2.4%, greater than about 2.3%, greater than about 2.2%, greater than about 2.1%, greater than about 2.0%, greater than about 1.9%, greater than about 1.8%, greater than about 1.7%, greater than about 1.6%, greater than about 1.5%, greater than about 1.4%, greater than about 1.3%, greater than about 1.2%, greater than about 1.1%, greater than about 1.0%, greater than about 0.9%, greater than about 0.8%, greater than about 0.7%, greater than about 0.6%, greater than about 0.5% or greater than about 0.4% of the total lisenqizumab material with N-glycosylation.

[0136] Embodiment 68. A composition as described in any of embodiments 65-67, wherein the level of lisenqizumab with M6 is about 0.4% to about 2.5%, about 0.4% to about 2.4%, about 0.4% to about 2.2%, about 0.4% to about 2.0%, about 0.4% to about 1.8%, about 0.4% to about 1.6%, about 0.4% to about 1.4%, about 0.4% to about 1.2%, about 0.4% to about 1.0%, about 0.4% to about 0.9%, about 0.4% to about 0.8%, about 0.4% to about 0.7%, about 0.4% to about 0.6%, about 0.4% to about 0.5% or about 0.6% to about 0.7% of the total lisenqizumab material with N-glycosylation.

[0137] Embodiment 69. A composition as described in any of embodiments 65-68, wherein the level of lisenqizumab with M6 is about 2.5%, about 2.4%, about 2.3%, about 2.2%, about 2.1%, about 2.0%, about 1.9%, about 1.8%, about 1.7%, about 1.6%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1.0%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5% or about 0.4% of the total lisenqizumab material with N-glycosylation.

[0138] Embodiment 70. The composition of embodiment 54, wherein the high mannose glycan is M7.

[0139] Embodiment 71. A composition as described in embodiment 70, wherein the level of lisenqizumab with M7 is less than 2.0%, less than about 1.9%, less than about 1.8%, less than about 1.7%, less than about 1.6%, less than about 1.5%, less than about 1.4%, less than about 1.3%, less than about 1.2%, less than about 1.1%, less than about 1.0%, less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, less than about 0.5% or greater than about 0.4% of the total lisenqizumab material with N-glycosylation.

[0140] Embodiment 72. A composition as described in embodiment 70 or 71, wherein the level of lisenqizumab with M7 is greater than about 1.9%, greater than about 1.8%, greater than about 1.7%, greater than about 1.6%, greater than about 1.5%, greater than about 1.4%, greater than about 1.3%, greater than about 1.2%, greater than about 1.1%, greater than about 1.0%, greater than about 0.9%, greater than about 0.8%, greater than about 0.7%, greater than about 0.6%, greater than about 0.5% or greater than about 0.4% of the total lisenqizumab material with N-glycosylation.

[0141] Embodiment 73. A composition as described in any of embodiments 70-72, wherein the level of lisenqizumab with M7 is about 0.4% to about 1.9%, about 0.4% to about 1.8%, about 0.4% to about 1.6%, about 0.4% to about 1.4%, about 0.4% to about 1.2%, about 0.4% to about 1.0%, about 0.4% to about 0.9%, about 0.4% to about 0.8%, about 0.4% to about 0.7%, about 0.4% to about 0.6%, about 0.4% to about 0.5% or about 0.5% to about 0.6% of the total lisenqizumab material with N-glycosylation.

[0142] Embodiment 74. A composition as described in any of embodiments 70-73, wherein the level of lisenqizumab with M7 is about 1.9%, about 1.8%, about 1.7%, about 1.6%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1.0%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5% or about 0.4% of the total lisenqizumab material with N-glycosylation.

[0143] Embodiment 75. The composition of any one of embodiments 51-74, wherein the level of risankizumab with high mannose N-glycans is determined by 2-AB and HILIC-FL chromatography.

[0144] Embodiment 76. The composition of any one of embodiments 51-74, wherein the level of risankizumab with high mannose N-glycans is determined by RapiFluor HILIC-FL chromatography.

[0145] Embodiment 77. The composition of any one of embodiments 51-76, wherein greater than about 84.4% of the total risankizumab species having N-glycosylation have fucosylated complex oligosaccharides.

[0146] Embodiment 78. The composition of embodiment 77, wherein about 88.0% to about 90.9% of the total risankizumab species having N-glycosylation have fucosylated complex oligosaccharides.

[0147] Embodiment 79. The composition of embodiment 77 or 78, wherein the level of risankizumab with fucosylated complex oligosaccharides is determined by 2-AB and HILIC-FL chromatography.

[0148] Embodiment 80. The composition of embodiment 77 or 78, wherein the level of risankizumab with high mannose N-glycans is determined by RapiFluor HILIC-FL chromatography.

[0149] Embodiment 81. The composition of any one of Embodiments 51-80, wherein the composition comprises about 0.8% to about 1.4% aglycosylated lisenqizumab.

[0150] Embodiment 82. The composition of embodiment 81, wherein the aglycosylated risankizumab is determined by tryptic peptide mapping.

[0151] Embodiment 83. A composition as described in embodiment 51 or 52, wherein the composition has at least feature (b): at least about 99.1% of the lisenqizumab is present as a monomer and / or no more than about 0.4% of the lisenqizumab is present as a high molecular weight (HMW) species as measured by ultra-high performance size exclusion chromatography (UP-SEC).

[0152] Embodiment 84. The composition of embodiment 83, wherein at least about 99.1% of the lisenqizumab is present as monomer and no more than about 0.4% of the lisenqizumab is present as high molecular weight (HMW) species as measured by ultra-high performance size exclusion chromatography (UP-SEC).

[0153] Embodiment 85. The composition of embodiment 83 or 84, wherein at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.6%, or at least about 99.7% of the lisenqizumab is present as a monomer.

[0154] Embodiment 86. The composition of any of embodiments 83-85, wherein about 99.1% to about 99.7%, 99.1% to about 99.6%, about 99.2% to about 99.7%, or about 99.2% to about 99.6% of the risenzizumab is present as a monomer.

[0155] Embodiment 87. The composition of any one of Embodiments 83-86, wherein no more than about 0.35%, no more than about 0.3%, no more than about 0.25%, no more than about 0.2%, no more than about 0.15%, or no more than about 0.1% of the risankizumab is present as high molecular weight (HMW) species.

[0156] Embodiment 88. The composition of any of Embodiments 83-87, wherein about 0.1% to about 0.4%, about 0.1% to about 0.3%, about 0.1% to about 0.2%, about 0.2% to about 0.4%, or about 0.2% to about 0.3% of the lisenqizumab is present as high molecular weight (HMW) species.

[0157] Embodiment 89. A composition as described in embodiment 51 or 52, wherein the composition has at least feature (c): greater than about 97.5% of the lisenqizumab is present as a major peak and / or less than about 2.2% of the lisenqizumab is present as a low molecular weight (LMW) substance as measured by capillary gel electrophoresis under non-reducing conditions (CGE-NR).

[0158] Embodiment 90. The composition of embodiment 89, wherein greater than about 97.5% of the lisenqizumab is present as a major peak and less than about 2.2% of the lisenqizumab is present as low molecular weight (LMW) species.

[0159] Embodiment 91. A composition as described in embodiment 89 or 90, wherein greater than about 97.6%, greater than about 97.7%, greater than about 97.8%, greater than about 97.9%, greater than about 98.0%, greater than about 98.1%, greater than about 98.2%, greater than about 98.3% or greater than about 98.4% of lisenqizumab is present as a major peak.

[0160] Embodiment 92. The composition of any of embodiments 89-91, wherein about 97.6% to about 98.4%, about 97.6% to about 98.3%, about 97.6% to about 98.2%, about 97.7% to about 98.4%, about 97.7% to about 98.3%, about 97.7% to about 98.2%, about 97.8% to about 98.4%, about 97.8% to about 98.3%, or about 97.8% to about 98.2% of liscenzumab is present as a major peak.

[0161] Embodiment 93. The composition of any one of embodiments 89-92, wherein less than about 2.1%, less than 2.0%, less than 1.9%, less than 1.8%, less than 1.7%, less than 1.6% or less than 1.5% of the risankizumab is present as low molecular weight (LMW) species.

[0162] Embodiment 94. The composition of any of Embodiments 89-93, wherein about 1.5% to about 2.1%, about 1.6% to about 2.1%, about 1.7% to about 2.1%, about 1.5% to about 2.0%, about 1.6% to about 2.0%, or about 1.7% to about 2.0% of the lisenqizumab is present as low molecular weight (LMW) species.

[0163] Embodiment 95. The composition of embodiment 51 or 52, wherein the composition has at least the following feature (d): after a single subcutaneous 150 mg dose of the composition is administered to a human, the incidence of anti-drug antibodies (ADA) that emerge during treatment in the human is less than about 4.7%.

[0164] Embodiment 96. The composition of embodiment 95, wherein the incidence of ADA occurring during treatment is less than about 4.5%, less than about 4.0%, less than about 3.5%, less than about 3.0%, less than about 2.5%, less than about 2.0%, less than about 1.5%, less than about 1.0%, less than about 0.5%, less than about 0.4%, less than about 0.3%, less than about 0.2%, less than about 0.1%, less than about 0.01%, less than about 0.001%, or less than about 0.0001%.

[0165] Embodiment 97. The composition of embodiment 95 or 96, wherein the incidence of ADA occurring during the treatment period is 0.0%.

[0166] Embodiment 98. The composition of any one of embodiments 95-97, wherein the incidence of ADA that emerges during treatment is measured following administration of a single subcutaneous injection of a 150 mg dose of the composition to a human.

[0167] Embodiment 99. The composition of any one of embodiments 95-98, wherein the presence of ADA is determined using a bridging electrochemiluminescent immunoassay.

[0168] Embodiment 100. The composition of any one of Embodiments 51-99, wherein the risenizumab is produced in a CHO cell line.

[0169] Embodiment 101. The composition of any one of Embodiments 51-100, further comprising a pharmaceutically acceptable excipient.

[0170] Embodiment 102. The pharmaceutical composition of embodiment 101, wherein the excipient is selected from the group consisting of a surfactant, a polyol, and a buffer.

[0171] Embodiment 103. The pharmaceutical composition of embodiment 102, wherein the polyol is trehalose.

[0172] Embodiment 104. The pharmaceutical composition of embodiment 103, wherein the amount of trehalose is about 150 to about 220 mM.

[0173] Embodiment 105. The pharmaceutical composition of embodiment 104, wherein the amount of trehalose is about 185 mM.

[0174] Embodiment 106. The pharmaceutical composition of any one of Embodiments 101-105, wherein the buffer is selected from the group consisting of acetate buffer and succinate buffer.

[0175] Embodiment 107. A pharmaceutical composition as described in Embodiment 106, wherein the buffer is acetate buffer.

[0176] Embodiment 108. The pharmaceutical composition of Embodiment 107, wherein the amount of acetate buffer is about 5 to about 50 mM.

[0177] Embodiment 109. The pharmaceutical composition of Embodiment 108, wherein the amount of acetate buffer is about 10 mM.

[0178] Embodiment 110. The pharmaceutical composition of any one of Embodiments 101-109, wherein the surfactant is polysorbate 20 (PS20).

[0179] Embodiment 111. A pharmaceutical composition as described in Embodiment 110, wherein the amount of PS20 is about 0.02 mg / ml.

[0180] Embodiment 112. The pharmaceutical composition of embodiment 111, wherein the amount of PS20 is about 0.2 mg / mL.

[0181] Embodiment 113. A pharmaceutical composition as described in Embodiment 112, comprising: 150 mg / ml risankizumab; 185 mM trehalose; 10 mM acetate; and 0.20 mg / mL polysorbate 20, wherein the pharmaceutical composition has a pH of about 5.7.

[0182] Embodiment 114. A pharmaceutical composition as described in Embodiment 112, comprising: 150 mg / ml risankizumab; 0.054 mg / mL acetic acid; 1.24 mg / mL sodium acetate trihydrate; 70 mg / mL trehalose dihydrate; 0.20 mg / mL polysorbate 20; and Water for Injection, USP; wherein the pharmaceutical composition has a pH of about 5.7.

[0183] Embodiment 115. The pharmaceutical composition of Embodiment 110, wherein the lisenqizumab is present at a concentration of 150 mg / ml.

[0184] Embodiment 116. The pharmaceutical composition of any one of Embodiments 101-115, wherein the pharmaceutical composition is packaged in a vial, a prefilled syringe, or an on-body device.

[0185] Embodiment 117. The pharmaceutical composition of any one of Embodiments 101-116, wherein the pharmaceutical composition is suitable for subcutaneous injection.

[0186] Embodiment 118. The pharmaceutical composition of any one of Embodiments 101-117, wherein the pharmaceutical composition is suitable for intravenous injection.

[0187] Embodiment 119. The pharmaceutical composition of any one of Embodiments 101-118, wherein the pharmaceutical composition is a liquid composition.

[0188] Embodiment 120. The pharmaceutical composition of any one of Embodiments 101-118, wherein the pharmaceutical composition is an aqueous liquid composition.

[0189] Embodiment 121. A method of treating an immune disease using the pharmaceutical composition of any one of Embodiments 101-120.

[0190] Embodiment 122. A method for producing a lisenqizumab drug product having an amount of PLA2 less than about 250 pg / mg of lisenqizumab, the method comprising: (1) culturing a host cell line expressing lisenqizumab in a growth medium under conditions that permit the production of lisenqizumab; (2) clarifying the growth medium by centrifugation and depth filtration; (3) contacting the clarified medium containing lisenqizumab with a protein A resin; (4) eluting lisenqizumab from the protein A resin; monoclonal antibody to obtain a first eluate; (5) filtering the first eluate through a depth filter; (6) contacting the filtered first eluate with a mixed mode resin to obtain a first flow-through containing lisenqizumab; (7) contacting the first flow-through with a cation exchange resin; (8) eluting lisenqizumab from the cation exchange resin to obtain a second eluate; (9) treating the second eluate by ultrafiltration and diafiltration to obtain a lisenqizumab drug product having an amount of PLA2 less than about 250 pg / mg of lisenqizumab.

[0191] Embodiment 123. A method as described in Embodiment 122, which also includes a virus inactivation step on the first eluate before step (5).

[0192] Embodiment 124. A method as described in Embodiment 122 or 123, further comprising filtering the second eluate for viruses before step (9).

[0193] Embodiment 125. A method for producing a lisenqizumab drug product, wherein the amount of lisenqizumab with high mannose N-glycans in the lisenqizumab drug product is less than about 5.4% of the total lisenqizumab material with N-glycosylation, the method comprising: (1) culturing a host cell line expressing lisenqizumab in a growth medium under conditions that allow the production of lisenqizumab; (2) clarifying the growth medium by centrifugation and depth filtration; (3) contacting the clarified medium containing lisenqizumab with a protein A resin; (4) eluting lisenqizumab from the protein A resin to obtain a first eluate; (5) filtering the first eluate through a depth filter; (6) contacting the filtered first eluate with a mixed mode resin to obtain a first flow-through containing levitra; (7) contacting the first flow-through with a cation exchange resin; (8) eluting levitra from the cation exchange resin to obtain a second eluate; (9) treating the second eluate by ultrafiltration and diafiltration to obtain a levitra drug product, wherein the amount of levitra with high mannose N-glycans in the levitra drug product is less than about 5.4% of the total levitra substance with N-glycosylation.

[0194] Embodiment 126. A method as described in Embodiment 125, which also includes a virus inactivation step on the first eluate before step (5).

[0195] Embodiment 127. A method as described in Embodiment 125 or 126, further comprising filtering the second eluate for viruses before step (9).

[0196] Embodiment 128. A composition comprising: (1) risankizumab; and (2) poloxamer 188 (P188), wherein the composition optionally does not comprise polysorbate 20 (PS20) and / or polysorbate 80 (PS80).

[0197] Embodiment 129. The composition of embodiment 128, comprising about 60 mg / ml to about 150 mg / ml of lisenqizumab.

[0198] Embodiment 130. The composition of embodiment 128 or 129, further comprising phospholipase A2 (PLA2).

[0199] Embodiment 131. The composition of any one of embodiments 128-130, wherein the PLA2 is PLA2G15.

[0200] Embodiment 132. The composition of any one of embodiments 128-131, wherein the level of PLA2 is greater than about 250 pg, wherein the level of PLA2 is greater than about 260 pg, greater than about 270 pg, greater than about 280 pg, greater than about 290 pg, greater than about 300 pg, greater than about 310 pg, greater than about 320 pg, greater than about 330 pg, greater than about 340 pg, greater than about 350 pg, greater than about 360 pg, greater than about 380 pg, greater than about 400 pg, greater than about 450 pg, greater than about 500 pg, greater than about 550 pg, greater than about 600 pg, greater than about 650 pg, greater than about 700 pg, greater than about 750 pg, greater than about 800 pg, greater than about 900 pg or greater than about 1000 pg / mg of lisenqizumab.

[0201] Embodiment 133. The composition of any one of embodiments 128-131, wherein the level of PLA2 is about 250 pg to about 1100 pg, about 260 pg to about 1100 pg, about 270 pg to about 1100 pg, about 280 pg to about 1100 pg, about 290 pg to about 1100 pg, about 300 pg to about 1100 pg, about 310 pg to about 1100 pg, about 320 pg to about 1100 pg, about 340 pg to about 1100 pg, about 3 From about 60pg to about 1100pg, from about 250pg to about 1000pg, from about 250pg to about 900pg, from about 250pg to about 800pg, from about 250pg to about 700pg, from about 250pg to about 600pg, from about 250pg to about 500pg, from about 250pg to about 400pg, from about 250pg to about 1030pg, from about 290pg to about 1090pg, from about 360pg to about 450pg or from about 310pg to about 920pg / mg of lisenqizumab.

[0202] Embodiment 134. A composition as described in any of embodiments 128-131, wherein the level of PLA2 is about 260 pg, about 270 pg, about 280 pg, about 290 pg, about 300 pg, about 310 pg, about 320 pg, about 330 pg, about 340 pg, about 350 pg, about 360 pg, about 380 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about 750 pg, about 800 pg, about 900 pg, about 1000 pg or about 1100 pg / mg of lisenqizumab.

[0203] Embodiment 135. The composition of any one of Embodiments 130-134, wherein the level of PLA2 is determined by ELISA.

[0204] Embodiment 136. The composition of any one of embodiments 128-135, wherein at least 85% of the initial amount of P118 is retained after storage at 5°C for 6 months.

[0205] Embodiment 137. The composition of any one of embodiments 128-135, wherein at least 80% of the initial amount of P118 is retained after storage at 5°C for 6 months.

[0206] Embodiment 138. The composition of any one of embodiments 128-135, wherein at least 65% of the initial amount of P118 is retained after storage at 25°C for 3 months.

[0207] Embodiment 139. The composition of any one of embodiments 128-135, wherein at least 60% of the initial amount of P118 is retained after storage at 25°C for 6 months.

[0208] Embodiment 140. The composition of any one of embodiments 128-135, wherein at least 60% of the initial amount of P118 is retained after storage at 40°C for 3 months.

[0209] Embodiment 141. The composition of any one of embodiments 128-135, wherein at least 60% of the initial amount of P118 is retained after storage at 40°C for 6 months.

[0210] Embodiment 142. The composition of any one of Embodiments 136-141, wherein the P188 is measured using a Pluronic F-68 colorimetric assay.

[0211] Other benefits of the present disclosure will be apparent to those skilled in the art by reading this patent application.The embodiments of the present disclosure described in the following paragraphs are intended to illustrate the present invention and should not be construed as narrowing the scope of the present invention.

[0212] definition

[0213] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0214] The term "and / or" as used herein in phrases such as "A and / or B" is intended to mean "A and B", "A or B", "A", or "B".

[0215] The term "about" generally refers to a numerical range that one skilled in the art would consider equivalent to the recited value (ie, having the same function or result). In many cases, the term "about" may include values ​​rounded to the nearest significant figure.

[0216] Unless the context requires otherwise, the terms “comprise,” “comprises,” and “comprising” are used with and without the clear understanding that they are to be interpreted as inclusive rather than exclusive, such that they indicate the inclusion of listed features but not the exclusion of one or more other such features.

[0217] The term "carrier" used in conjunction with a pharmaceutical excipient refers to any and all solvents, dispersion media, preservatives, coatings, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.

[0218] The terms "patient," "subject," "individual," and the like refer to a human being.

[0219] Risenqizumab

[0220] According to USAN, levitra has the following chemical name:

[0221] 1. IgG1, anti-(human interleukin 23 subunit p19) (human-Mus musculus heavy chain), disulfide bonded to human-Mus musculus kappa chain, dimer

[0222] 2. Immunoglobulin G1-κ, anti-(human interleukin-23 subunit α (IL-23-A, interleukin-23 subunit p19, IL-23p19)); humanized monoclonal antibody; gamma 1 heavy chain (1-449) [humanized VH (Homo sapiens IGHV1-69*08 (79%)-(IGHD)-IGHJ6*01 (91%)) [8.8.13] (1-120)-Homo sapiens IGHG1*03 {CH2 L 4 >A(237),L 5 >A(238), CH3 K 107 >-(450)}(121-449)], (223-214')-disulfide bonded to kappa light chain (1'-214')[humanized V-KAPPA(homo sapiens IGKV1-27*01(80%)-IGKJ2*02(91%))[6.3.9](1'-107')-homo sapiens IGKC*01(108'-214')]; dimer (229-229":232-232")-bis-disulfide

[0223] According to INN (see WHO Drug Information, 29(2), 254-255), Risenzizumab has the chemical name:

[0224] Immunoglobulin G1-κ, anti-[Homo sapiens IL23A (interleukin 23 subunit α, IL-23A, IL23 subunit p19IL23p19)], humanized monoclonal antibody; γ1 heavy chain (1-449) [humanized VH (Homo sapiens IGHV1-69*02 (79.40%)-(IGHD)-IGHJ5*01) [8.8.13] (1-120)-Homo sapiens IGHG1*01, G1m17,1 (CH1 (121-218), hinge (219-233), CH2L1.3>A (237), L1.2>A (238) (234-343), CH3 (344-448), CHS K2>del(449))(121-449)], (223-214')-disulfide bonded to kappa light chain (1'-214') [humanized V-KAPPA (homo sapiens IGKV1-27*01(80.00%)-IGKJ2*01)[6.3.9](1'-107')-homo sapiens IGKC*01, Km3(108'-214')]-disulfide bonded to dimer (229-229":232-232")-bis-disulfide

[0225] Lisenqizumab binds human IL-23 with high affinity and at an inhibitory concentration (IC) below 10 pM. 50 The results showed that IL-23-stimulated IL-17 production was inhibited at 1 pM, compared to 167 pM for ustekinumab in the same system. Risenkizumab did not affect IL-12 at the maximum tested concentration (33 nM), and it did not inhibit IL-12-stimulated IFN-γ production.

[0226] Lisenqizumab amino acid sequence

[0227] Risenqizumab has the CDRs shown in Tables 1 and 2. The variable regions of Risenqizumab are shown in Table 3.

[0228] Table 1

[0229]

[0230] Table 2

[0231]

[0232] Table 3

[0233]

[0234] Risenqizumab comprises the heavy chain and light chain sequences shown in Table 4.

[0235] Table 4

[0236] Lisenqizumab light chain sequence (SEQ ID NO: 9)

[0237]

[0238] Lisenqizumab heavy chain sequence (SEQ ID NO: 10)

[0239]

[0240] Risenqizumab compositions with reduced accompanying proteins

[0241] In one aspect, the present disclosure relates to a liquid composition comprising: (1) lisenqizumab; and (2) PLA2 in an amount less than about 250 pg / mg lisenqizumab.

[0242] In one embodiment, the liquid composition described herein comprises about 60 mg / ml to about 150 mg / ml risankizumab. For example, but not limited to, the liquid compositions described herein contain about 70 mg / ml to about 150 mg / ml, about 80 mg / ml to about 150 mg / ml, about 90 mg / ml to about 150 mg / ml, about 100 mg / ml to about 150 mg / ml, about 110 mg / ml to about 150 mg / ml, about 120 mg / ml to about 150 mg / ml, about 130 mg / ml to about 150 mg / ml, about 140 mg / ml to about 150 mg / ml, 60 mg / ml to about 70 mg / ml, 60 mg / ml to about 80 mg / ml, 60 mg / ml to about 90 mg / ml, 60 mg / ml to about 100 mg / ml, 60 mg / ml to about 110 mg / ml, 60 mg / ml to about 120 mg / ml, 60 mg / ml to about 130 mg / ml or 60 mg / ml to about 140 mg / ml of lisenqizumab, as well as ranges and amounts between any of these concentrations.

[0243] In one embodiment, the liquid composition described herein comprises about 60 mg / ml, about 70 mg / ml, about 80 mg / ml, about 90 mg / ml, about 100 mg / ml, about 110 mg / ml, about 120 mg / ml, about 130 mg / ml, about 140 mg / ml or about 150 mg / ml of lisenqizumab.

[0244] As used herein, the term "phospholipase A2" or "PLA2" refers to a family of enzymes that catalyze the hydrolysis of membrane phospholipids. PLA2 catalyzes the hydrolysis of the sn-2 position of membrane glycerophospholipids to release arachidonic acid (AA), which is a precursor of eicosanoids, including prostaglandins (PG) and leukotrienes (LT). The same reaction also produces lysophospholipids (Murakami and Kudo (2002) J. Biochem 131: 285-292), which represent another class of lipid mediators. There are at least sixteen classes of phospholipase A2. Dennis and colleagues classified these into six classes based on their properties: secretory phospholipase A2 (sPLA2 classes I, II, III, V, IX, X, XI, XII, XIII, and XIV); cytosolic phospholipase A2 (group IV cPLA2); calcium-independent phospholipase A2 (class VI iPLA2); PAF acetylhydrolases (GVII and GVIIIPAF-AH PLA2s); lysosomal phospholipase A2 (class XV LPLA2); and fat-specific phospholipase A2 (GXVIAdPLA) (Shayman and Tesmer (2019) Molecular and Cell Biology of Lipids 1864:932-940). In some embodiments, PLA2 according to the present disclosure is capable of catalyzing the hydrolysis of surfactants such as polysorbate 20 (PS20), polysorbate 80 (PS80), polysorbate 40 (PS40), polysorbate 60 (PS60), polysorbate 65 (PS65), or poloxamer 188, etc. Exemplary PLA2 according to the present disclosure include, but are not limited to, PLA2G15, PLA2G7, and PLA2G2.

[0245] As used herein, the term "PLA2G15", also referred to as "PLA2 XV class", refers to a unique member of the PLA2 family (Shayman et al. (2011) Prog. Lipid Res. 50: 1-13). PLA2G15 is localized in lysosomes and late endosomes within the cell, has an acidic pH optimum and acts as PLA2 (Abe and Shayman (2007) J. Lipid Res. 48: 2255-2263). The primary structure of PLA2G15 is highly conserved between mice, cattle and humans. There are six exons in the gene. The primary structure of the human and mouse enzyme consists of 412 amino acids (407 amino acids for the cattle enzyme). The enzyme contains a consensus sequence including a signal peptide cleavage site and a lipase motif AXSXG, which is unique to serine hydrolases. Serine is part of a catalytic triad that also includes aspartic acid and histidine. The amino-terminal 33 amino acid signal peptide is present together with a cleavage site between proline 33 and alanine 34 on mouse and human peptides. In addition, there are four N-linked glycosylation sites in mouse and human proteins (three in bovine proteins) (Hiraoka and Shayman (2005) J. Lipid Res. 46: 2441-2447). The structure and function of PLA2G15 are further described in Shayman and Tesmer, Molecular and Cell Biology of Lipids (2019) 1864: 932-940, the contents of which are incorporated herein by reference in their entirety.

[0246] Representative human PLA2G15 cDNA and human PLA2G15 protein sequences are well known in the art and are publicly available from the National Center for Biotechnology Information (NCBI). For example, two different human PLA2G15 isoforms are known. Human PLA2G15 isoform 1 (NP_036452.1) can be encoded by transcript variant 1 (NM_012320.4), which is a longer transcript. Human PLA2G15 isoform 2 (NP_001350480.1) can be encoded by transcript variant 2 (NM_001363551.2), which has a shorter and different C-terminus compared to isoform 1. The nucleic acid and polypeptide sequences of PLA2G15 orthologs in organisms other than humans are well known, and include, for example, chimpanzee PLA2G15 (XM_001167383.5 and XP_001167383.1), rhesus monkey PLA2G15 (NM_001265818.1 and NP_001252747.1), bovine PLA2G15 (NM_174560.2 and NP_776985.2), dog PLA2G15 (NM_001002940.1 and NP_001002940.1), rat PLA2G15 (NM_001004277.2 and NP_001004277.1), mouse PLA2G15 (NM_001357319.1 and NM_001357319.2), and mouse PLA2G15 (NM_001357319.1 and NM_001357319.1). P_001344248.1; NM_133792.3 and NP_598553.1), Chinese hamster PLA2G15 (XM_003504311.5 and XP_003504359.1; XM_027437910.2 and XP_027293711.1), chicken PLA2G15 (XM_001231518 .7 and XP_001231519.1), tropical clawed frog PLA2G15 (XM_012962222.3 and XP_012817676.2; XM_031900713.1 and XP_031756573.1) and zebrafish PLA2G15 (NM_001386706.1 and NP_001373635.1). Representative sequences of PLA2G15 orthologs are shown in Table 5.

[0247] Anti-PLA2G15 antibodies suitable for detecting PLA2G15 protein are well known in the art, including, for example, antibody catalog numbers NBP1-92089, H00023659-M01 and NBP2-17193, NBP1-92088 and NBP2-17192 (Novus Biologicals, Littleton, CO), antibody orb185108 (biorbyt, St. Louis, MO), antibody catalog numbers ABIN7004525, A BIN2580837, ABIN2580838 and ABIN2580836 (available on the World Wide Web at antibodies-online.com), antibody catalog numbers sc-376078, sc-529817, sc-543705, sc-522840, sc-376078AC, sc-376078HRP, sc-376078FITC, sc-376078PE and sc-376078AF488 (Santa Cruz Biotechnology, Dallas, TX), etc. In addition, reagents for detecting PLA2G15 expression are well known. Multiple clinical trials for PLA2G15 are available at the NIH Gene Testing Registry. Obtain (eg, GTR Test ID: GTR000543805.3, provided by Fulgent Clinical Diagnostic Laboratories, Temple City, CA)).

[0248] Table 5

[0249] SEQ ID NO:11 Mouse PLA2G15 isoform 1, amino acid sequence (NP_598553.1)

[0250]

[0251] SEQ ID NO:12 Mouse PLA2G15 isoform 2 amino acid sequence, (NP_001344248.1)

[0252]

[0253] SEQ ID NO: 13 Chinese hamster PLA2G15 isoform X1 amino acid sequence (XP_003504359.1)

[0254]

[0255] SEQ ID NO:14 Chinese hamster PLA2G15 isoform X2 amino acid sequence (XP_027293711.1)

[0256]

[0257] * Table 5 includes polypeptide molecules comprising an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical over its entire length to the amino acid sequence of any SEQ ID NO listed in Table 5, or a portion thereof. Such polypeptides may function as full-length polypeptides as further described herein. Also included are polypeptides with or without signal peptides, and / or polypeptides that include or only include preproteins, and / or polypeptides that include or only include mature proteins.

[0258] In one embodiment, the liquid compositions described herein contain a detectable amount of PLA2, wherein the detectable amount is less than 250 pg / mg of lisdex. For example, but not limited to, the amount of PLA2 in the liquid compositions described herein is less than about 240, less than about 220, less than about 200, less than about 180, less than about 160, less than about 140, less than about 120, less than about 100, less than about 90, less than about 80, less than about 70, less than about 60, less than about 50, less than about 40, less than about 30, less than about 25, less than about 20, less than about 15, less than about 10, less than about 9, less than about 8, less than about 7, less than about 6, less than about 5, less than about 4.4, less than about 3, less than about 2, less than about 1, less than about 0.5, less than about 0.1, less than about 0.05, less than about 0.06, less than about 0.08, less than about 0.10, less than about 0.09, less than about 0.11, less than about 0.09, less than about 0.01 .05 or less than about 0.01 pg / mg lishengqizumab, or any range between including the end values, such as about 200 to about 249, about 160 to about 200, about 120 to about 160, about 100 to about 120, about 80 to about 100, about 60 to about 80, about 40 to about 60, about 25 to about 40, about 10 to about 25, about 5 to about 10, about 4 to about 10, about 1 to about 5, about 1 to about 4, about 1 to about 3, about 1 to about 2, about 0.5 to about 1, about 0.1 to about 0.5, about 0.05 to about 0.1, or about 0.01 to about 0.5 pg / mg lishengqizumab. In some embodiments, the amount of PLA2 is less than or equal to the detection limit of the PLA2 detection assay, for example, less than or equal to about 9 pg / mg lishengqizumab; or less than or equal to about 4.4 pg / mg lishengqizumab. In one embodiment, the amount of PLA2 is from about 70 to about 240 pg / mg of risankizumab.

[0259] In one embodiment, the liquid composition described herein comprises PLA2 in an amount of about 240, about 220, about 200, about 180, about 160, about 140, about 120, about 100, about 90, about 80, about 70, about 60, about 50, about 40, about 30, about 25, about 20, about 15, about 10, about 9, about 8, about 7, about 6, about 5, about 4.4, about 3, about 2, about 1, about 0.5, about 0.1, about 0.05 or about 0.01 pg / mg of lisenqizumab.

[0260] In one embodiment, the liquid compositions described herein contain PLA2 in an amount of less than about 250 pg, but greater than about 240 pg, greater than about 220 pg, greater than about 200 pg, greater than about 180 pg, greater than about 160 pg, greater than about 140 pg, greater than about 120 pg, greater than about 100 pg, greater than about 90 pg, greater than about 80 pg, greater than about 70 pg, greater than about 60 pg, greater than about 50 pg, greater than about 40pg, greater than about 30pg, greater than about 25pg, greater than about 20pg, greater than about 15pg, greater than about 10pg, greater than about 9pg, greater than about 8pg, greater than about 7pg, greater than about 6pg, greater than about 5pg, greater than about 4pg, greater than about 3pg, greater than about 2pg, greater than about 1pg, greater than about 0.5pg, greater than about 0.1pg, greater than about 0.05pg or greater than about 0.01pg / mg of lisenqizumab.

[0261] In one embodiment, the present disclosure relates to a liquid composition comprising: (1) about 150 mg / ml of lisenqizumab, lisenqizumab comprising a light chain having an amino acid sequence of SEQ ID NO:9 and a heavy chain having an amino acid sequence of SEQ ID NO:10; and (2) an amount of PLA2 less than about 250 (e.g., not greater than 240, about 70 to about 240, less than about 9, or less than about 4.4) pg / mg of lisenqizumab.

[0262] The PLA2 in the liquid composition described herein can be PLA2G2, PLA2G15 or a combination thereof. In one embodiment, the PLA2 is PLA2G15.

[0263] The amount of PLA2 in the liquid compositions described herein can be determined using methods known in the art, such as by mass spectrometry or by ELISA. In one embodiment, the amount of PLA2 in the liquid compositions described herein is determined by ELISA, such as the ELISA method described in Example 9.

[0264] In one embodiment, the PLA2 in the liquid composition described herein is derived from a CHO cell line.

[0265] In one embodiment, the liquid composition described herein further comprises one or more of a surfactant, a polyol, and a buffer.

[0266] The polyol may be selected from the group consisting of trehalose, mannitol, sucrose and sorbitol. In one embodiment, the polyol is trehalose, and the amount of trehalose is about 150 to about 220 mM (eg, about 185 mM).

[0267] The buffer may be selected from the group consisting of acetate buffer, histidine buffer, citrate buffer, phosphate buffer, glycine buffer and arginine buffer. In one embodiment, the buffer is acetate buffer, and the amount of acetate buffer is about 5 to about 100 mM (e.g., about 10 mM).

[0268] The surfactant may be selected from the group consisting of polysorbate 20 (PS20), polysorbate 80 (PS80), polysorbate 40 (PS40), polysorbate 60 (PS60), polysorbate 65 (PS65), and poloxamer 188. In one embodiment, the surfactant is PS20, and the amount of PS20 is at most 1.0 mg / ml (e.g., about 1.0 mg / ml, about 0.8 mg / ml, about 0.6 mg / ml, about 0.4 mg / ml, about 0.2 mg / mL, or about 0.1 mg / ml). In another embodiment, the surfactant is PS80, and the amount of PS80 is at most 1.0 mg / ml (e.g., about 1.0 mg / ml, about 0.8 mg / ml, about 0.6 mg / ml, about 0.4 mg / ml, about 0.2 mg / ml, or about 0.1 mg / ml).

[0269] In one embodiment, the liquid compositions described herein have a pH of about 5.0 to about 6.5 (eg, about 5.7).

[0270] In one embodiment, the liquid composition described herein comprises (1) 150 mg / mL of lisenqizumab, lisenqizumab comprising a light chain having an amino acid sequence of SEQ ID NO:9 and a heavy chain having an amino acid sequence of SEQ ID NO:10; 185 mM trehalose; 10 mM acetate; and 0.20 mg / mL polysorbate 20, wherein the liquid composition has a pH of about 5.7; and (2) an amount of PLA2 (e.g., PLA2G15) less than about 250 pg / mg of lisenqizumab.

[0271] In one embodiment, the liquid composition described herein comprises (1) 150 mg / ml of lisenqizumab, lisenqizumab comprising a light chain having an amino acid sequence of SEQ ID NO: 9 and a heavy chain having an amino acid sequence of SEQ ID NO: 10; 0.054 mg / mL acetic acid; 1.24 mg / mL sodium acetate trihydrate; 70 mg / mL trehalose dihydrate; 0.20 mg / mL polysorbate 20; and wherein the liquid composition has a pH of about 5.7. and (2) an amount of PLA2 (e.g., PLA2G15) less than about 250 pg / mg of lisenqizumab.

[0272] In one embodiment, the liquid composition described herein comprises (1) 150 mg / ml of lisenqizumab, lisenqizumab comprising a light chain having an amino acid sequence of SEQ ID NO: 9 and a heavy chain having an amino acid sequence of SEQ ID NO: 10; 0.054 mg / mL acetic acid; 1.24 mg / mL sodium acetate trihydrate; 70 mg / mL trehalose dihydrate; 0.20 mg / mL polysorbate 20; and wherein the liquid composition has a pH of about 5.7. and (2) an amount of PLA2 (e.g., PLA2G15) less than about 250 (e.g., no more than 240, about 70 to about 240, less than about 9, or less than about 4.4) pg / mg of lisenqizumab.

[0273] In one embodiment, the liquid composition described herein comprises (1) 150 mg / ml of lisenqizumab, lisenqizumab comprising a light chain having an amino acid sequence of SEQ ID NO: 9 and a heavy chain having an amino acid sequence of SEQ ID NO: 10; 0.054 mg / mL acetic acid; 1.24 mg / mL sodium acetate trihydrate; 70 mg / mL trehalose dihydrate; 0.20 mg / mL polysorbate 20; and wherein the liquid composition has a pH of about 5.7. and (2) an amount of PLA2 (e.g., PLA2G15) less than about 250 (e.g., no more than 240, about 70 to about 240, less than about 9, or less than about 4.4) pg / mg of lisenqizumab, wherein the amount of PLA2 is determined by ELISA (e.g., the ELISA method described in Example 9).

[0274] Liquid formulations contemplated by the present disclosure may include added water, such as USP grade water.

[0275] In some embodiments, the liquid drug formulations described herein are packaged in vials, pre-filled syringes, or on-body devices.

[0276] In some embodiments, the liquid pharmaceutical formulations described herein are suitable for parenteral administration. Parenteral administration includes, for example, subcutaneous, intramuscular, intradermal, intramedullary injection, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, and intravitreal. In one embodiment, the disclosed liquid formulations are injectable formulations. In one embodiment, the liquid formulations disclosed herein are suitable for subcutaneous injection or intravenous injection.

[0277] Risenzizumab with reduced high-mannose N-glycans, increased purity and / or reduced immunogenicity Composition

[0278] In another aspect, the present disclosure relates to a composition comprising lisenqizumab, wherein the composition has one or more of the following characteristics: (a) less than about 5.4% of the total lisenqizumab species with N-glycosylation have high mannose N-glycans; (b) at least about 99.1% of the lisenqizumab is present as a monomer and / or no more than about 0.4% of the lisenqizumab is present as a high molecular weight (HMW) species as measured by ultra-high performance size exclusion chromatography (UP-SEC); (c) greater than about 97.5% of the lisenqizumab is present as a major peak and / or less than about 2.2% of the lisenqizumab is present as a low molecular weight (LMW) species as measured by capillary gel electrophoresis under non-reducing conditions (CGE-NR); and / or (d) the incidence of anti-drug antibodies (ADA) arising during treatment is less than about 4.7% after a single subcutaneous 150 mg dose of the composition is administered to humans.

[0279] In one embodiment, the composition is a pharmaceutical composition.

[0280] In one embodiment, the pharmaceutical composition is a liquid composition.

[0281] In one embodiment, the pharmaceutical composition is an aqueous composition.

[0282] In one embodiment, the disclosure relates to a composition comprising risankizumab, wherein the composition has characteristic (a): less than about 5.4% of the total risankizumab species having N-glycosylation have high mannose N-glycans.

[0283] In one embodiment, the present disclosure relates to a composition comprising lisenqizumab, wherein the composition has characteristic (b): at least about 99.1% of the lisenqizumab is present as a monomer and / or no more than about 0.4% of the lisenqizumab is present as a high molecular weight (HMW) species as measured by ultra-high performance size exclusion chromatography (UP-SEC).

[0284] In one embodiment, the present disclosure relates to a composition comprising lisenqizumab, wherein the composition has characteristic (c) as measured by capillary gel electrophoresis under non-reducing conditions (CGE-NR), greater than about 97.5% of the lisenqizumab is present as a major peak and / or less than about 2.2% of the lisenqizumab is present as low molecular weight (LMW) species.

[0285] In one embodiment, the present disclosure relates to a composition comprising risankizumab, wherein the composition has characteristic (d) the incidence of anti-drug antibodies (ADA) arising during treatment is less than about 4.7% after a single subcutaneous 150 mg dose of the composition is administered to humans.

[0286] In one embodiment, the present disclosure relates to a composition comprising lisenqizumab, wherein the composition has the characteristics that (a) less than about 5.4% of the total lisenqizumab species with N-glycosylation have high mannose N-glycans; and (b) at least about 99.1% of the lisenqizumab is present as monomers and / or no more than about 0.4% of the lisenqizumab is present as high molecular weight (HMW) species as measured by ultra-high performance size exclusion chromatography (UP-SEC).

[0287] In one embodiment, the present disclosure relates to a composition comprising lixinqizumab, wherein the pharmaceutical composition has the characteristics that (a) less than about 5.4% of the total lixinqizumab species with N-glycosylation have high mannose N-glycans; and (c) as measured by capillary gel electrophoresis under non-reducing conditions (CGE-NR), greater than about 97.5% of the lixinqizumab is present as a major peak and / or less than about 2.2% of the lixinqizumab is present as low molecular weight (LMW) species.

[0288] In one embodiment, the present disclosure relates to a composition comprising levitra, wherein the composition has the characteristics of (a) less than about 5.4% of the total levitra substance with N-glycosylation has high mannose N-glycans; and (d) the incidence of anti-drug antibodies (ADA) arising during treatment is less than about 4.7% after administration of a single subcutaneous 150 mg dose of the composition.

[0289] In one embodiment, the present disclosure relates to a composition comprising lishengqizumab, wherein the composition has the characteristics (b) at least about 99.1% of the lishengqizumab is present as a monomer and / or no more than about 0.4% of the lishengqizumab is present as a high molecular weight (HMW) species as measured by ultra-high performance size exclusion chromatography (UP-SEC); and (c) greater than about 97.5% of the lishengqizumab is present as a major peak and / or less than about 2.2% of the lishengqizumab is present as a low molecular weight (LMW) species as measured by capillary gel electrophoresis under non-reducing conditions (CGE-NR).

[0290] In one embodiment, the present disclosure relates to a composition comprising lisenqizumab, wherein the composition has the characteristics (b) at least about 99.1% of the lisenqizumab is present as monomer and / or no more than about 0.4% of the lisenqizumab is present as high molecular weight (HMW) species as measured by ultra-high performance size exclusion chromatography (UP-SEC); and (d) after a single subcutaneous dose of the composition, the incidence of anti-drug antibodies (ADA) occurring during treatment is less than about 4.7%.

[0291] In one embodiment, the present disclosure relates to a composition comprising lisenqizumab, wherein the composition has the characteristics (c) greater than about 97.5% of the lisenqizumab is present as a major peak and / or less than about 2.2% of the lisenqizumab is present as a low molecular weight (LMW) substance as measured by capillary gel electrophoresis under non-reducing conditions (CGE-NR); and (d) after a single subcutaneous dose of the composition, the incidence of anti-drug antibodies (ADA) occurring during the treatment period is less than about 4.7%.

[0292] In one embodiment, the present disclosure relates to a composition comprising lishengqizumab, wherein the composition has the characteristics that (a) less than about 5.4% of the total lishengqizumab substances with N-glycosylation have high mannose N-glycans; (b) at least about 99.1% of the lishengqizumab is present as a monomer and / or no more than about 0.4% of the lishengqizumab is present as a high molecular weight (HMW) species as measured by ultra-high performance size exclusion chromatography (UP-SEC); and (c) greater than about 97.5% of the lishengqizumab is present as a major peak and / or less than about 2.2% of the lishengqizumab is present as a low molecular weight (LMW) species as measured by capillary gel electrophoresis under non-reducing conditions (CGE-NR).

[0293] In one embodiment, the present disclosure relates to a composition comprising lisenqizumab, wherein the composition has the characteristics that (a) less than about 5.4% of the total lisenqizumab species with N-glycosylation have high mannose N-glycans; (b) at least about 99.1% of the lisenqizumab is present as monomers and / or no more than about 0.4% of the lisenqizumab is present as high molecular weight (HMW) species as measured by ultra-high performance size exclusion chromatography (UP-SEC); and (d) the incidence of anti-drug antibodies (ADA) arising during treatment is less than about 4.7% after a single subcutaneous 150 mg dose of the composition is administered to humans.

[0294] In one embodiment, the present disclosure relates to a composition comprising lisenqizumab, wherein the composition has the characteristics (b) at least about 99.1% of the lisenqizumab is present as a monomer and / or no more than about 0.4% of the lisenqizumab is present as a high molecular weight (HMW) species as measured by ultra-high performance size exclusion chromatography (UP-SEC); (c) greater than about 97.5% of the lisenqizumab is present as a major peak and / or less than about 2.2% of the lisenqizumab is present as a low molecular weight (LMW) species as measured by capillary gel electrophoresis under non-reducing conditions (CGE-NR); and (d) after administration of a single subcutaneous dose of the composition to humans, the incidence of anti-drug antibodies (ADA) arising during treatment is less than about 4.7%.

[0295] In one embodiment, the present disclosure relates to a composition comprising lisenqizumab, wherein the composition has the characteristics that (a) less than about 5.4% of the total lisenqizumab species with N-glycosylation have high mannose N-glycans; (b) at least about 99.1% of the lisenqizumab is present as monomers and / or no more than about 0.4% of the lisenqizumab is present as high molecular weight (HMW) species as measured by ultra-high performance size exclusion chromatography (UP-SEC); (c) greater than about 97.5% of the lisenqizumab is present as a major peak and / or less than about 2.2% of the lisenqizumab is present as low molecular weight (LMW) species as measured by capillary gel electrophoresis under non-reducing conditions (CGE-NR); and (d) after administration of a single subcutaneous dose of the composition to humans, the incidence of anti-drug antibodies (ADA) arising during treatment is less than about 4.7%.

[0296] In one embodiment, the compositions described herein comprise about 60 mg / ml to about 150 mg / ml risankizumab. For example, but not limited to, the compositions described herein contain about 70 mg / ml to about 150 mg / ml, about 80 mg / ml to about 150 mg / ml, about 90 mg / ml to about 150 mg / ml, about 100 mg / ml to about 150 mg / ml, about 110 mg / ml to about 150 mg / ml, about 120 mg / ml to about 150 mg / ml, about 130 mg / ml to about 150 mg / ml, about 140 mg / ml to about 150 mg / ml, 60 mg / ml to about 70 mg / ml, 60 mg / ml to about 80 mg / ml, 60 mg / ml to about 90 mg / ml, 60 mg / ml to about 100 mg / ml, 60 mg / ml to about 110 mg / ml, 60 mg / ml to about 120 mg / ml, 60 mg / ml to about 130 mg / ml, or 60 mg / ml to about 140 mg / ml of lisenqizumab, as well as ranges and amounts between any of these concentrations.

[0297] In one embodiment, the composition described herein comprises about 60 mg / ml, about 70 mg / ml, about 80 mg / ml, about 90 mg / ml, about 100 mg / ml, about 110 mg / ml, about 120 mg / ml, about 130 mg / ml, about 140 mg / ml or about 150 mg / ml of lisenqizumab.

[0298] In one embodiment, risankizumab is produced in a CHO cell line.

[0299] In one embodiment, the compositions described herein further comprise a pharmaceutically acceptable excipient.

[0300] In one embodiment, a pharmaceutical composition described herein comprises 150 mg / ml risankizumab; 185 mM trehalose; 10 mM acetate; and 0.20 mg / mL polysorbate 20, wherein the pharmaceutical composition has a pH of about 5.7.

[0301] In one embodiment, the pharmaceutical composition described herein comprises 150 mg / ml risankizumab; 0.054 mg / mL acetic acid; 1.24 mg / mL sodium acetate trihydrate; 70 mg / mL trehalose dihydrate; 0.20 mg / mL polysorbate 20; and Water for Injection, USP; wherein the pharmaceutical composition has a pH of about 5.7.

[0302] In one embodiment, a pharmaceutical composition described herein has a pH of about 5.0 to about 6.5 (eg, about 5.7).

[0303] In some embodiments, the pharmaceutical compositions described herein are packaged in vials, prefilled syringes, or on-body devices.

[0304] In some embodiments, the pharmaceutical compositions described herein are suitable for parenteral administration. Parenteral administration includes, for example, subcutaneous, intramuscular, intradermal, intramedullary injection, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, and intravitreal. In one embodiment, the disclosed liquid formulations are injectable formulations. In one embodiment, the liquid formulations disclosed herein are suitable for subcutaneous injection or intravenous injection.

[0305] In some aspects, provided herein is a method of treating an immune disease using a composition described herein. Immune diseases include, but are not limited to, autoimmune diseases and inflammatory diseases (eg, psoriasis, inflammatory bowel disease, ulcerative colitis, psoriatic arthritis, and Crohn's disease).

[0306] a. Lisenqizumab pharmaceutical composition with reduced high-mannose N-glycans

[0307] In one embodiment, the compositions provided herein have at least characteristic (a): less than about 5.4% of the total risankizumab species having N-glycosylation have high mannose N-glycans.

[0308] In one embodiment, the high mannose N-glycans include one or more high mannose N-glycans selected from mannose 5 N-glycans (M5), mannose 6 N-glycans (M6), and mannose 7 N-glycans (M7). For example, in one embodiment, the high mannose N-glycans are M5, M6, and M7.

[0309] In one embodiment, the composition described herein comprises a plurality of lisenqizumab species with or without N-glycosylation, wherein the sum of lisenqizumab with M5, lisenqizumab with M6, and lisenqizumab with M7 is less than 5.4% of the total lisenqizumab species with N-glycosylation. For example, but not limited to, the sum of lisenqizumab with M5, lisenqizumab with M6, and lisenqizumab with M7 in the compositions described herein is a detectable amount that is less than about 5.3%, less than about 5.2%, less than about 5.1%, less than about 5.0%, less than about 4.9%, less than about 4.8%, less than about 4.7%, less than about 4.6%, less than about 4.5%, less than about 4.4%, less than about 4.3%, less than about 4.2%, less than about 4.1%, less than about 4.0%, less than about 3.9%, less than about 3. 8% or less than about 3.7%, or any range therebetween, inclusive, such as about 3.6% to about 5.3%, about 3.6% to about 5.0%, about 3.6% to about 4.8%, about 3.6% to about 4.5%, about 3.6% to about 4.1%, about 3.6% to about 3.8%, about 3.8% to about 5.3%, about 4.1% to about 5.3%, about 4.5% to about 5.3%, about 4.8% to about 5.3%, about 5.0% to about 5.3%, about 4.3% to about 4.9%, or about 3.6% to about 4.9% of the total lisenqizumab material with N-glycosylation.

[0310] In one embodiment, the composition described herein comprises the sum of lisenqizumab with M5, lisenqizumab with M6, and lisenqizumab with M7, the sum being less than about 5.4% but greater than about 5.3%, greater than about 5.2%, greater than about 5.1%, greater than about 5.0%, greater than about 4.9%, greater than about 4.8%, greater than about 4.7%, greater than about 4.6%, greater than about 4.5%, greater than about 4.4%, greater than about 4.3%, greater than about 4.2%, greater than about 4.1%, greater than about 4.0%, greater than about 3.9%, greater than about 3.8%, greater than about 3.7% or greater than about 3.6% of the total lisenqizumab material with N-glycosylation.

[0311] In one embodiment, the composition described herein comprises the sum of lisenqizumab with M5, lisenqizumab with M6, and lisenqizumab with M7, the sum being about 5.3%, about 5.2%, about 5.1%, about 5.0%, about 4.9%, about 4.8%, about 4.7%, about 4.6%, about 4.5%, about 4.4%, about 4.3%, about 4.2%, about 4.1%, about 4.0%, about 3.9%, about 3.8%, about 3.7% or about 3.6% of the total lisenqizumab material with N-glycosylation.

[0312] In one embodiment, the disclosure relates to a composition comprising about 150 mg / ml of an antibody comprising a light chain having the amino acid sequence of SEQ ID NO:9 and a heavy chain having the amino acid sequence of SEQ ID NO:10, with or without N-glycosylation, wherein less than about 5.4% (e.g., about 3.6% to about 4.1%, about 4.3% to about 4.9%, or about 3.6% to about 4.9%) of the total antibody material with N-glycosylation has high mannose N-glycans (e.g., M5, M6, and M7).

[0313] In one embodiment, the high mannose N-glycan is M5.

[0314] In one embodiment, the compositions described herein comprise a plurality of lisenqizumab species with or without N-glycosylation, wherein the level of lisenqizumab with M5 is less than 5.3% of the total lisenqizumab species with N-glycosylation. For example, but not limited to, the level of lisenqizumab with M5 in the compositions described herein is a detectable amount of less than about 5.2%, less than about 5.1%, less than about 5.0%, less than about 4.9%, less than about 4.8%, less than about 4.7%, less than about 4.6%, less than about 4.5%, less than about 4.4%, less than about 4.3%, less than about 4.2%, less than about 4.1%, less than about 4.0%, less than about 3.9%, less than about 3.8%, less than about 3.7%, less than about 3.6%, less than about 3.5%, less than about 3.4%, less than about 3.3%, less than about 3.2% of the total lisenqizumab species with N-glycosylation. , less than about 3.1%, less than about 3.0%, less than about 2.9%, or less than about 2.8%, or any range therebetween, including the end values, such as about 2.7% to about 5.2%, about 3.1% to about 5.2%, about 3.5% to about 5.2%, about 4.0% to about 5.2%, about 4.5% to about 5.2%, about 5% to about 5.2%, about 2.7% to about 5.0%, about 2.7% to about 4.5%, about 2.7% to about 4.0%, about 2.7% to about 3.5%, about 2.7% to about 3.1%, about 3.2% to about 3.7%, or about 2.7% to about 3.7% of the total lisenqizumab material with N-glycosylation.

[0315] In one embodiment, the compositions described herein comprise a detectable amount of lisenqizumab having M5, the detectable amount being less than about 5.3% of the total lisenqizumab material having N-glycosylation, but greater than about 5.2%, greater than about 5.1%, greater than about 5.0%, greater than about 4.9%, greater than about 4.8%, greater than about 4.7%, greater than about 4.6%, greater than about 4.5%, greater than about 4.4%, greater than about 4.3%, greater than about 4.2%, greater than about 4.1%, greater than about 4.0%, greater than about 3.9%, greater than about 3.8%, greater than about 3.7%, greater than about 3.6%, greater than about 3.5%, greater than about 3.4%, greater than about 3.3%, greater than about 3.2%, greater than about 3.1%, greater than about 3.0%, greater than about 2.9% or greater than about 2.8% or greater than about 2.7%.

[0316] In one embodiment, the composition described herein comprises lisenqizumab having M5 in an amount of about 2.7%, about 2.8%, about 2.9%, about 3.0%, about 3.1%, about 3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4.0%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, about 5.0%, about 5.1% or about 5.2% of the total lisenqizumab material having N-glycosylation.

[0317] In one embodiment, the present disclosure relates to a composition comprising about 150 mg / ml of lisenqizumab with or without N-glycosylation, wherein less than about 5.3% (e.g., about 2.7% to about 3.1%, about 3.2% to about 3.7%, or about 2.7% to about 3.7%) of the total lisenqizumab material with N-glycosylation has M5.

[0318] In one embodiment, the high mannose N-glycan is M6.

[0319] In one embodiment, the compositions described herein comprise a plurality of lishengqizumab species with or without N-glycosylation, wherein the level of lishengqizumab with M6 is less than 2.6% of the total lishengqizumab species with N-glycosylation. For example, but not limited to, the level of lishengqizumab with M6 in the compositions described herein is a detectable amount of less than about 2.5%, less than about 2.4%, less than about 2.3%, less than about 2.2%, less than about 2.1%, less than about 2.0%, less than about 1.9%, less than about 1.8%, less than about 1.7%, less than about 1.6%, less than about 1.5%, less than about 1.4%, less than about 1.3%, less than about 1.2%, less than about 1.1%, less than about 1.0%, less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, or less than about 0. .5%, or any range therebetween inclusive, such as about 0.4% to about 2.5%, about 0.4% to about 2.4%, about 0.4% to about 2.2%, about 0.4% to about 2.0%, about 0.4% to about 1.8%, about 0.4% to about 1.6%, about 0.4% to about 1.4%, about 0.4% to about 1.2%, about 0.4% to about 1.0%, about 0.4% to about 0.9%, about 0.4% to about 0.8%, about 0.4% to about 0.7%, about 0.4% to about 0.6%, about 0.4% to about 0.5%, or about 0.6% to about 0.7% of the total lisenqizumab material with N-glycosylation.

[0320] In one embodiment, the compositions described herein comprise a detectable amount of lisenqizumab with M6, the detectable amount being less than about 2.6%, but greater than about 2.5%, greater than about 2.4%, greater than about 2.3%, greater than about 2.2%, greater than about 2.1%, greater than about 2.0%, greater than about 1.9%, greater than about 1.8%, greater than about 1.7%, greater than about 1.6%, greater than about 1.5%, greater than about 1.4%, greater than about 1.3%, greater than about 1.2%, greater than about 1.1%, greater than about 1.0%, greater than about 0.9%, greater than about 0.8%, greater than about 0.7%, greater than about 0.6%, greater than about 0.5% or greater than about 0.4% of the total lisenqizumab material with N-glycosylation.

[0321] In one embodiment, the composition described herein comprises lisenqizumab with M6 in an amount of about 2.5%, about 2.4%, about 2.3%, about 2.2%, about 2.1%, about 2.0%, about 1.9%, about 1.8%, about 1.7%, about 1.6%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1.0%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5% or about 0.4% of the total lisenqizumab material with N-glycosylation.

[0322] In one embodiment, the present disclosure relates to a composition comprising about 150 mg / ml of lisenqizumab with or without N-glycosylation, wherein less than about 2.6% (e.g., about 0.4% to about 0.5%, about 0.6% to about 0.7%, or about 0.4% to about 0.7%) of the total lisenqizumab material with N-glycosylation has M6.

[0323] In one embodiment, the high mannose N-glycan is M7.

[0324] In one embodiment, the compositions described herein comprise a plurality of lisenqizumab species with or without N-glycosylation, wherein the level of lisenqizumab with M7 is less than 2.0% of the total lisenqizumab species with N-glycosylation. For example, but not limited to, the level of lisenqizumab with M6 in the compositions described herein is a detectable amount of less than about 1.9%, less than about 1.8%, less than about 1.7%, less than about 1.6%, less than about 1.5%, less than about 1.4%, less than about 1.3%, less than about 1.2%, less than about 1.1%, less than about 1.0%, less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, less than about 0.5%, or greater than about 0.4% of the total lisenqizumab species with N-glycosylation. %, or any range therebetween, including the end values, such as about 0.4% to about 1.9%, about 0.4% to about 1.8%, about 0.4% to about 1.6%, about 0.4% to about 1.4%, about 0.4% to about 1.2%, about 0.4% to about 1.0%, about 0.4% to about 0.9%, about 0.4% to about 0.8%, about 0.4% to about 0.7%, about 0.4% to about 0.6%, about 0.4% to about 0.5%, or about 0.5% to about 0.6% of the total lisenqizumab material with N-glycosylation.

[0325] In one embodiment, the composition described herein comprises a detectable amount of lisenqizumab with M7, the detectable amount being less than about 2.0% but greater than about 1.9%, greater than about 1.8%, greater than about 1.7%, greater than about 1.6%, greater than about 1.5%, greater than about 1.4%, greater than about 1.3%, greater than about 1.2%, greater than about 1.1%, greater than about 1.0%, greater than about 0.9%, greater than about 0.8%, greater than about 0.7%, greater than about 0.6%, greater than about 0.5% or greater than about 0.4% of the total lisenqizumab material with N-glycosylation.

[0326] In one embodiment, the composition described herein comprises lisenqizumab with M7 in an amount of about 1.9%, about 1.8%, about 1.7%, about 1.6%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1.0%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5% or about 0.4% of the total lisenqizumab material with N-glycosylation.

[0327] In one embodiment, the present disclosure relates to a composition comprising about 150 mg / ml of lisenqizumab with or without N-glycosylation, wherein less than about 2.0% (e.g., about 0.4% to about 0.5%, about 0.5% to about 0.6%, or about 0.4% to about 0.6%) of the total lisenqizumab material with N-glycosylation has M7.

[0328] In one embodiment, the amount of lisenqizumab having high mannose N-glycans in the compositions described herein can be determined using methods known in the art, such as by 2-AB and HILIC-FL chromatography (e.g., using 2-AB and HILIC-FL chromatography described in Example 12) or by RapiFluor HILIC-FL chromatography (using RapiFluor HILIC-FL chromatography described in Example 13).

[0329] In one embodiment, a pharmaceutical composition described herein comprises about 150 mg / mL levitra; 185 mM trehalose; 10 mM acetate; and 0.20 mg / mL polysorbate 20, wherein the pharmaceutical composition has a pH of about 5.7; and wherein less than about 5.4% of the total levitra material having N-glycosylation has high mannose N-glycans (e.g., M5, M6, and / or M7).

[0330] In one embodiment, the pharmaceutical composition described herein comprises about 150 mg / ml lisenqizumab; 0.054 mg / mL acetic acid; 1.24 mg / mL sodium acetate trihydrate; 70 mg / mL trehalose dihydrate; 0.20 mg / mL polysorbate 20; and wherein the pharmaceutical composition has a pH of about 5.7. And wherein less than about 5.4% of the total lisenqizumab material having N-glycosylation has high mannose N-glycans (e.g., M5, M6 and / or M7).

[0331] In one embodiment, the pharmaceutical composition described herein comprises about 150 mg / ml lisenqizumab; 0.054 mg / mL acetic acid; 1.24 mg / mL sodium acetate trihydrate; 70 mg / mL trehalose dihydrate; 0.20 mg / mL polysorbate 20; and, wherein the pharmaceutical composition has a pH of about 5.7; and wherein less than about 5.4% of the total lisenqizumab material having N-glycosylation has high mannose N-glycans (e.g., M5, M6 and / or M7); wherein the amount of lisenqizumab having high mannose N-glycans is determined by 2-AB and HILIC-FL chromatography (e.g., using the 2-AB and HILIC-FL chromatography described in Example 12) or by RapiFluorHILIC-FL chromatography (e.g., using the RapiFluor HILIC-FL chromatography described in Example 13).

[0332] In one embodiment, the pharmaceutical composition described herein comprises about 150 mg / ml lisenqizumab; 0.054 mg / mL acetic acid; 1.24 mg / mL sodium acetate trihydrate; 70 mg / mL trehalose dihydrate; 0.20 mg / mL polysorbate 20; and wherein the pharmaceutical composition has a pH of about 5.7; and wherein the sum of lisenqizumab with M5, lisenqizumab with M6, and lisenqizumab with M7 is less than 5.4% (e.g., about 3.6% to about 4.1%, about 4.3% to 4.9%, or about 3.6% to about 4.9%) of the total lisenqizumab material with N-glycosylation.

[0333] In one embodiment, the pharmaceutical composition described herein comprises about 150 mg / ml risankizumab; 0.054 mg / mL acetic acid; 1.24 mg / mL sodium acetate trihydrate; 70 mg / mL trehalose dihydrate; 0.20 mg / mL polysorbate 20; and wherein the pharmaceutical composition has a pH of about 5.7. And wherein the sum of lisenqizumab with M5, lisenqizumab with M6, and lisenqizumab with M7 is less than 5.4% (e.g., about 3.6% to about 4.1%, about 4.3% to 4.9%, or about 3.6% to about 4.9%) of the total lisenqizumab material with N-glycosylation, wherein the amount of lisenqizumab with high mannose N-glycans is determined by 2-AB and HILIC-FL chromatography (e.g., using 2-AB and HILIC-FL chromatography described in Example 12) or by RapiFluor HILIC-FL chromatography (e.g., using RapiFluor HILIC-FL chromatography described in Example 13).

[0334] Liquid formulations contemplated by the present disclosure may include added water, such as USP grade water.

[0335] In some embodiments, greater than about 84.4% of the total lisenqizumab material with N-glycosylation has fucosylated complex oligosaccharides. For example, but not limited to, the level of lisenqizumab with fucosylated complex oligosaccharides in the compositions described herein is an amount greater than about 85%, greater than about 85.5%, greater than about 86%, greater than about 86.5%, greater than about 87%, greater than about 87.5%, greater than about 88%, greater than about 88.5%, greater than about 89%, greater than about 89.5%, greater than about 90%, or greater than about 90.5% of the total lisenqizumab material with N-glycosylation.

[0336] In some embodiments, the level of lisenqizumab with fucosylated complex oligosaccharides in the compositions described herein is an amount of about 85% to about 91%, about 88.0% to about 88.9%, about 89.8% to about 90.9%, or 88.0% to 90.9% of the total lisenqizumab species with N-glycosylation. In some embodiments, the level of lisenqizumab with fucosylated complex oligosaccharides in the compositions described herein is an amount of about 88.0%, about 88.3%, about 88.4%, about 88.9%, about 89.8%, about 90.2%, or about 90.9% of the total lisenqizumab species with N-glycosylation.

[0337] In some embodiments, the level of risankizumab with fucosylated complex oligosaccharides is determined by 2-AB and HILIC-FL chromatography or by RapiFluor HILIC-FL chromatography.

[0338] In some embodiments, the composition comprises about 0.8% to about 1.4% (e.g., about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, or about 1.4%) of aglycosylated lisenkizumab.

[0339] In some embodiments, aglycosylated lisanzumab is determined by tryptic peptide mapping (e.g., using the tryptic peptide mapping analysis described in Example 14).

[0340] b. Risenqizumab compositions with increased purity

[0341] In one embodiment, the compositions provided herein have at least feature (b): at least about 99.1% of the lishengqizumab is present as a monomer and / or no more than about 0.4% of the lishengqizumab is present as a high molecular weight (HMW) species as measured by ultra-high performance size exclusion chromatography (UP-SEC). For example, the compositions described herein contain lishengqizumab, wherein at least about 99.1% of the lishengqizumab is present as a monomer and no more than about 0.4% of the lishengqizumab is present as a high molecular weight (HMW) species as measured by UP-SEC.

[0342] In one embodiment, at least about 99.1% of the lishengqizumab is present as a monomer in the composition provided herein as measured by UP-SEC. For example, but not limited to, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.6%, or at least about 99.7% of the lishengqizumab is present as a monomer as measured by UP-SEC, or any range between, including the end values, such as about 99.1% to about 99.7%, 99.1% to about 99.6%, about 99.2% to about 99.7%, or about 99.2% to about 99.6% of the lishengqizumab is present as a monomer as measured by UP-SEC.

[0343] In one embodiment, no more than about 0.4% of the lisenqizumab is present in the compositions provided herein as high molecular weight (HMW) species as measured by UP-SEC. For example, but not limited to, no more than about 0.35%, no more than about 0.3%, no more than about 0.25%, no more than about 0.2%, no more than about 0.15%, or no more than about 0.1% of the lisenqizumab is present as high molecular weight (HMW) species as measured by UP-SEC, or any range between, including the end values, such as about 0.1% to about 0.4%, about 0.1% to about 0.3%, about 0.1% to about 0.2%, about 0.2% to about 0.4%, or about 0.2% to about 0.3% of the lisenqizumab is present as high molecular weight (HMW) species as measured by UP-SEC.

[0344] In one embodiment, the present disclosure relates to a composition comprising about 150 mg / ml of lisenqizumab, and wherein at least about 99.1% (about 99.1% to about 99.7%, 99.1% to about 99.6%, about 99.2% to about 99.7%, or about 99.2% to about 99.6%) of the lisenqizumab is present as a monomer and / or no more than about 0.4% (about 0.1% to about 0.4%, about 0.1% to about 0.3%, about 0.1% to about 0.2%, about 0.2% to about 0.4%, or about 0.2% to about 0.3%) of the lisenqizumab is present as high molecular weight (HMW) species as measured by ultra-performance size exclusion chromatography (UP-SEC).

[0345] In one embodiment, a pharmaceutical composition described herein comprises about 150 mg / mL risankizumab; 185 mM trehalose; 10 mM acetate; and 0.20 mg / mL polysorbate 20, wherein the pharmaceutical composition has a pH of about 5.7; and as measured by ultra-high performance size exclusion chromatography (UP-SEC) (e.g., using the UP-SEC method described in Example 15), at least about 99.1% (about 99.1 % to about 99.7%, 99.1% to about 99.6%, about 99.2% to about 99.7% or about 99.2% to about 99.6%) of the lisenqizumab is present as a monomer and / or no more than about 0.4% (about 0.1% to about 0.4%, about 0.1% to about 0.3%, about 0.1% to about 0.2%, about 0.2% to about 0.4% or about 0.2% to about 0.3%) of the lisenqizumab is present as high molecular weight (HMW) species.

[0346] In one embodiment, the pharmaceutical composition described herein comprises about 150 mg / ml risankizumab; 0.054 mg / mL acetic acid; 1.24 mg / mL sodium acetate trihydrate; 70 mg / mL trehalose dihydrate; 0.20 mg / mL polysorbate 20; and wherein the pharmaceutical composition has a pH of about 5.7. and wherein at least about 99.1% (about 99.1% to about 99.7%, 99.1% to about 99.6%, about 99.2% to about 99.7%, or about 99.2% to about 99.6%) of the lisenqizumab is present as a monomer and / or no more than about 0.4% (about 0.1% to about 0.4%, about 0.1% to about 0.3%, about 0.1% to about 0.2%, about 0.2% to about 0.4%, or about 0.2% to about 0.3%) of the lisenqizumab is present as high molecular weight (HMW) species as measured by ultra-performance size exclusion chromatography (UP-SEC) (e.g., using the UP-SEC method described in Example 15).

[0347] Liquid formulations contemplated by the present disclosure may include added water, such as USP grade water.

[0348] In one embodiment, the composition described herein has at least feature (c): greater than about 97.5% of the lishengqizumab is present as a major peak and / or less than about 2.2% of the lishengqizumab is present as a low molecular weight (LMW) substance as measured by capillary gel electrophoresis under non-reducing conditions (CGE-NR). For example, the composition described herein comprises lishengqizumab, wherein greater than about 97.5% of the lishengqizumab is present as a major peak and less than about 2.2% of the lishengqizumab is present as a low molecular weight (LMW) substance as measured by CGE-NR.

[0349] In one embodiment, greater than about 97.5% of the lisenqizumab is present as a major peak as measured by CGE-NR. For example, but not limited to, greater than about 97.6%, greater than about 97.7%, greater than about 97.8%, greater than about 97.9%, greater than about 98.0%, greater than about 98.1%, greater than about 98.2%, greater than about 98.3%, or greater than about 98.4% of the lisenqizumab is present as a major peak as measured by CGE-NR, or any value between, including the end values, as measured by CGE-NR. Range, such as about 97.6% to about 98.4%, about 97.6% to about 98.3%, about 97.6% to about 98.2%, about 97.7% to about 98.4%, about 97.7% to about 98.3%, about 97.7% to about 98.2%, about 97.8% to about 98.4%, about 97.8% to about 98.3%, or about 97.8% to about 98.2% of liscenzumab is present as a major peak.

[0350] In one embodiment, less than about 2.2% of the lisenqizumab is present as low molecular weight (LMW) species as measured by CGE-NR. For example, but not limited to, less than about 2.1%, less than 2.0%, less than 1.9%, less than 1.8%, less than 1.7%, less than 1.6%, or less than 1.5% of the lisenqizumab is present as low molecular weight (LMW) species as measured by CGE-NR, or any range between, including the end values, such as about 1.5% to about 2.1%, about 1.6% to about 2.1%, about 1.7% to about 2.1%, about 1.5% to about 2.0%, about 1.6% to about 2.0%, or about 1.7% to about 2.0% of the lisenqizumab is present as low molecular weight (LMW) species as measured by CGE-NR.

[0351] In one embodiment, the present disclosure relates to a composition comprising about 150 mg / ml risankizumab, and wherein greater than about 97.5% (e.g., about 97.6% to about 98.4%, about 97.6% to about 98.3%, about 97.6% to about 98.2%, about 97.7% to about 98.4%, about 97.7% to about 98.3%, about 97.7% to about 98.2%) is present in the presence of a 5- or 6-ounce lipase inhibitor, as measured by capillary gel electrophoresis under non-reducing conditions (CGE-NR). , about 97.8% to about 98.4%, about 97.8% to about 98.3% or about 97.8% to about 98.2%) of lisenqizumab is present as a major peak and / or less than about 2.2% (e.g., about 1.5% to about 2.1%, about 1.6% to about 2.1%, about 1.7% to about 2.1%, about 1.5% to about 2.0%, about 1.6% to about 2.0% or about 1.7% to about 2.0%) of lisenqizumab is present as a low molecular weight (LMW) substance.

[0352] In one embodiment, a pharmaceutical composition described herein comprises about 150 mg / mL risankizumab; 185 mM trehalose; 10 mM acetate; and 0.20 mg / mL polysorbate 20, wherein the pharmaceutical composition has a pH of about 5.7; and as measured by capillary gel electrophoresis under non-reducing conditions (CGE-NR) (e.g., using the CGE-NR method described in Example 15), wherein greater than about 97.5% (e.g., about 97.6% to about 98.4%, about 97.6% to about 98.3%, about 97.6% to about 98.2%, about 97 .7% to about 98.4%, about 97.7% to about 98.3%, about 97.7% to about 98.2%, about 97.8% to about 98.4%, about 97.8% to about 98.3%, or about 97.8% to about 98.2%) of the lisenqizumab is present as a major peak and / or less than about 2.2% (e.g., about 1.5% to about 2.1%, about 1.6% to about 2.1%, about 1.7% to about 2.1%, about 1.5% to about 2.0%, about 1.6% to about 2.0%, or about 1.7% to about 2.0%) of the lisenqizumab is present as a low molecular weight (LMW) substance.

[0353] In one embodiment, the pharmaceutical composition described herein comprises about 150 mg / ml risankizumab; 0.054 mg / mL acetic acid; 1.24 mg / mL sodium acetate trihydrate; 70 mg / mL trehalose dihydrate; 0.20 mg / mL polysorbate 20; and wherein the pharmaceutical composition has a pH of about 5.7. And as measured by capillary gel electrophoresis under non-reducing conditions (CGE-NR) (e.g., using the CGE-NR method described in Example 15), wherein greater than about 97.5% (e.g., about 97.6% to about 98.4%, about 97.6% to about 98.3%, about 97.6% to about 98.2%, about 97.7% to about 98.4%, about 97.7% to about 98.3%, about 97.7% to about 98.2%, about 97.8% About 98.4%, about 97.8% to about 98.3%, or about 97.8% to about 98.2%) of the lisenqizumab is present as a major peak and / or less than about 2.2% (e.g., about 1.5% to about 2.1%, about 1.6% to about 2.1%, about 1.7% to about 2.1%, about 1.5% to about 2.0%, about 1.6% to about 2.0%, or about 1.7% to about 2.0%) of the lisenqizumab is present as a low molecular weight (LMW) substance.

[0354] Liquid formulations contemplated by the present disclosure may include added water, such as USP grade water.

[0355] c. Risenqizumab compositions with reduced immunogenicity

[0356] In one embodiment, the compositions described herein have at least feature (d): following administration of a single subcutaneous dose of the pharmaceutical composition to humans, the incidence of anti-drug antibodies (ADA) during treatment is less than about 4.7%.

[0357] For example, but not limited to, the incidence of ADA occurring during treatment can be less than about 4.5%, less than about 4.0%, less than about 3.5%, less than about 3.0%, less than about 2.5%, less than about 2.0%, less than about 1.5%, less than about 1.0%, less than about 0.5%, less than about 0.4%, less than about 0.3%, less than about 0.2%, less than about 0.1%, less than about 0.01%, less than about 0.001% or less than about 0.0001%.

[0358] In one embodiment, the incidence of ADA that develops during treatment is about 0.0%.

[0359] In one embodiment, the incidence of ADA that emerges during treatment is measured following administration of a single subcutaneous injection of a 150 mg dose of the pharmaceutical composition to a human.

[0360] In one embodiment, the presence of ADA is determined by using a validated bridging electrochemiluminescent immunoassay.

[0361] In one embodiment, the incidence of ADA occurring during treatment is less than about 4.7%, e.g., about 0.0%, following administration of a single subcutaneous injection of a 150 mg dose of the pharmaceutical composition as measured by using a bridging electrochemiluminescent immunoassay (e.g., by using the bridging electrochemiluminescent immunoassay described in Example 16).

[0362] In one embodiment, the present disclosure relates to a composition comprising about 150 mg / ml of risankizumab, and wherein the incidence of anti-drug antibodies (ADA) arising during treatment is less than about 4.7% (e.g., about 0.0%) following administration of a single subcutaneous injection of a 150 mg dose of the composition to humans.

[0363] In one embodiment, the pharmaceutical composition described herein comprises about 150 mg / mL risankizumab; 185 mM trehalose; 10 mM acetate; and 0.20 mg / mL polysorbate 20, wherein the pharmaceutical composition has a pH of about 5.7; and wherein the incidence of anti-drug antibodies (ADA) arising during treatment is less than about 4.7% (e.g., about 0.0%) following administration of a single subcutaneous injection of a 150 mg dose of the pharmaceutical composition to humans.

[0364] In one embodiment, the pharmaceutical composition described herein comprises about 150 mg / mL risankizumab; 185 mM trehalose; 10 mM acetate; and 0.20 mg / mL polysorbate 20, wherein the pharmaceutical composition has a pH of about 5.7; and as measured by using a bridging electrochemiluminescent immunoassay (e.g., by using the bridging electrochemiluminescent immunoassay described in Example 16), wherein the incidence of anti-drug antibodies (ADA) arising during treatment is less than about 4.7% (e.g., about 0.0%) following administration of a single subcutaneous injection of a 150 mg dose of the pharmaceutical composition.

[0365] In one embodiment, the pharmaceutical composition described herein comprises about 150 mg / ml of risankizumab; 0.054 mg / mL of acetic acid; 1.24 mg / mL of sodium acetate trihydrate; 70 mg / mL of trehalose dihydrate; 0.20 mg / mL of polysorbate 20; and wherein the pharmaceutical composition has a pH of about 5.7. And wherein after a single subcutaneous injection of a 150 mg dose of the pharmaceutical composition is administered to a human, the incidence of anti-drug antibodies (ADA) that occur during treatment is less than about 4.7% (e.g., about 0.0%).

[0366] In one embodiment, the pharmaceutical composition described herein comprises about 150 mg / ml of risankizumab; 0.054 mg / mL acetic acid; 1.24 mg / mL sodium acetate trihydrate; 70 mg / mL trehalose dihydrate; 0.20 mg / mL polysorbate 20; and wherein the pharmaceutical composition has a pH of about 5.7. And the incidence of anti-drug antibodies (ADA) that appear during treatment is less than about 4.7% (e.g., about 0.0%) after administering a single subcutaneous injection of a 150 mg dose of the pharmaceutical composition as measured by using a bridging electrochemiluminescent immunoassay (e.g., by using a bridging electrochemiluminescent immunoassay described in Example 16).

[0367] Liquid formulations contemplated by the present disclosure may include added water, such as USP grade water.

[0368] Risenqizumab composition with poloxamer 188

[0369] In one aspect, the present disclosure relates to a composition comprising: (1) risankizumab; and (2) poloxamer 188 (P188), wherein the composition does not comprise polysorbate 20 (PS20) and / or polysorbate 80 (PS80).

[0370] In one embodiment, the compositions described herein comprise about 60 mg / ml to about 150 mg / ml risankizumab. For example, but not limited to, the compositions described herein contain about 70 mg / ml to about 150 mg / ml, about 80 mg / ml to about 150 mg / ml, about 90 mg / ml to about 150 mg / ml, about 100 mg / ml to about 150 mg / ml, about 110 mg / ml to about 150 mg / ml, about 120 mg / ml to about 150 mg / ml, about 130 mg / ml to about 150 mg / ml, about 140 mg / ml to about 150 mg / ml, 60 mg / ml to about 70 mg / ml, 60 mg / ml to about 80 mg / ml, 60 mg / ml to about 90 mg / ml, 60 mg / ml to about 100 mg / ml, 60 mg / ml to about 110 mg / ml, 60 mg / ml to about 120 mg / ml, 60 mg / ml to about 130 mg / ml, or 60 mg / ml to about 140 mg / ml of lisenqizumab, as well as ranges and amounts between any of these concentrations.

[0371] In one embodiment, the composition described herein comprises about 60 mg / ml, about 70 mg / ml, about 80 mg / ml, about 90 mg / ml, about 100 mg / ml, about 110 mg / ml, about 120 mg / ml, about 130 mg / ml, about 140 mg / ml or about 150 mg / ml of lisenqizumab.

[0372] In one embodiment, the compositions described herein further comprise phospholipase A2 (PLA2) in an amount greater than about 250 pg / mg of risankizumab.

[0373] In one embodiment, the composition described herein comprises PLA2 in an amount of greater than about 260 pg, greater than about 270 pg, greater than about 280 pg, greater than about 290 pg, greater than about 300 pg, greater than about 310 pg, greater than about 320 pg, greater than about 330 pg, greater than about 340 pg, greater than about 350 pg, greater than about 360 pg, greater than about 380 pg, greater than about 400 pg, greater than about 450 pg, greater than about 500 pg, greater than about 550 pg, greater than about 600 pg, greater than about 650 pg, greater than about 700 pg, greater than about 750 pg, greater than about 800 pg, greater than about 900 pg, or greater than about 1000 pg / mg of lisenqizumab, or any range therebetween, including the end values, such as about 250 pg to about 1100 pg, about 260 pg to about 1100 pg, about 2 0pg, about 270pg to about 1100pg, about 280pg to about 1100pg, about 290pg to about 1100pg, about 300pg to about 1100pg, about 310pg to about 1100pg, about 320pg to about 1100pg, about 340pg to about 1100pg, about 360pg to about 1100pg, about 250pg to about 10 ... To about 900pg, about 250pg to about 800pg, about 250pg to about 700pg, about 250pg to about 600pg, about 250pg to about 500pg, about 250pg to about 400pg, about 250pg to about 1030pg, about 290pg to about 1090pg, about 360pg to about 450pg or about 310pg to about 920pg / mg of lisenqizumab.

[0374] In one embodiment, the composition described herein comprises PLA2 in an amount of about 260 pg, about 270 pg, about 280 pg, about 290 pg, about 300 pg, about 310 pg, about 320 pg, about 330 pg, about 340 pg, about 350 pg, about 360 pg, about 380 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about 750 pg, about 800 pg, about 900 pg, about 1000 pg or about 1100 pg / mg of lisenqizumab.

[0375] The PLA2 in the compositions described herein can be PLA2G2, PLA2G15, or a combination thereof. In one embodiment, the PLA2 is PLA2G15.

[0376] The amount of PLA2 in the compositions described herein can be determined using methods known in the art, such as by mass spectrometry or by ELISA. In one embodiment, the amount of PLA2 in the compositions described herein is determined by ELISA, such as using the ELISA method described in Example 9.

[0377] In some embodiments, the composition does not include PS80.

[0378] Purification of Lisenqizumab

[0379] In one embodiment, risankizumab can be produced recombinantly in various host cells (e.g., CHO cells or NS0 cells) using the methods described in the examples (e.g., Example 3) or using methods known in the art, such as cell culture methods using hydrolysate-based culture media or chemically defined culture media containing a specific range of manganese and / or galactose (see, e.g., U.S. Pat. No. 9,062,106) or by using recombinant host cells that overexpress β1,4 galactosyltransferase or host cells with β-galactosidase knockdown (U.S. Pat. No. 9,550,826). U.S. Pat. Nos. 9,062,106 and 9,550,826 are incorporated herein by reference in their entirety.

[0380] The lisenqizumab composition described herein can be used in Example 3 and Fig.14 The exemplary optimized purification process described in and below was generated.

[0381] Once a clear solution or mixture containing antibodies is obtained, a combination of different purification techniques can be used, including but not limited to affinity separation steps, ion exchange separation steps, mixed mode separation steps and hydrophobic interaction separation steps, which are separated from other proteins such as HP produced by cells. The separation step is based on a mixture of biophysical characteristics to separate proteins, such as but not limited to charge, hydrophobicity degree and / or size, depending on the specific form of separation, including chromatographic separation. In one aspect of the present disclosure, separation can be carried out using chromatography, including but not limited to cation chromatography, anion chromatography, hydrophobic interaction and / or mixed mode chromatography. For each of these techniques, several different chromatographic resins are commercially available, allowing for precise customization of purification schemes for the specific proteins involved. The essence of each described separation method is that proteins can be passed through chromatographic media at different rates, such as resins in columns, to achieve increased physical separation when they further pass through chromatographic media, or proteins can be selectively adhered to chromatographic media, such as separation resins in columns, and then eluted using different eluents. In some cases, the antibody is separated from the HP when the HP specifically adheres to the chromatographic medium such as the resin of the column and the antibody does not, i.e., the antibody is contained in the eluent, while in other cases, the antibody of interest may adhere to the chromatographic medium such as the resin of the column and the HP is squeezed out of the column during the wash cycle.

[0382] a. Initial recovery

[0383] In certain embodiments, it may be desirable to subject samples produced according to the present disclosure to at least a first stage of clarification and preliminary recovery.

[0384] The initial recovery may include one or more centrifugation steps to further clarify the sample mixture, thereby facilitating purification of the protein of interest. Centrifugation of the sample may be performed, for example, but not limited to, at 7,000 × g to about 12,750 × g. In the case of large-scale purification, such centrifugation may be performed online, with a flow rate set, for example, but not limited to, to achieve a turbidity level of 150 NTU in the resulting supernatant. Such supernatant may then be collected for further purification.

[0385] In certain embodiments, the initial recovery may also include the use of one or more depth filtration steps to further clarify the sample matrix, thereby facilitating the purification of antibodies produced using the cell culture techniques of the present disclosure. The depth filter contains a filter medium with a graded density. Such graded density allows larger particles to be captured near the filter surface, while smaller particles penetrate the larger open area of ​​the filter surface and are only captured in smaller openings closer to the center of the filter. In certain embodiments, the depth filtration step may be a defatting depth filtration step. Although certain embodiments only employ a depth filtration step in the initial recovery stage, other embodiments may employ depth filters, including defatting depth filters, in one or more additional purification stages. Non-limiting examples of depth filters that can be used in the context of the present disclosure include XOHC depth filters, DOHC depth filters, Cuno TM 30 / 60ZA depth filter (3M Corp.) and 0.45 / 0.2μm Sartopore TM Double layer filter cartridge.

[0386] b. Affinity chromatography

[0387] In certain embodiments, it may be advantageous to subject the lisenqizhumab produced according to the present disclosure to affinity chromatography to further purify the antibody from HP (e.g., lipase). In certain embodiments, the chromatographic material is capable of selectively or specifically binding to lisenqizhumab. Non-limiting examples of such chromatographic materials include: protein A, protein G, chromatographic materials comprising, for example, antigens bound by an antibody of interest, and chromatographic materials comprising Fc-binding proteins. In some embodiments, the affinity chromatography step may involve subjecting the preliminary recovered sample to a column comprising a suitable protein A resin. In certain embodiments, protein A resin can be used for affinity purification and separation of a variety of antibody isotypes, particularly IgG1, IgG2, and IgG4. Protein A is a bacterial cell wall protein that binds primarily to mammalian IgG through its Fc region. In its native state, protein A has five IgG binding domains and other domains of unknown function.

[0388] Protein A resins are available from several commercial sources. One suitable resin may be MabSelect from GE Healthcare TM Another suitable resin may be MabSelect SuRe TM . Using MabSelect TMA non-limiting example of a suitable column for packing is a column of about 1.0 cm diameter x about 21.6 cm long (about 17 ml bed volume). A column of this size can be used for small-scale purification and can be compared with other columns for scale-up. For example, a 20 cm x 21 cm column with a bed volume of about 6.6 L can be used for larger purifications. Regardless of the column, the column can use a suitable resin such as MabSelect TM or MabSelect SuRe TM Stuffing.

[0389] c. Ion exchange chromatography

[0390] In certain embodiments, it may be advantageous to subject the lisenqizumab produced according to the present disclosure to ion exchange chromatography to purify the lisenqizumab from HP (e.g., lipase). Ion exchange separation includes any method for separating two substances based on the difference in the ionic charge of each of the two substances, and cation exchange materials or anion exchange materials can be used. For example, the use of cation exchange materials relative to anion exchange materials is based on the local charge of the protein. Therefore, the present disclosure encompasses the use of anion exchange steps before using a cation exchange step, or the use of cation exchange steps before using anion exchange steps. In addition, the present disclosure encompasses the use of only cation exchange steps, only anion exchange steps, or any continuous combination of the two.

[0391] When separation is carried out, the initial protein mixture can be contacted with an ion exchange material using any of a variety of techniques, for example, batch purification techniques or chromatographic techniques.

[0392] Anionic substituents or cationic substituents can be attached to the matrix to form anionic or cationic supports for chromatography. Non-limiting examples of anionic exchange substituents include diethylaminoethyl (DEAE), quaternary aminoethyl (QAE) and quaternary amine (Q) groups. Cationic substituents include carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P) and sulfonate (S). Cellulose ion exchange resins such as DE23 TM 、DE32 TM DE52 TM CM-23 TM CM-32 TM and CM-52 TM Available from Whatman Ltd. Based in Maidstone, Kent, UK And cross-linked ion exchangers are also known. For example, DEAE-, QAE-, CM- and and DEAE-, Q-, CM-, and S- and Fast Fe is available from Pharmacia AB. In addition, DEAE and CM derived ethylene glycol-methacrylate copolymers such as TO YOPEARL TM DEAE-6505 or M and TOYOPEARL TM CM-650S or M is available from Toso Haas Co., Philadelphia, Pa. In some embodiments, a Poros TM Cation exchange chromatography with XS resin.

[0393] d.Ultrafiltration / diafiltration

[0394] In certain embodiments, it may be advantageous to subject the lisenqizumab produced according to the present disclosure to ultrafiltration / diafiltration to purify the lisenqizumab from HP (e.g., lipase). Ultrafiltration is described in detail in: Microfiltration and Ultrafiltration: Principles and Applications, L. Zeman and A. Zydney (Marcel Dekker, Inc., New York, NY, 1996), and Ultrafiltration Handbook, Munir Cheryan (Techno mic Publishing, 1986; ISBN No. 87762-456-9). One filtration process is tangential flow filtration as described in the Millipore catalog entitled "Pharmaceutical Process Filtration Catalogue" on pages 177-202 (Bedford, Mass., 1995 / 96). Ultrafiltration is generally considered to mean filtration using a filter with a pore size of less than 0.1 μm. By employing filters with such small pore sizes, the volume of the sample can be reduced by permeation of the sample buffer through the filter, while the antibody remains behind the filter.

[0395] Diafiltration is a method of using an ultrafilter to remove and exchange salts, sugars, and non-aqueous solvents, separate free substances from bound substances, remove low molecular weight materials, and / or cause rapid changes in ions and / or pH environments. Microsolutes are most effectively removed by adding solvent to the solution being ultrafiltered at a rate approximately equal to the ultrafiltration rate. This washes tiny substances from the solution at a constant volume, effectively purifying the retained protein. In certain embodiments of the present disclosure, a diafiltration step may be used to exchange various buffers used in conjunction with the present disclosure, as well as to remove impurities from the protein formulation, optionally prior to further chromatography or other purification steps.

[0396] e. Hydrophobic Interaction Chromatography

[0397] In certain embodiments, it may be advantageous to subject the lisenqizumab produced according to the present disclosure to hydrophobic interaction chromatography to purify the lisenqizumab from HP (lipase). For example, the first eluate obtained from the ion exchange column can be subjected to a hydrophobic interaction material so that a second eluate with a reduced HP level is obtained. Hydrophobic interaction chromatography (HIC) steps, such as those disclosed herein, are typically performed to purify proteins, including the removal of HP.

[0398] When performing a HIC-based separation, the sample mixture is contacted with the HIC material, for example using a batch purification technique or using a column. Prior to HIC purification, it may be desirable to remove any chaotropic or very hydrophobic substances, for example by passing the mixture through a pre-column.

[0399] While ion exchange chromatography relies on the charge of proteins to separate them, hydrophobic interaction chromatography uses the hydrophobic properties of proteins. The hydrophobic groups on the protein interact with the hydrophobic groups on the column. The more hydrophobic the protein, the stronger its interaction with the column. Thus, the HIC step removes host cell-derived impurities (e.g., DNA and other high and low molecular weight product-related substances).

[0400] HIC columns typically comprise a base matrix (e.g., cross-linked agarose or synthetic copolymer material) coupled to a hydrophobic ligand (e.g., an alkyl or aryl group). Suitable HIC columns comprise agarose resin substituted with phenyl groups (e.g., Phenyl Sepharose TM Many HIC columns are commercially available. Examples include, but are not limited to, Phenyl Sepharose with low or high substitution. TM 6 Fast Flow Column (Pharmacia LKB Biotechnology, AB, Sweden); Phenyl Sepharose TM High-efficiency column (Pharmacia LKB Biotechnology, AB, Sweden); Octyl Sepharose TM High-efficiency column (Pharmacia LKB Biotechnology, AB, Sweden); Fractogel TM EMD Propyl or Fractogel TM EMD phenyl column (E. Merck, Germany); Macro-Prep TM Methyl or Macro-Prep TM Tert-butyl support (Bio-Rad, California); WP HI-Propyl (C3)TM Column (JT Baker, New Jersey); and Toyopearl TM Ether, phenyl or butyl column (TosoHaas, Pa.).

[0401] f. Multimodal (mixed-mode) chromatography

[0402] In certain embodiments, it may be advantageous to subject the lisenqizumab produced according to the present disclosure to multimodal chromatography to purify the lisenqizumab from HP (e.g., lipase). Multimodal chromatography is a chromatography utilizing a multimodal medium resin. Such resins comprise multimodal chromatography ligands. In certain embodiments, such ligands refer to ligands that are capable of providing at least two different but synergistic sites for interacting with a substance to be bound. One of these sites produces an attractive charge-charge interaction between the ligand and the substance of interest. Other sites typically provide electron acceptor-donor interactions and / or hydrophobic and / or hydrophilic interactions. Electron donor-acceptor interactions include interactions such as hydrogen bonds, π-π, cation-π, charge transfer, dipole-dipole, induced dipole, etc. Multimodal chromatography ligands are also referred to as "mixed-mode" chromatography ligands.

[0403] In certain embodiments, multimodal chromatographic resins can be composed of multimodal ligands that are coupled to an organic or inorganic support (sometimes represented as a basic matrix) directly or via a spacer. The support can be in the form of particles, such as substantially spherical particles, monoliths, filters, membranes, surfaces, capillaries, etc. In certain embodiments, the support can be prepared by natural polymers, such as cross-linked carbohydrate materials, such as agarose, agar, cellulose, dextran, , polysaccharides, konjac, carrageenan, gellan gum, alginate, etc. In order to obtain high adsorption capacity, the support can be porous, and then the ligand is coupled to the outer surface and the pore surface. Such natural polymer supports can be prepared according to standard methods, such as reverse phase suspension gelation (S Hjerten: Biochim Biophys Acta 79 (2), 393-398 (1964). Alternatively, the support can be prepared from a synthetic polymer, such as a cross-linked synthetic polymer, for example styrene or styrene derivatives, divinylbenzene, acrylamide, acrylate, methacrylate, vinyl ester, vinyl amide, etc. Such synthetic polymers can be prepared according to standard methods, see, for example, "Styrene based polymer supports developed by suspension polymerization" (R Arshady: Chimica e L'Industria 70 (9), 70-75 (1988)). Porous natural or synthetic polymer supports can also be obtained from commercial sources, such as Amersham Biosciences, Uppsala, Sweden. In some embodiments, mixed mode chromatography combines anion exchange (AEX) and hydrophobic interaction (HIC) functions. A non-limiting example of such mixed mode chromatography suitable for the present disclosure is Capto TM Adhere mixed mode chromatography.

[0404] Characterization of Risenqizumab Compositions

[0405] Reducing the concomitant protein of the lishengqizumab formulation beneficially increases the stability of the formulation (e.g., reducing particle formation, increasing the shelf life of the lishengqizumab drug product, etc.). In one embodiment, at 2°C to 40°C (e.g., 4°C to 35°C, 4°C to 25°C, 4°C to 15°C, 4°C to 10°C, 2°C to 8°C, or any temperature in the foregoing range, such as about 2°C, about 4°C, about 5°C, about 8°C, about 25°C, about 40°C, etc.), no visible or shiny particles are observed in the liquid lishengqizumab composition described herein within at least 3 months (e.g., at least 6 months, at least 9 months, at least 12 months, at least 15 months, at least 18 months, at least 21 months, at least 24 months, at least 27 months, at least 30 months, at least 33 months, or at least 36 months). In one embodiment, no visible or shiny particles are observed in the liquid lishengqizumab composition described herein within 24 months at about 4°C.

[0406] In some embodiments, the liquid risanqizumab composition described herein comprises a surfactant with increased stability. The surfactant may be selected from the group consisting of polysorbate 20 (PS20), polysorbate 80 (PS80), polysorbate 40 (PS40), polysorbate 60 (PS60), polysorbate 65 (PS65) and poloxamer 188.

[0407] The stability of the surfactant in the liquid lishengqizumab composition described herein can be evaluated by directly measuring the amount of the surfactant in the liquid lishengqizumab composition after storage for a period of time (e.g., 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, 30 months, 36 months, etc.) at a certain temperature (e.g., 2°C, 4°C, 5°C, 8°C, 10°C, 12°C, 25°C, 30°C, 35°C or 40°C). Alternatively, the stability of the surfactant in the liquid lishengqizumab composition described herein can be evaluated by measuring the amount of degradation products of the surfactant in the lishengqizumab composition (e.g., the amount of free fatty acids) after storage for a period of time (e.g., 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, 30 months, 36 months, etc.) at a certain temperature (e.g., 2°C, 4°C, 5°C, 8°C, 10°C, 12°C, 25°C, 30°C, 35°C or 40°C). In some embodiments, the time period is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or more months, or any range therebetween, including the end values, such as 3 months to 36 months, 12 months to 24 months, etc.

[0408] In some embodiments, the liquid levitra compositions described herein comprise PS20 at a concentration of, for example, 0.20 mg / mL, and the stability of PS20 in such liquid levitra compositions is increased.

[0409] In one embodiment, the stability of PS20 is assessed by directly measuring the amount of PS20 in the risanqizumab composition after storage at a certain temperature (e.g., 2°C, 4°C, 5°C, 8°C, 10°C, 12°C, 25°C, 30°C, 35°C or 40°C) for a period of time (e.g., 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, 30 months, 36 months, etc.). The amount of PS20 can be measured using any method known in the art, such as using a high performance liquid chromatography charged aerosol detector (HPLC-CAD), such as the HPLC-CAD described in Example 10. In some embodiments, the time period is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or more months, or any range therebetween, including the end values, such as 3 months to 36 months, 12 months to 24 months, etc.

[0410] For example, in some embodiments, after 6 months of storage at 5°C, at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or about 100%) of the initial amount of PS20 is retained. In some embodiments, after 24 months of storage at 5°C, at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or about 100%) of the initial amount of PS20 is retained. In some embodiments, after 6 months of storage at 25°C, at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or about 100%) of the initial amount of PS20 is retained. In some embodiments, after storage at 40° C. for 6 months, at least 40% (e.g., at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or about 100%) of the initial amount of PS20 is retained.

[0411] In one embodiment, the stability of PS20 is evaluated by measuring the amount of free acid (FFA), i.e., degradation products of PS20, in the risanqizumab composition formulated with PS20 after storage for a period of time (e.g., 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, 30 months, 36 months, etc.) at a certain temperature (e.g., 2°C, 4°C, 5°C, 8°C, 10°C, 12°C, 25°C, 30°C, 35°C, or 40°C). The amount of FFA can be measured using any method known in the art, such as an enzyme FFA or LC-FFA assay, such as the reverse phase high performance liquid chromatography UV (RP-HPLC-UV) method described in Example 10. In some embodiments, the time period is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or more months, or any range therebetween, including the end values, such as 3 months to 36 months, 12 months to 24 months, etc.

[0412] For example, in some embodiments, the total amount of FFA in the liquid composition prepared with PS20 described herein after storage for 6 months at 5°C increases by no more than 1.75 times (e.g., no more than 1.5 times, no more than 1.25 times, or no more than 1.1 times), or does not increase. In some embodiments, the total amount of FFA in the liquid composition prepared with PS20 described herein is no more than 20 nmol / ml (e.g., no more than 18 nmol / ml, no more than 15 nmol / ml, no more than 12 nmol / ml, no more than 10 nmol / ml, no more than 8 nmol / ml, or no more than 5 nmol / ml, or any range between the end values, such as 5 nmol / ml to 10 nmol / ml) after storage for 6 months at 5°C. In some embodiments, the amount of FFA is less than or equal to the detection limit of the FFA detection assay.

[0413] In some embodiments, the total amount of FFA in the liquid composition formulated with the PS20 described herein increases by no more than 3.5 times (e.g., no more than 3.2 times, no more than 3.0 times, no more than 2.5 times, no more than 2.0 times, no more than 1.8 times, no more than 1.6 times, no more than 1.4 times, no more than 1.2 times, or no more than 1.1 times) after storage for 6 months at 25°C, or does not increase. In some embodiments, the total amount of FFA in the liquid composition formulated with the PS20 described herein is no more than 25 nmol / ml (e.g., no more than 20 nmol / ml, no more than 18 nmol / ml, no more than 15 nmol / ml, no more than 12 nmol / ml, no more than 10 nmol / ml, no more than 8 nmol / ml, or no more than 5 nmol / ml, or any range between the end values, such as 5 nmol / ml to 10 nmol / ml) after storage for 6 months at 25°C. In some embodiments, the amount of FFA is less than or equal to the detection limit of the FFA detection assay.

[0414] In some embodiments, the total amount of FFA in the liquid composition formulated with the PS20 described herein increases by no more than 3 times (e.g., no more than 2.8 times, no more than 2.5 times, no more than 2.0 times, no more than 1.8 times, no more than 1.6 times, no more than 1.4 times, no more than 1.2 times, or no more than 1.1 times) after storage at 40°C for 6 months, or does not increase. In some embodiments, the total amount of FFA in the liquid composition formulated with the PS20 described herein is no more than 35 nmol / ml (e.g., no more than 30 nmol / ml, no more than 25 nmol / ml, no more than 20 nmol / ml, no more than 18 nmol / ml, no more than 15 nmol / ml, no more than 12 nmol / ml, no more than 10 nmol / ml, no more than 8 nmol / ml, or no more than 5 nmol / ml, or any range between the end values, such as 5 nmol / ml to 10 nmol / ml) after storage at 40°C for 6 months. In some embodiments, the amount of FFA is less than or equal to the detection limit of the FFA detection assay, eg, less than or equal to 1 nmol / ml.

[0415] In some embodiments, the liquid levitra compositions described herein comprise PS80, and the stability of PS80 in such liquid levitra compositions is increased.

[0416] In one embodiment, the stability of PS80 is assessed by directly measuring the amount of PS80 in the risankizumab composition after storage at a certain temperature (e.g., 2°C, 4°C, 5°C, 8°C, 10°C, 12°C, 25°C, 30°C, 35°C or 40°C) for a period of time (e.g., 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, 30 months, 36 months, etc.). The amount of PS80 can be measured using any method known in the art, for example, using HPLC-CAD, such as HPLC-CAD described in Example 10. In some embodiments, the time period is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or more months, or any range therebetween, including the end values, such as 3 months to 36 months, 12 months to 24 months, etc.

[0417] For example, in some embodiments, after 6 months of storage at 5°C, at least 80% (e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or about 100%) of the initial amount of PS80 is retained. In some embodiments, after 6 months of storage at 25°C, at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or about 100%) of the initial amount of PS80 is retained. In some embodiments, after 6 months of storage at 40°C, at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or about 100%) of the initial amount of PS80 is retained.

[0418] In one embodiment, the stability of PS80 is evaluated by measuring the amount of free acid (FFA), i.e., degradation products of PS80, in the risanqizumab composition formulated with PS80 after storage for a period of time (e.g., 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, 30 months, 36 months, etc.) at a certain temperature (e.g., 2°C, 4°C, 5°C, 8°C, 10°C, 12°C, 25°C, 30°C, 35°C, or 40°C). The amount of FFA can be measured using any method known in the art, such as an enzymatic FFA or LC-FFA assay, such as the RP-HPLC-UV method described in Example 10. In some embodiments, the time period is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or more months, or any range therebetween, including the end values, such as 3 months to 36 months, 12 months to 24 months, etc.

[0419] For example, in some embodiments, the total amount of FFA in a liquid composition formulated with PS80 described herein increases no more than 8 times (e.g., no more than 7 times, no more than 6 times, no more than 5 times, no more than 4 times, no more than 3 times, no more than 2 times, no more than 1.5 times, no more than 1.2 times, or no more than 1.1 times) after storage at 5°C for 6 months, or does not increase. In some embodiments, the total amount of FFA in a liquid composition formulated with PS80 described herein is no greater than 45 nmol / ml (e.g., no greater than 40 nmol / ml, no greater than 35 nmol / ml, no greater than 30 nmol / ml, no greater than 25 nmol / ml, no greater than 20 nmol / ml, no greater than 18 nmol / ml, no greater than 15 nmol / ml, no greater than 12 nmol / ml, no greater than 10 nmol / ml, no greater than 8 nmol / ml, or no greater than 5 nmol / ml, or any range including the end values, such as 5 nmol / ml to 10 nmol / ml, 5 nmol / ml to 20 nmol / ml, 5 nmol / ml to 30 nmol / ml, or 5 nmol / ml to 45 nmol / ml) after storage for 6 months at 5°C. In some embodiments, the amount of FFA is less than or equal to the detection limit of the FFA detection assay, for example, less than or equal to 1 nmol / ml.

[0420] In some embodiments, the total amount of FFA in a liquid composition formulated with PS80 described herein increases by no more than 12 times (e.g., no more than 11 times, no more than 10 times, no more than 9 times, no more than 8 times, no more than 7 times, no more than 6 times, no more than 5 times, no more than 4 times, no more than 3 times, no more than 2 times, no more than 1.5 times, no more than 1.2 times, or no more than 1.1 times) after storage at 25°C for 6 months, or does not increase. In some embodiments, the total amount of FFA in a liquid composition formulated with PS80 described herein is no greater than 65 nmol / ml (e.g., no greater than 60 nmol / ml, no greater than 55 nmol / ml, no greater than 50 nmol / ml, no greater than 45 nmol / ml, no greater than 40 nmol / ml, no greater than 35 nmol / ml, no greater than 30 nmol / ml, no greater than 25 nmol / ml, no greater than 20 nmol / ml, no greater than 18 nmol / ml, no greater than 15 nmol / ml, no greater than 12 nmol / ml, no greater than 10 nmol / ml, no greater than 8 nmol / ml, or no greater than 5 nmol / ml, or any range therebetween, including the end values, such as 5 nmol / ml to 10 nmol / ml, 5 nmol / ml to 20 nmol / ml, 5 nmol / ml to 30 nmol / ml, or 5 nmol / ml to 65 nmol / ml) after storage at 25°C for 6 months. In some embodiments, the amount of FFA is less than or equal to the detection limit of the FFA detection assay, eg, less than or equal to 1 nmol / ml.

[0421] In some embodiments, the total amount of FFA in the liquid composition prepared with PS80 described herein after storage for 6 months at 40°C is no more than 2.5 times (e.g., no more than 2 times, no more than 1.5 times, no more than 1.2 times, or no more than 1.1 times), or does not increase. In some embodiments, the total amount of FFA in the liquid composition prepared with PS80 described herein is no more than 15nmol / ml (e.g., no more than 12nmol / ml, no more than 10nmol / ml, no more than 8nmol / ml, no more than 5nmol / ml, or no more than 3nmol / ml, or any range between the end values, such as 3nmol / ml to 5nmol / ml, 3nmol / ml to 10nmol / ml, 5nmol / ml to 10nmol / ml, or 3nmol / ml to 15nmol / ml) after storage for 6 months at 40°C. In some embodiments, the amount of FFA is less than or equal to the detection limit of the FFA detection assay.

[0422] In some aspects, the risenqizumab compositions described herein comprise poloxamer 188. In some embodiments, the risenqizumab compositions comprising P188 do not comprise PS20 and / or PS80. In some embodiments, the stability of P188 in the risenqizumab compositions is increased compared to the stability of PS20 or PS80.

[0423] In some embodiments, the stability of P188 is assessed by directly measuring the amount of P188 in the levitra monoclonal antibody composition after storage at a certain temperature (e.g., 2°C, 4°C, 5°C, 8°C, 10°C, 12°C, 25°C, 30°C, 35°C, or 40°C) for a period of time (e.g., 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, 30 months, 36 months, etc.). The amount of P188 can be measured using any method known in the art, for example, using a Pluronic F-68 colorimetric assay, such as the Pluronic F-68 colorimetric assay described in Example 17. In some embodiments, the time period is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or more months, or any range therebetween, including the end values, such as 3 months to 36 months, 12 months to 24 months, 3 months to 6 months, etc.

[0424] For example, in some embodiments, after storing for 3 months at 5 ℃, at least 85% (for example, at least 90%, at least 95%, at least 98%, at least 99% or about 100%) of the initial amount of P188 is retained. In some embodiments, after storing for 3 months at 25 ℃, at least 65% (for example, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or about 100%) of the initial amount of P188 is retained. In some embodiments, after storing for 3 months at 40 ℃, at least 60% (for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or about 100%) of the initial amount of P188 is retained.

[0425] For example, in some embodiments, after storing for 6 months at 5 ℃, at least 80% (for example, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or about 100%) of the initial amount of P188 is retained. In some embodiments, after storing for 6 months at 25 ℃, at least 60% (for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or about 100%) of the initial amount of P188 is retained. In some embodiments, after storing for 6 months at 40 ℃, at least 60% (for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or about 100%) of the initial amount of P188 is retained.

[0426] Example 1: Particles were observed after dilution of the risankizumab drug products DP1 and DP2.

[0427] The 90 mg / ml Risenqizumab drug product 1 (DP1) was developed using the methods and formulations described in International Application PCT / US2013 / 038109. Risenqizumab drug product 2 (DP2) was subsequently developed at a concentration of 150 mg / ml and was approved by the FDA. The formulations of DP1 and DP2 are shown in Table 6 below.

[0428] Table 6

[0429]

[0430] Lisenqizumab formulations DP1 and DP2 contained highly purified Lisenqizumab API and were stable. However, particles containing free fatty acids (FFA) were unexpectedly observed in DP1 and DP2, especially when DP2 was diluted (e.g., diluted to 51 mg / ml and 60 mg / ml) and used to study the feasibility of on-body device display and placed under certain storage conditions, as shown in Table 7 below.

[0431] Table 7

[0432]

[0433] Example 2: Proteomic analysis identified candidate chaperones.

[0434] The inventors hypothesized that the formation of particles in the levitra product was due to the degradation of the surfactant polysorbate 20 (PS20), which could lead to antibody aggregation. It was hypothesized that the degradation of PS20 was caused by residual CHO cell esterases that had been co-purified with the levitra drug substance. To identify putative companion proteins (HPs) that could cause polysorbate-20 degradation in the levitra drug product, host cell proteins (HCPs) from DP1 and DP2 bulk drug substance (BDS) samples were enriched and then subjected to LC-MS / MS analysis.

[0435] Multiple levitra BDS samples from each DS process were pooled to generate a representative BDS material for HCP enrichment and identification. In total, six BDS batches from process 1 were pooled to generate a representative DP1 BDS sample, and four DP2 BDS batches were mixed to prepare a DP2 BDS sample for HCP enrichment and identification studies.

[0436] The protein concentration of each representative risankizumab BDS sample was measured and shown in Table 8.

[0437] Table 8 Lisenqizumab concentration in pooled samples

[0438]

[0439] method

[0440] Immunoaffinity purification for enrichment of HCPs from lisdexizumab

[0441] 1. Anti-HCP antibody coupled to agarose beads

[0442] a. Buffer preparation:

[0443] Acidification buffer: 1 mM HCl;

[0444] Coupling buffer: 200 mM NaHCO3, 500 mM NaCl, pH 9.0;

[0445] Blocking buffer: 1 M ethanolamine; pH 8.0;

[0446] Wash buffer: 100 mM sodium acetate; 500 mM NaCl, pH 4.0.

[0447] b. Buffer exchange of anti-HCP antibodies

[0448] Anti-total HCP antibody (CRO: ): 1.99 mg / mL;

[0449] Anti-LMW HCP Antibody (CRO: ): 5.19mg / ml;

[0450] Dialysis apparatus: Thermo Scientific Pro #66810.

[0451] 3 mL of anti-total HCP antibody and 3 mL of anti-LMWHCP antibody were buffer exchanged separately into 4 L of coupling buffer (described above) by dialysis overnight in a cold room using slow mixing (about 60 rpm).

[0452] c. Conditioning of CNBR-activated agarose beads (Cytiva Cat. No. 71-5000-15AF)

[0453] Weigh 0.5 g of CNBR agarose beads into a poly-Prep chromatography column (Bio-Rad catalog number 731-1550). Suspend the agarose beads in 5 mL of 1 mM ice-cold HCl. Invert the column to ensure that the agarose beads are completely wet (about 10 minutes); place the column in a 15 mL centrifuge tube and centrifuge at 200 g for 7 minutes to dry the beads (Beckman Avanti J-15R); wash the CNBR agarose beads 3 more times using 1 mM ice-cold HCl.

[0454] d. Anti-HCP antibody coupling and washing

[0455] 4 mL of buffer-exchanged anti-total HCP antibody and anti-LMW HCP antibody were added to two separate poly-prep columns containing the conditioned CNBR-activated agarose beads obtained in c. The two columns were kept in a shaker in a cold room overnight, mixing at low speed to couple the anti-LMW or anti-total HCP antibody to the CNBR-activated agarose beads. The poly-prep columns were centrifuged to remove unbound anti-HCP antibodies from the agarose beads by centrifugation at 200 g for 7 minutes (Beckman Avanti J-15R).

[0456] 5mL blocking buffer is added to each column and mixed overnight at low speed on a table shaker in a cold room to block the agarose beads fixed with antibodies. Blocking buffer (same as described in step c) is removed by centrifugation. The column is washed by alternating coupling buffer and washing buffer (4 times each, 5mL each time). Finally, the column is conditioned in 1×PBS buffer by adding and eluting 5mL of 1×PBS twice. Agarose beads (antibody coupled) are suspended and stored in 4°C PBS buffer in the column until use.

[0457] 2. Packing of immunoaffinity columns for HCP purification / enrichment

[0458] The suspended agarose beads (antibody coupled) were transferred from the poly-prep chromatography column to a Tricorn 5 / 50 column (Cytiva product code: 28406409; approximately 1 mL column volume (CV)). Agitation was minimized to avoid the introduction of bubbles into the agarose bead packing in the Tricorn column. The Tricorn column was connected to a peristaltic pump P1 (Cytiva) to establish an immunoaffinity purification system. The Tricorn column was conditioned by running 20 mL (20 CV) of PBS buffer using the P1 pump, driving the liquid flow at a flow rate of 0.5 mL / min. The agarose bead-filled Tricorn column was stored at 4°C.

[0459] 3. HCP coupling, washing and elution from purification column

[0460] Two Tricorn immunoaffinity columns (anti-total HCP and anti-LMW HCP columns) packed as described above, working with two separate P1 pumps, were used to sequentially purify the pooled Risenqizumab BDS samples (DP1 and DP2). For each Risenqizumab BDS sample, the following procedures and materials were used for HCP coupling, washing, and elution from the immunoaffinity column.

[0461] a. Buffer Preparation

[0462] Protein binding buffer: 1× PBS (pH 7.4);

[0463] Washing buffer: 1× PBS, 0.05% Tween 20 (pH 7.4);

[0464] Elution buffer: 100 mM glycine, 400 mM arginine (HCl) (pH 2.7);

[0465] Neutralization buffer: 1 M Tris-HCl (pH 8.5).

[0466] A total of 4.5 g of Risenqizhumab BDS sample (50 mL of 90 mg / mL, 30 mL of 150 mg / mL, depending on the Risenqizhumab concentration in the pooled BDS sample) was prepared for purification on each Tricorn immunoaffinity column (two columns in total). For each cycle of Risenqizhumab BDS sample loading, 50 (30) mL of DP1 or DP2 pooled BDS sample was divided into 5 aliquots (10 mL or 6 mL each) and washed on the immunoaffinity column (5 cycles in total, each aliquot was used for one cycle). In each cycle, 10 mL (or 6 mL) of Risenqizhumab BDS sample was circulated through the Tricorn immunoaffinity column at a flow rate of about 0.5 mL / min for 40 minutes. The Tricorn column was washed with about 20 CV of wash buffer delivered by the P1 pump. The circulation and washing steps were repeated for another 4 aliquots of Risenqizhumab BDS sample in each Tricorn immunoaffinity column. After 5 cycles of loading and washing the lishengqizhumab BDS sample on each Tricorn column, each column was washed with another 10CV wash buffer. The bound proteins (HCP and lishengqizhumab molecules) were eluted from each Tricorn column using 15mL (CV) elution buffer delivered at the same flow rate of about 0.5mL / min by a P1 pump. The eluent was neutralized with neutralization buffer (1mL eluent was mixed with 200μL neutralization buffer) to about pH 7 (measured with a pH probe). Neutralization should be performed after the purified eluent comes out of the column. The protein concentration in each portion of the neutralized eluent from the Tricorn immunoaffinity column of each lishengqizhumab BDS sample was measured by a Lunatic spectrophotometer. The concentration should be below the detection limit.

[0467] 4. Concentration and purification of the eluate from the immunoaffinity column

[0468] The purified eluate (about 15 mL) was concentrated to a total volume of about 0.5 mL using 15 mL 3k MWCO (Millipore, catalog number UFC900324). The 0.5 mL eluate was further concentrated to a final volume of about 100 μL using 0.5 mL size, 3K MWCO (Millipore, catalog number UFC500324). The concentration of total protein in each 100 μL concentrated eluate was measured using a Lunatic spectrophotometer. The total protein concentration should be about 1 mg / mL. Similar purification and concentration procedures were used for all 4 representative lishengqizhumab BDS samples using the same immunoaffinity purification column (anti-LMW and anti-total HCP column). A total of 8 eluted samples were collected from the purification and concentration of all four pooled lishengqizhumab BDS samples for LC-MS / MS analysis.

[0469] LC-MS / MS Method for HP Identification

[0470] 1. Sample Denaturation and Reduction

[0471] 60 μL of 8 M urea and 8.8 μL of 1 M DTT were added to each 20 μL sample of the eluate. Each sample was incubated at 37°C with shaking at 450 rpm for 30 minutes.

[0472] 2. Sample Alkylation and Digestion

[0473] 200 μL of 8M urea was added to a 0.5mL 30kDa MWCO centrifugal filter (Millipore, catalog number, MRCF0R030). The reduced sample was then added to the filter and centrifuged at 14,000 × g for 15 minutes. 400 μL of 8M urea was added to the filter and centrifuged at 14,000 × g for 15 minutes. The flow-through from the collection tube was discarded. 100 μL of 50mM iodoacetamide solution was added and mixed at 600rpm for 1 minute in a thermomixer and incubated at room temperature for 20 minutes without mixing. The filter was centrifuged at 14,000 × g for 10 minutes. 100 μL of 8M urea was added to the filter and centrifuged at 14,000 × g for 15 minutes. This step was repeated once. 100 μL of 50mM ammonium bicarbonate was added to the filter device and centrifuged at 14,000 × g for 10 minutes. This step was repeated once. The filter was transferred to a new collection tube.

[0474] 35 μL of 50 mM ammonium bicarbonate was added, followed by 1 μL of 0.4 μg / uL trypsin (ThermoScientific, catalog number 90057, enzyme to protein ratio 1:50), and mixed at 600 rpm in a thermomixer for 1 minute. The filter was incubated overnight at 37 ° C in a thermomixer. The tube was wrapped with parafilm to avoid evaporation. The next day, the filter was centrifuged at 14,000 × g for 10 minutes. 40 μL of 50 mM ammonium bicarbonate was added and the filter was centrifuged at 14,000 × g for 10 minutes. The sample was acidified with formic acid to ensure pH <1. Peptide concentration was measured by Bradford colorimetric assay (Thermo Scientific, catalog number 23250).

[0475] 3. LC-MS / MS Analysis

[0476] For each digested sample, 800 ng was injected onto a Bruker nanoElute™ ultrahigh pressure liquid chromatography (UHPLC) system with an Aurora series UHPLC C18 column maintained at 50 °C. 25 cm x 75 μm ID, 1.6 μm (Ionopticks, catalog number AUR2-25075C18A-CSI). Peptides were eluted into the mass spectrometer at a flow rate of 250 nL / min using a gradient of mobile phase A (0.1% formic acid in water) and mobile phase B (0.1% formic acid in acetonitrile) as shown in Table 9.

[0477] Table 9 Gradients used for LC-MS / MS analysis

[0478] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 98 2 60 87 13 90 80 20 100 70 30 110 15 85 120 15 85

[0479] Data were acquired using a Bruker timsTOF pro QTOF mass spectrometer operating in positive ion mode, scanning from 100 to 1700 m / z in PASEF mode. The ion mobility resolution was set to 0.60–1.35 V·s / cm2 with a ramp time of 100 ms. For each cycle, 10 PASEF MS / MS scans were performed on the precursor with 14,500 intensity units to achieve a duty cycle close to 100%. Polygonal filters were applied in terms of m / z and ion mobility to exclude low m / z, singly charged ions from MS / MS fragmentation. The collision energy was stepped up as a function of ion mobility. The data were searched against the CHO proteome database by MSFragger V17.1.

[0480] result

[0481] Table 10 Potential associated proteins (HP) identified in pooled Risenkizumab BDS samples.

[0482] Protein name Putative phospholipase B-like protein 2 Acid ceramidase-like protein Isoamyl acetate-hydrolyzing esterase 1 homolog Sphingomyelin phosphodiesterase Sialyl O-acetylesterase-like protein Hepatic carboxylesterase-like isoform 1 Hepatic carboxylesterase 4-like protein Ester hydrolase C11orf54 homolog isoform 1 Lipoprotein lipase Calcineurin-like phosphatase domain-containing protein 1-like protein Peroxiredoxin-6-like protein

[0483] By affinity purification, various chaperone proteins were identified in the pooled risankizumab BDS samples, including putative phospholipase B-like protein 2 (PLBL2), acid ceramidase, isopentyl acetate-hydrolyzing esterase 1, sphingomyelin phosphodiesterase, liver carboxylesterase-like isoform 1 (CES1), liver carboxylesterase 4, ester hydrolase C11orf54 homolog isoform 1, sialyl O-acetylesterase-like protein (SIAE), calcineurin-like phosphatase domain-containing protein 1, and peroxiredoxin-6-like partial protein (Prdx6) ( Figure 1 and Table 10). Some of these proteins, such as PLBL2 and acid ceramidase, have also been previously reported to be present in another antibody drug substance (Graf et al. (2021) J. Pharm. Sci. 110:3358-3567).

[0484] Example 3: Development of a levitra drug product with improved stability

[0485] The inventors hypothesized that the presence of certain types of chaperone proteins from the host cells (e.g., host cell lipases) led to particle formation in the diluted levofloxacin drug product. To address this issue and improve the stability and shelf life of the levofloxacin product, as well as to further improve its quality, a new levofloxacin drug substance, process 4DS, was developed. An exemplary purification process for production process 4DS is described below.

[0486] The CHO cells expressing Lishengqizhu monoclonal antibody are thawed and cultured in a shaking bottle, a cell bag and a seed bioreactor stage with an increasing volume to provide enough cells to inoculate the production bioreactor. The cell culture broth is harvested by centrifugation and filtration to effectively remove cells, and a clarified harvest is provided to further purify the product. The clarified harvest is then processed by a series of chromatographic steps, virus inactivation, virus filtration, concentration and buffer exchange by tangential flow filtration, and final preparation. Develop a purification process to reduce host cell lipase by screening various reagents and conditions, including but not limited to, for example, protein A chromatography washing schemes and washing buffer, depth filters, chromatographic column resins (e.g., AEX resins, CEX resins, MM resins and / or HIC resins) and / or conditions for ultrafiltration and diafiltration (UF / DF) processes. As measured by ELISA with an acceptable yield compromise, the reagents and conditions that effectively reduce the levels of certain host cell lipases in the purified Lishengqizhu monoclonal antibody bulk drug (DS) are suitable for establishing two optimized purification processes, referred to herein as process 3 and process 4. In addition to the purification process, the upstream cell culture process of Process 4 was further improved to increase culture longevity, productivity and robustness.

[0487] A general overview of the purification process of Process 4 is shown in Fig.14 A and Fig.14 B. Specifically, cell culture broth was harvested and clarified by centrifugation and depth filtration using X0HC depth filters. TM The clarified harvest was purified by affinity chromatography with protein A resin. The eluate was low pH inactivated using phosphoric acid and then depth filtered using XOHC and DOHC depth filters. The Capto TM Purification of levitra monoclonal antibody samples by Adhere mixed mode chromatography. TM The XS resin further purifies the flow-through by cation exchange chromatography. The eluate is subjected to virus filtration. Ultrafiltration / diafiltration (UF / DF) is then performed by doping the load directly with a high salt solution followed by doping with salt-free 8DV. The purified bulk drug substance (BDS) is then formulated and stored appropriately.

[0488] Detailed parameters for the different steps of Process 4 are described below.

[0489] Initial recovery

[0490] Initial recovery by centrifugation and depth filtration was used to remove cells and cell debris from the production bioreactor tank. The 3000 L production bioreactor was used as the feed tank for a 710 Alpha Laval centrifuge. The centrifuge was operated at a set point of 5555 rpm, a feed rate of 30 L / min, and a discharge interval of 215 seconds for GMP1 to GMP3 and 252 seconds for GMP4, respectively. The centrate was then passed through eighteen 1.1 m 2 The filter bank of the Millipore X0HC media Pod unit was then passed through two Sartopore 2 30-inch (1.8 m each) 2 )0.45μm / 0.2μm capsule filter.

[0491] After centrifugation and depth filtration of the bioreactor harvest, the filter was rinsed with a target weight of 396 kg of 50 mM sodium acetate (pH 5.5) buffer. The centrifugation and filtration of the harvest were performed as a stand-alone operation. Filtration was performed in the fermentation kit at ambient temperature (18-25°C). The harvest temperature was cooled to 18-22°C before filtration, with a set value of 20°C. The filtrate was collected in a 3000L harvest tank, cooled to 2-8°C, and can be kept for up to 3 days.

[0492] MabSelect SuRe Protein A Chromatography

[0493] MabSelect SuRe protein A chromatography was used to capture the 4DS of the lisenqizumab process from the clarified harvest and to reduce the amount of process-related impurities. The diameter of the MabSelect SuRe self-plugging column (GE Healthcare) was 60 cm and the target volume was about 62.0 L (bed height was 21 to 23 cm). The operation was carried out at ambient temperature (18-25° C.) in a fermentation kit with the process parameters shown in Table 11A below.

[0494] MabSelcet SuRe post is operated in binding and elution mode. Three MabSelectSuRe chromatographic cycles are needed to process each batch. The post is balanced with 50mM sodium acetate pH 5.5 and then loaded into 13 to 35g of Risen Qizhu monoclonal antibody / L resin. There are three washing steps after loading. Washing 1 is 50mM sodium acetate pH 5.5. Washing 2 is 50mM Tris, 1M arginine (pH8.0), and washing 3 is 50mM sodium acetate pH 5.5. Elution is performed with 50mM sodium acetate pH 3.5. Then the post is regenerated with 0.2M sodium hydroxide, and rebalanced with equilibrium buffer before loading in the next cycle.

[0495] The loading material (2-8°C) was not warmed before loading onto the column. The elution peak collection started at 0.2 OD up to 0.2 OD down (280 nm wavelength, 1 mm path length). The eluate entered a 300L portable stainless steel tank or a 500L disposable mixing (SUM) system and then passed offline through a 0.45 / 0.2 μm filter and into a collection container. A Sartopore 2 30-inch (1.8 m each) 2 ) 0.45μm / 0.2μm capsule filter for eluate filtration. Each eluate filtration is used for 3 cycles of daily MabSelect SuRe chromatography. The MabSelect SuRe eluate is collected in a 700L portable stainless steel tank or a 1000L single-use mixed (SUM) system and can be kept at 9-25°C for up to 1 day or cooled to 2-8°C for up to 3 days before low pH inactivation.

[0496] Table 11A. Summary of MabSelect SuRe Protein A capture parameters

[0497]

[0498] pH inactivation and POD filtration

[0499] The purpose of the pH inactivation step is to inactivate adventitious viruses that may be present. The pH inactivation step is performed in the fermentation kit at ambient temperature (18-25°C).

[0500] The pH of the Protein A eluate was adjusted to 3.5 ± 0.1 (measured at 18-25°C) with 0.5 M phosphoric acid. After a storage period of 60-90 minutes, the inactivated material was neutralized to pH 8.0 ± 0.1 (measured at 18-25°C) using 2.0 M Tris. For subsequent filtration, the conductivity of the material should be in the range of 3.8 to 4.8 mS / cm (measured at 24-26°C), so no dilution is required before POD filtration.

[0501] Depth filtration is used to remove particles and reduce impurity levels in process streams. In GMP1 and GMP2 the filter bank consists of two 1.1m 2 Millipore D0HC media Pod unit, followed by five 1.1m 2 Millipore X0HC Media Pod Unit and Sartopore 2 30-inch (1.8m each) 2 )0.45μm / 0.2μm capsule filter. In GMP3 and GMP4, the number of DOHC filters and XOHC filters were reduced to one and four, respectively, to increase the step yield. 2The filter set was equilibrated with 25 mM Tris, 25 mM NaCl (pH 8.0) and the contents of the feed tank were then filtered. 2 The filter set was rinsed with 25 mM Tris, 25 mM NaCl, pH 8.0. The filtrate was collected in a 700 L portable stainless steel tank or a 1000 L SUM system and subjected to Capto Adhere chromatography on the same day.

[0502] Capto Adhere Chromatography

[0503] The Capto Adhere chromatography step was used to reduce the impurity levels in the process stream. The column packed with Capto Adhere resin had a diameter of 45 cm and a target volume of 19.1 L (bed height of 12 cm). The operation was carried out at ambient temperature (18-25°C) in a purification suite with the process parameters shown in Table 11B below.

[0504] Capto Adhere column is operated in flow-through mode. One Capto Adhere chromatography cycle is required to process each batch. First, the column is pre-balanced with 2M sodium chloride, and then balanced with 25mM Tris, 25mM sodium chloride (pH 8.0). The column is loaded with 150 to 300g of Risen Qizhu monoclonal antibody / L resin, and then washed with 260mM Tris (pH 8.0). The column is regenerated with 0.1M acetic acid (pH 2.9) and 2M sodium chloride. After each batch, the column is disinfected with 1M sodium hydroxide and stored in 0.1M sodium hydroxide.

[0505] The loading material was kept at 18-25°C before loading onto the column. During loading, the product flow-through was collected starting at 1 OD from the front of the peak during washing and ending at 5 OD from the tail of the peak (280 nm wavelength, 1 mm path length). The CaptoAdhere flow-through was collected in a 1000L portable stainless steel tank or a 1000L SUM system.

[0506] On the day of Capto Adhere chromatography, Capto Adhere FTW was adjusted to a target pH of 5.25. To prepare the Poros XS loading, the Capto Adhere FTW material was titrated to pH 5.25 + 0.1 (measured at 18-25 °C) using 2M acetic acid and the conductivity was adjusted to 4.5 to 7.5 mS / cm with WFI if necessary. 2) 0.45 μm / 0.2 μm capsule filter Filter the conditioned Capto Adhere FTW. The filtered conditioned Capto Adhere FTW can be kept at 9-25°C for up to 1 day or cooled to 2-8°C for up to 3 days prior to Poros XS chromatography.

[0507] Table 11B. Summary of Capto Adhere chromatographic parameters

[0508]

[0509] Poros XS Chromatography

[0510] The Poros XS chromatography step was used to reduce basic substances and process-related impurities such as host cell proteins and leached protein A. The column packed with Poros XS resin had a diameter of 60 cm and a target volume of 56.5 L (bed height of 20 cm). The operation was carried out at ambient temperature (18-25° C.) in a purification suite with the process parameters shown in Table 11C below.

[0511] Poros XS column is operated in binding and elution mode. Two Poros XS chromatography cycles are required to process each batch. The column is balanced with 50mM sodium acetate, 31mM sodium chloride (pH 5.25). The loading range of the column is 25 to 50g of lishengqizhumab / L resin. The washing step is 50mM sodium acetate, 31mM sodium chloride, pH 5.25. Elution is performed with 50mM sodium acetate, 181mM sodium chloride (pH5.25). When the elution buffer pH of GMP1 to GMP3 is close to the target, the elution buffer pH of GMP4 is adjusted to 5.34, and the higher end of the elution pH batch record range (5.15-5.35) of 5M sodium hydroxide is used to improve the Poros XS step yield. Before the next cycle, the column is regenerated with 25mM Tris, 3M sodium chloride (pH 8.5). Finally, the column was sanitized with 1.0 M sodium hydroxide and stored in 0.1 M sodium hydroxide. The column was sanitized and stored at the end of the last cycle of each batch run.

[0512] On the day of the Poros XS chromatography, a Sartopore 2 30-inch (1.8 m 2 ) 0.45 μm / 0.2 μm capsule filter Poros XS load was filtered. During elution, the elution peak was collected starting at 1 OD from the front of the peak and ending at 5 OD from the tail of the peak (280 nm wavelength, 1 mm path length). The eluate was passed through a 0.45 / 0.2 μm filter when leaving the chromatography plate and was filtered using two Sartopore 2 30-inch (1.8 m each) 2) 0.45μm / 0.2μm capsule filter into a collection vessel for eluent filtration. Use the filter for 2 Poros XS chromatography cycles on the same processing day. The Poros XS eluate is collected in a 500L portable stainless steel tank or a 1000L SUM system and can be kept at 2-25°C for up to 5 days before nanofiltration.

[0513] Table 11C. Summary of Poros XS chromatographic parameters

[0514]

[0515] Virus Filtration

[0516] Virus filtration provides the ability to remove adventitious viruses larger than 20 nm in size. The process is performed in the purification suite at ambient temperature (18-25°C).

[0517] The virus filtration filter set consists of two parallel Millipore Virosolve Pro Magnus2.1Shield (0.51m each) 2 ) 0.1 μm capsule filter, two parallel Millipore Virosolve Pro Magnus 2.1 filters (each 0.51 μm 2 ) and a single Sartopore 2 30 inches (1.8m 2 )0.45μm / 0.2μm capsule filter. Before product filtration, each pre-filter and Virosolve nanofilter were rinsed with ≥102L of WFI, and then rinsed with ≥26L of 50mM sodium acetate, 181mM sodium chloride (pH 5.25). The product was filtered using a quattroflow pump, with a nanofilter target pressure of 23psig and an upper limit of 32psig. The post-filtration rinse was 20L of 50mM sodium acetate, 181mM sodium chloride, pH 5.25. The virus filtrate can be kept at 2-25°C for up to 5 days.

[0518] UF / DF

[0519] The product was concentrated using the UF / DF step and diafiltered into the final formulation buffer.The process utilized a 30 kD Millipore Pellicon3 Biomax UF module, D screen, and was performed in a purification suite at ambient temperature (18-25°C) with the process parameters shown in Table 1 ID below.

[0520] The load material was diluted to a 10X dilution (9 parts nanofiltrate to 1 part 5M sodium chloride) with 5M sodium chloride, and the pH of the load material was then adjusted to 5.45 ± 0.1 (measured at 18-25°C) with 2M sodium acetate. Prior to loading onto the UF / DF membrane, the filtrate was passed through a single Sartopore2 30-inch (1.8 m 2 ) 0.45 μm / 0.2 μm capsule filter filters the conditioned load material into the recirculation tank.

[0521] UF / DF has eight 1.14m 2 The membrane was tested on Skid Z-2300 with a total membrane area of ​​9.12m 2 The UF / DF load was concentrated to a 50 g / L target and then diafiltered with 0.002% (w / v) sodium chloride and subsequently concentrated to 235 g / L. The retentate was passed through a single Sartopore 2 10 inch 0.45 / 0.2 μm (0.45 m 2 ) sterile filter. Rinse the ultrafiltration system with approximately 5 kg of 0.002% sodium chloride to recover the product retained in the system. Rinse 1 and Rinse 2 are passed through the same Sartopore 2 10-inch 0.45 / 0.2 μm (0.45 m 2 ) The sterile filter was transferred to a collection bag for rinse 1 and a collection bag for rinse 2. After the rinse was recovered, the retentate was diluted with an appropriate amount of rinse 1 and rinse 2 to achieve a target concentration of 200 g / L.

[0522] The retentate pool was prepared by adding 5X formulation buffer, 50 mM acetate, 925 mM trehalose, 0.1% Tween 20 (pH 5.70). The final bulk drug substance was diluted to 150 g / L lisenqizumab in 1X formulation buffer, 10 mM acetate, 185 mM trehalose, 0.02% Tween 20 (pH 5.70). The prepared UF / DF retentate was then filtered through a 0.22 μm Millipak200 sterile filter (0.1 m 2 The final formulated UF / DF retentate can be kept at 9-25°C for up to 1 day and cooled to 2-8°C for up to 5 days before the final bagging step.

[0523] Table 11D. Ultrafiltration / diafiltration process description

[0524]

[0525] a. UF2 flow rate needs to be reduced throughout the process to keep TMP within range.

[0526] Bagging

[0527] The purpose of bagging is to package and store the final bulk drug substance. The operation is carried out in the purification suite at ambient temperature (18-25°C). The filtered formulated UF / DF retentate is pumped into a sterile 6L Celsius FFT bag. The bag is filled to a volume of approximately 6kg.

[0528] The Process 4 parameter targets are summarized in Table 11E below.

[0529] Table 11E. Process 4 parameter targets

[0530]

[0531]

[0532]

[0533] According to Table 12 shown below, lisanzumab produced by Process 3 and Process 4 were formulated to produce Drug Product 3 (DP3) and Drug Product 4 (DP4), respectively.

[0534] Table 12

[0535]

[0536]

[0537] For the 60 mg / ml display of DP3, no visible or shiny particles were observed during the 24-month stability period at 4°C.

[0538] The 51 mg / ml display further demonstrated the increased stability of DP3 compared to DP2.

[0539] Sparkle particles were observed in the 51 mg / mL presentation of DP2 at 3 months (Table 7), whereas no visible product-related particles were observed in the 51 mg / mL presentation of DP3 at 6 months.

[0540] Similarly, for the 60 mg / mL presentation of the DP4 vial, no visible or shiny particles were observed during the 18 month stability period at 4°C.

[0541] Example 4: Identification of PLA2 by LC-MS proteomics

[0542] Several types of phospholipases, including phospholipase A2 class XV (PLA2 G15), have been identified as potential contributors to polysorbate 20 degradation. These lipases can be present at very low abundance in DS, complicating detection by LC-MS / MS. Enrichment of lipase levels by immunoaffinity purification using immobilized antibodies against specific lipases can increase the abundance levels required for detection by LC-MS / MS. In this study, PLA2 was enriched in DP1, DP2, DP3, and DP4 DS using immunoaffinity purification. The enriched DS were analyzed by enzyme-linked immunosorbent assay (ELISA) and LC-MS / MS.

[0543] Multiple batches of DS were pooled to generate material for PLA2 enrichment. The protein concentration of each DS pool is shown in Table 13.

[0544] Table 13. Lisenqizumab concentration in DS pool

[0545]

[0546]

[0547] 1. Immunoaffinity purification of PLA2 (chromatography)

[0548] A. Buffer exchange of PLA2 antibody

[0549] (a) Buffer:

[0550] (1) Acidification buffer: 1 mM HCl;

[0551] (2) Coupling buffer: 200 mM NaHCO3; 500 mM NaCl, pH = 9.0;

[0552] (3) Blocking buffer: 1 M ethanolamine; pH = 8.0;

[0553] (4) Washing buffer: 100 mM sodium acetate; 500 mM NaCl, pH = 4.0;

[0554] (5) PLA2 antibody: 1 mg / mL.

[0555] (b) Buffer exchange of PLA2 antibody

[0556] Thaw 3.5 μg of PLA2 antibody at room temperature. TMDialysis Cassette 10K MWCO (ThermoFisher Scientific, catalog number 66810) was used to exchange the PLA2 antibody buffer with the coupling buffer. The dialysis cassette was floated in 4 L of coupling buffer at 60 rpm at 4°C overnight. The buffer-exchanged PLA2 antibody was collected into a clean microtube and proceeded to Part B below.

[0557] B. Conditioning of CNBR-activated Sepharose beads

[0558] Weigh 0.5g CNBR activated Sepharose beads (Cytiva, catalog number 71-5000-15AF) and fill the beads into a poly-Prep chromatographic column (Bio-Rad, catalog number 731-1550). The beads are suspended in 5mL ice-cold 1mM HCl. The column is inverted to ensure that the beads are fully hydrated (approximately 10 minutes). The column is placed in a 15mL conical tube and centrifuged (Beckman Avanti J-15R) at 200g for 7 minutes to dry the beads. Repeat the previous two steps to wash the beads three more times in an ice-cold 1mM HCl solution. Keep the beads dry after the washing steps.

[0559] C. PLA2 Antibody Conjugation

[0560] The concentration of the buffer-exchanged PLA2 antibody was determined from Part A using a Lunatic UV / Vis multicolor spectrophotometer with an extinction coefficient of 1.0 M-1 cm-1 (E1%=10). The concentration of the PLA2 antibody was 0.78 mg / mL before coupling to the CNBR Sepharose beads. 4 mL of PLA2 antibody was added to the column filled with CNBR Sepharose beads. If necessary, the volume of the PLA2 antibody was increased to 4 mL using coupling buffer. The column was placed on a bench shaker and shaken overnight at low speed at 4°C to complete the coupling of the antibody to the beads. The column was centrifuged at 200g for 7 minutes to remove unbound antibody. The concentration of the PLA2 antibody remaining in the column filtrate was determined. The concentration of the PLA2 antibody remaining in the filtrate was 0.01 mg / mL. 5 mL of blocking buffer was added to the column, and the column was shaken overnight at low speed at 4°C. The column was centrifuged at 200g for 7 minutes to remove the blocking buffer. Add 5 mL of coupling buffer to the column and centrifuge the column at 200 g for 7 minutes to remove the buffer, and repeat this step three more times. Add 5 mL of wash buffer I to the column and centrifuge the column at 200 g for 7 minutes to remove the buffer. Repeat this step three more times for further washing. Add 5 mL of PBS buffer to condition the column, and centrifuge the column at 200 g for 7 minutes to remove the buffer, and repeat this step and keep the beads suspended in 5 mL of PBS buffer. If the next step is not performed, keep the column at 4 ° C.

[0561] D. Packing of immunoaffinity columns for PLA2 enrichment

[0562] A Tricorn 5 / 50 column (Cytiva, Cat. No. 28406409) was rinsed in 20% ethanol for at least 1 minute at room temperature. The column volume (CV) was approximately 1 mL.

[0563] The beads (coupled with PLA2 antibody) were transferred from the poly-Prep chromatography column to a Tricorn 5 / 50 column. While the Tricorn column was packed with beads, agitation was minimized to avoid the introduction of air bubbles. The immunoaffinity column was equilibrated with 20 CV PBS (pH 7.4) at 0.5 mL / min. The immunoaffinity column was used in the next step described below or stored at 4°C until use.

[0564] E. PLA2 elution of DS samples

[0565] a. PLA2 enrichment

[0566] Prepare 5 aliquots of 4.5 g of DS sample to load onto the immunoaffinity column. Use one aliquot for each cycle. DP1: 10 mL per aliquot, 50 mL in total; DP2: 6 mL per aliquot, 30 mL in total; DP3: 6 mL per aliquot, 30 mL in total; DP4: 6 mL per aliquot, 30 mL in total. The column was balanced with 10CV PBS (pH 7.4). One aliquot of DP1 BDS was recirculated through the immunoaffinity column at 0.5 mL / min for 40 minutes. The column was washed with 20CV PBS, 0.05% Tween 20 (pH 7.4) at 0.5 mL / min. Repeat the recirculation and washing steps for another 4 aliquots of DS in this section. The column was eluted into a 10 mL conical tube with 10CV 100 mM glycine, 400 mM arginine-HCl (pH 2.7) at 0.5 mL / min. Immediately neutralize the eluate with 1.5 mL 1 M Tris-HCl (pH 8.5). Keep the enriched sample on ice during this procedure. Use pH strips to verify that the neutralized eluate is approximately pH 7.0. Repeat the elution and neutralization steps for each DS pool.

[0567] F. Concentration of Enriched Samples

[0568] Each eluate was transferred to an Amicon concentrator (3K MWCO, 15 mL; Millipore, Cat. No. UFC900324) and centrifuged at 4,000 g for one hour at 4° C. The protein concentration in the concentrated neutralized eluates was measured by A280 using a Lunatic UV / Vis multicolor spectrophotometer (E1%=15.2).

[0569] 2. PLA2 ELISA

[0570] As described in other embodiments described herein, PLA2 concentration is measured by ELISA.

[0571] A. ELISA results analysis

[0572] The results showed that PLA2 was enriched 92-fold in DP1, 57-fold in DP2, and 80-fold in DP3. PLA2 was not detected in the DP4 sample (Table 14).

[0573] Table 14. PLA2 concentration and enrichment factor

[0574]

[0575] *The lower limit of quantitation for this assay is 0.328 ng / mL. Samples were diluted at the minimum required dilution (2-fold) included in the LOQ calculation.

[0576] **Protein concentration not provided (absorbance 280 ), therefore normalized PLA2 concentrations (ng / mg) cannot be reported.

[0577] 3. LC-MS / MS Method for PLA2 Identification

[0578] A. Sample Denaturation and Reduction

[0579] 8 M urea was added to each 20 μg eluate sample to reach a urea concentration of 6 M. 1 M DTT was added to reach 10 mM. Each eluate sample was incubated at 37° C. with shaking at 450 rpm for 30 minutes.

[0580] B. Sample Alkylation and Digestion

[0581] 200 μL of 8M urea was added to a 0.5mL 30kDa MWCO centrifugal filter (Millipore, catalog number MRCF0R030). The reduced sample was then added to the filter and centrifuged at 14,000 × g for 15 minutes. 400 μL of 8M urea was added to the filter, and the filter was centrifuged at 14,000 × g for 15 minutes. The flow-through was discarded from the collection tube. 100 μL of 50mM iodoacetamide solution was added and mixed at 600rpm for 1 minute in a thermomixer, and incubated at room temperature for 20 minutes without mixing. The filter was centrifuged at 14,000 × g for 10 minutes. 100 μL of 8M urea was added to the filter and centrifuged at 14,000 × g for 15 minutes. This step was repeated once. 100 μL of 50mM ammonium bicarbonate was added to the filter device and centrifuged at 14,000 × g for 10 minutes. This step was repeated once. The filter was transferred to a new collection tube. 35 μL 50mM ammonium bicarbonate was added, followed by 1 μL 0.4 μg / uL trypsin (Thermo Scientific, catalog number 90057, enzyme to protein ratio 1:50), and mixed at 600 rpm for 1 minute in a thermomixer. The filter was incubated overnight at 37°C in a thermomixer. The tube was wrapped with parafilm to avoid evaporation. The next day, the filter was centrifuged at 14,000 × g for 10 minutes. 40 μL 50mM ammonium bicarbonate was added, and the filter was centrifuged at 14,000 × g for 10 minutes. The sample was acidified with formic acid to ensure pH <1. Peptide concentration was measured by Bradford colorimetric assay (Thermo Scientific, catalog number 23250).

[0582] C. LC-MS / MS Analysis

[0583] For each digestion sample, 400 ng was injected into a DoublenanoViper maintained at 50 °C. TM PepMapTM Neo column 2μm, C18, 75μm×500mm (Thermo Scientific, catalog number DNV75500PN) on a Waters AQCUITY UPLC M system. The peptides were eluted into the mass spectrometer at a flow rate of 200nL / min using a gradient of mobile phase A (0.1% formic acid in water) and mobile phase B (0.1% formic acid in acetonitrile) (as shown in Table 15).

[0584] Table 15. Gradients used for LC-MS / MS analysis

[0585] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 98 2 5 95 5 10 95 5 180 65 35 200 30 70 201 0 100 210 0 100

[0586] Using the Thermo Scientific Orbitrap TM Fusion TM Lumos TM The mass spectrometer acquires data operated in positive ion mode. A survey scan was performed from 400 to 1500 m / z with a resolution of 240,000, an automatic gain control (AGC) target of 4e6 and a maximum injection time of 50 ms. Monoisotopic masses with 2 to 7 positive charges were selected with a minimum intensity threshold of 2.5e4, and then fragmented by high energy collision dissociation (HCD). The cycle time was ~ 3 seconds. Data were searched in the CHO proteome database by Proteome Discoverer3.0.

[0587] As shown in Table 16, nanoLC-MS / MS analysis identified PLA2G15 (UniProt ID: G3HKV9) in the DS-enriched eluates of DP1 and DP2 (≥2 unique peptides for protein identification).

[0588] Table 16. Identification of PLA2G15 by IP-MS in the levitra BDS of processes DP1, DP2, DP3 and DP4

[0589] Technology Unique peptides of PLA2G15 DP1 8 DP2 6 DP3 1 DP4 0

[0590] in conclusion

[0591] In this study, PLA2G15 protein was successfully enriched by immunoaffinity purification and subsequently detected in DP1 and DP2 DS using LC-MS / MS. A single PLA2G15 unique peptide was detected in DP3, which was below the standard required for protein identification, and no PLA2G15 unique peptide was detected in DP4 DS. These findings are consistent with the PS20 degradation observed in DP1 and DP2 DS during long-term storage or under accelerated stability conditions, while no PS20 degradation was observed in DP3 and DP4.

[0592] Example 5: Identification of PLA2 by Western Blot Analysis

[0593] Phospholipase A2 class XV (PLA2G15) levels in different Risenqizumab DS samples were also evaluated by Western blotting.

[0594] The lisenqizumab BDS sample was obtained by pooling lisenqizumab DS batches produced by the same lisenqizumab process. The lisenqizumab in the sample was largely depleted by ultrafiltration using an Amicon filter with a molecular weight cutoff of 100 kDa. 200 microliters of the pooled BDS sample was added to the Amicon filter and spun until 100 microliters of filtrate was obtained. The filtrate containing proteins with a molecular weight less than 100 kDa, as well as some residual lisenqizumab, was evaluated by Western blot analysis.

[0595] Table 17

[0596]

[0597]

[0598] SDS-PAGE and western blotting

[0599] Run the risankizumab samples and CHO PLA2G15 protein on 4-12% SDS-PAGE. Transfer the proteins to PVDF membrane and probe with rabbit anti-PLA2G15 antibody. Anti-rabbit antibody conjugated with horseradish peroxidase was used as detection reagent. Development was done with chromogenic TMB substrate.

[0600] result

[0601] A PLA2G15 band with a molecular weight of 47 kDa on Western blot was observed in all Risenqizumab BDS lots except DP4, where slightly less was observed in DP3 lot ( Figure 2 ).

[0602] Example 6: Knockout cell line data indicate that PLBL2 is not a problematic chaperone.

[0603] To identify specific chaperones responsible for PS20 degradation, CHO cell lines depleted of specific chaperones PLBL2, PLA2 or LPL were generated and characterized.

[0604] Cell line development and generation of cell culture material

[0605] Establish CHO clones expressing lishengqizhumab. The first two clones were pooled in equal parts and used as the starting cell source for CRISPR / Cas9-mediated gene knockout (KO) experiments using a ribonucleoprotein (RNP)-based method. In a parallel workflow, three proteins of interest were targeted individually, and unique guidance was designed for their corresponding genes in the CHO genome: phospholipase B-like 2 protein (PLBL2; NCBI: 100769512), phospholipase A2 XV class (PLA2G15; NCBI: 100760699) and lipoprotein lipase (LPL; NCBI: 100689191). After CRISPR / cas9 RNP transfection, the corresponding KO library was recovered, and then single cells were cloned by limiting dilution plating method. The first few clones were selected based on phenotypic (growth and productivity) and next-generation sequencing (NGS) data, one clone for each knockout target. The results of NGS analysis indicate that the first few PLBL2, LPL and PLA2G15 knockout clones have 0%, 0.13% and 20% wild-type sequences present in NGS preparations, respectively. The clones are then used for antibody production. Parental cell lines (referred to as wild-type, wt) are used in parallel to produce relevant control materials. Cell culture harvests are clarified by centrifugation and frozen on dry ice (for all conditions).

[0606] Purification development

[0607] PLBL2 KO, PLA2G15 KO, LPL KO and wild-type process control materials were thawed and purified using process 1 (see Example 1), while ultrafiltration / diafiltration (UF / DF) followed process 4 (see Example 3). The material from each process stream was clarified by depth filtration, followed by protein A affinity chromatography, depth filtration, anion exchange (AEX) chromatography, cation exchange (CEX) chromatography, UF / DF and formulated with PS20 added at a target 150 g / L lisenqizumab protein concentration. The intermediates in the process were submitted for product quality assessment. In addition to the four formulations from the purification process (3 KO and wild-type controls), placebo controls (negative controls; 10 mM acetate, 185 mM trehalose, 0.02% Tween 20, pH 5.70) and DP4 BDS controls (positive controls purified by process 4, see Example 3) were also included in the PS20 stability study for up to 12 months at storage temperatures of 5°C, 25°C and 40°C, respectively. High molecular weight (HMW) obtained by size exclusion chromatography (SEC), charge change obtained by weak cation exchange chromatography (WCX), low molecular weight (LMW) obtained by non-reducing capillary electrophoresis sodium dodecyl sulfate (CE-SDS), free fatty acid (FFA) and PS20 levels were monitored at different time points during the stability study.

[0608] Analytical Development

[0609] The presence of three lipases in antibody preparations derived from knockout samples and controls was tested by a specific ELISA method: PLBL2 and PLA2G15 were measured using a CHO-specific internal method, and LPL was measured using a commercial anti-mouse ELISA kit. Two different methods were used to measure the stability of PS20 in samples incubated for different time lengths (several time points during 12 months) and at different temperatures (5°C, 25°C, 40°C). One method measures the total amount of PS20 present in the sample; PS20 is directly quantified by using the RP-HPLC-CAD method. Another method evaluates the decomposition of PS20 by measuring the amount of free fatty acids (mainly lauric acid released by hydrolysis). This is performed using an RP-HPLC method with UV detection. Free fatty acids (FFA) were labeled with PDAM (1-pyrenyldiazomethane) for detection before analysis. The signal generated is proportional to the amount of FFA released. The product quality of levitra was also assessed using SEC to measure %HMW, non-reducing CE-SDS to measure %LMW, and CEX to measure charge change for samples incubated at different time points and at different temperatures.

[0610] result

[0611] In-process mapping results have demonstrated no impact on process performance and product quality under all conditions tested. Observations at the 3-month (5°C, 25°C, and 40°C) and 6-month time points (5°C, 25°C, and 40°C storage conditions) compared to the start of the study (0 months) indicated that the absence of PLBL2 in the BDS did not improve PS20 stability compared to the relevant process control conditions ( Figure 3A-3D These trends were observed in both CAD and FFA results reported by the assay development group ( Figure 3A-3D These data indicate that PLBL2 is unlikely to be a key chaperone responsible for PS20 degradation in the risankizumab formulation.

[0612] Table 18

[0613]

[0614] *Results from Poros XS eluate

[0615] Example 7: Chaperone protein doping studies indicate that PLA2 is a problematic chaperone protein.

[0616] PS20 is mainly composed of polyoxyethylene sorbitan laurate. However, due to the nature of the manufacturing process, commercial PS20 is a mixture of oligomers, including polyethylene glycol, polyethylene glycol esters, isosorbide polyethoxylates, sorbitan polyethoxylates, polysorbate monoesters, polysorbate diesters and sorbitol polyethoxylate esters, etc. (Ayorinde et al. (2000) Rapid Commun. Mass Spectrom 14: 2116-2124; Li et al. (2014) Anal. Chem. 86: 5150-5157; Martos et al. (2017) J. Pharm. Sci., 106: 1722-1735). Commercial PS20 batches typically contain more than 3000 different chemical components.

[0617] Enzymes are companion proteins (HPs) present in bulk drugs, such as lipases, esterases, etc. The active site of the enzyme is composed of a combination of amino acid residues with a certain structure, and the structure is different in different types of enzymes. Therefore, enzymes usually have different activities and specificities for different substrates. Since PS20 is a chemical mixture, different enzymes can have different degradation rates for the various components in PS20. Therefore, different enzymes can produce different PS20 degradation patterns (curves). Therefore, degradation pattern analysis can provide useful information for identifying certain classes of enzymes that are potential root causes of PS20 degradation.

[0618] Study on PLA2G15 doping in DP3 material and Lishengqizumab buffer

[0619] 1. Methods

[0620] Use PS20-CAD subspecies method.Originally develop this method to qualitatively determine subspecies composition and relative to PS20 manufacturing standard quantitative PS20 subspecies.The HPLC system used in this research is Agilent 1260II infinity HPLC, which is equipped with quaternary pump, mobile phase degasser, freezing automatic sampler, temperature-controlled column chamber and Thermo Scientific charged aerosol detector (CAD).Collect data by Waters Empower acquisition system.In embodiment 10, also describe the method using charged aerosol detector (CAD) to detect PS20.

[0621] 2. Study Design

[0622] The study design for the PLA2G15 doping study is shown in Table 19 below.

[0623] Table 19. PLA2G15 doping studies at 25°C.

[0624]

[0625]

[0626] *1 μg / mL is the final PLA2G15 concentration in solution.

[0627] Because the system required 70 minutes to complete each injection (labeled as "Injection" in Table 19), the injections were repeated to measure the degradation pattern (curve) at different incubation times. All samples were kept in the autosampler at 25°C during the entire test.

[0628] 3. Results

[0629] The degradation profile of PS20 in DP3 doped with PLA2G15 matches the PS20 degradation pattern observed in the diluted DP2 material ( Figure 4A ).

[0630] Study on doping of PLA2G15 at different doping levels in DP4 material

[0631] 1. Methods

[0632] The PS20-CAD subspecies method described above was used.

[0633] 2. Study Design

[0634] The study design for the PLA2G15 doping study is shown in Table 20 below.

[0635] Table 20. Study design of 8 arms doped with different levels of PLA2G15

[0636]

[0637]

[0638] **PLA2G15 doping levels were labeled in different ways (eg, 0.3 ng / mg x 150 mg / mL risankizumab = 0.9 μg / mL final PLA2G15 concentration in DP4 solution); two types of labeling were used here to compare ELISA data with doping levels in this study. All samples were incubated at 25°C.

[0639] 3. Results

[0640] PS20 subspecies data from T0, day 1, day 4 and day 11 25°C incubation samples have been collected. All samples were kept at 5°C in an autosampler during data collection. PS20 concentration data from T0 to one month (1M) have also been collected. PLA2G15 doping concentration-dependent PS20 degradation rates were observed. PLA2G15-induced PS20 degradation patterns were very similar at all PLA2G15 doping levels ( Figure 4B ).

[0641] PLBL2 doping studies

[0642] 1. Background

[0643] Phospholipase B-like 2 protein (PLBL2) was detected in DP2 material. Early literature reported that PLBL2 can induce PS20 degradation in antibody formulations. However, very high concentrations were used in the study (Dixit et al., Journal of pharmaceutical science, 2016, 105: 1657-1666). Recent publications show that due to its low activity, it is very unlikely that PLBL2 causes PS20 degradation in antibody formulations (Zhang et al. Journal of pharmaceutical science, 2020, 109: 2710-2718).

[0644] 2. Methods

[0645] The PS20-CAD subspecies method described above was used.

[0646] 3. Study Design

[0647] The DP3 drug solution was doped with 5 μg / mL PLBL2 enzyme. The solutions were mixed and tested. All samples were studied at 25°C. The study design was similar to the PLA2G15 doping study in DP3 material as described above. More information on the study design is shown in Table 21A below.

[0648] Table 21A. Study Design of PLBL2, CES and SIAE Doping Studies

[0649]

[0650] 4. Results

[0651] No detectable degradation of PS20 was observed after incubation for approximately 30 hours at 25°C. Figure 5 Typical results are shown. The results confirm the low activity of PLBL2 on PS20 degradation. The doping level (5 μg / mL) is much higher than the PLBL2 concentration detected in DP2 material by the PLBL2 ELISA method. No detectable PS20 degradation after incubation for about 30 hours at 25°C indicates that PLBL2 is unlikely to be the main root cause of PS20 degradation in DP2 material.

[0652] CES doping study

[0653] 1. Background

[0654] Carboxylesterase 1 (CES1) was detected in DP2 material. Literature reports that CES1 can induce PS20 degradation in antibody formulations (Zhang et al. Pharmaceutical Research, 2022, 39: 75-87).

[0655] 2. Methods

[0656] The PS20-CAD subspecies method described above was used.

[0657] 3. Study Design of CES Doping Study

[0658] The DP3 drug solution was doped with 5 μg / mL CES1 enzyme. The solutions were mixed and tested. All samples were studied at 25°C. The study design was similar to the PLA2G15 doping study in the Risenqizumab DP3 material. More information can be found in Table 21A.

[0659] 4. Results

[0660] Significant PS20 degradation was observed after only a few hours of incubation at 25°C. The PS20 degradation patterns (curves) for different incubation times were similar (different levels of degradation). CES1 can cause significant degradation of PS20 in the Risenqizumab formulation. However, the PS20 degradation pattern of CES 1 in the Risenqizumab formulation is very different from the PS20 degradation pattern / curve observed in the DP2 material. The main PS20 peak (polyoxyethylene sorbitan monolaurate) was significantly degraded by CES1, but the PS20 subspecies region with retention time >50 minutes showed only little degradation. Fig. 6A Typical results are shown. Figure 6BThe overlay of the PS20 degradation patterns (curves) observed in DP2 and caused by CES 1 is shown in Figure 3 and they are very different. Therefore, CES1 is unlikely to be the main contributor to PS20 degradation in DP2 material.

[0661] Study on doping with sialic acid O-acetylesterase

[0662] 1. Background

[0663] Sialyl O-acetylesterase (SIAE) was detected in DP2 materials. The literature reports that SIAE can induce PS20 degradation in antibody formulations, and moderate levels of PS20 degradation were observed after incubation at 45 ° C for 5 days, but high enzyme concentrations (5 μg / mL) were used in the study (Zhang et al. Journal of pharmaceutical sciences, 2021, 110: 3899-3873). The reported PS20 degradation pattern is different from the pattern observed in DP2 materials. In the literature, SIAE causes a significant decrease in the main peak of PS20 (polyoxyethylene sorbitan monolaurate) (Zhang et al. Journal of pharmaceutical sciences, 2021, 110: 3899-3873), which is stable in the DP2 materials tested herein.

[0664] 2. Methods

[0665] The PS20-CAD subspecies method described above was used.

[0666] 3. Sialyl O-acetylesterase doping study design

[0667] DP3 drug solution was spiked with 5 μg / mL SIAE enzyme. The solution was mixed and tested. More information on the study design can be found in Table 21A.

[0668] 4. Results

[0669] Figure 7 Typical results are shown. The results confirm the low activity of SIAE on PS20 degradation. At a doping level of 5 μg / mL, no detectable PS20 degradation was observed after incubation for about 30 hours at 25°C. The absence of detectable PS20 degradation after incubation for about 30 hours at 25°C indicates that SIAE is unlikely to be the main contributing factor to PS20 degradation in DP2 materials.

[0670] Peroxiredoxin 6 (PRDX6) doping study

[0671] 1. Background

[0672] Peroxiredoxin 6 (PRDX6) has been detected in DP2 material using LC-MS. Literature reports that PRDX6 may have PLA2-like activity. However, the PLA2-like activity of native proteins is limited at neutral pH. Oxidized phospholipids are more active in acidic environments and neutral pH (Fisher (2018) Journal of lipid research 59: 1132-1147).

[0673] 2. Materials

[0674] Recombinant human peroxiredoxin 6 protein (PRDX6) (ab87631, AB87631-100UG, 1 mg / ml. Abcam, kept at -80°C before use)

[0675] 3. Methods

[0676] The PS20-CAD subspecies method described above was used.

[0677] 4. Study Design of PRDX6 Doping Study

[0678] Risenqizumab DP3 was doped with 5 μg / mL PRDX6 enzyme. The solution was mixed and tested. More information on the study design can be found in Table 21B below.

[0679] Table 21B Study Design of PRDX6 and PLA2G7 Doping Study

[0680]

[0681] *Note: Data were collected through the 8th injection, but due to issues with low volume of solution remaining in the vial at the time of the 8th injection, the 7th injection is plotted in the summary.

[0682] 5. Results

[0683] Figure 8 Typical results are shown. The results show low activity of PRDX6 on PS20 degradation. At a doping level of 5 μg / mL, no detectable PS20 degradation was observed after incubation for about 30 hours at 25°C. The absence of detectable PS20 degradation after incubation for about 30 hours at 25°C indicates that PRDX6 is unlikely to be a major contributor to PS20 degradation in the DP2 material.

[0684] Study on doping of PLA2G7

[0685] 1. Background

[0686] Phospholipase A2 class VII (PLA2G7) has been detected in DP2 material using LC-MS. Literature reports that PLA2G7 can degrade PS20 in antibody formulation solution (Li et al. (2021) Analytical chemistry 93: 8161-8169).

[0687] 2. Materials

[0688] Recombinant human PLA2G7 / PAF-AH / LP-PLA2 protein, (5106-PL-010; 0.44 mg / mL, Bio-techne, kept at -80°C before use)

[0689] 3. Methods

[0690] The PS20-CAD subspecies method described above was used.

[0691] 4. Study Design of PLA2G7 Doping Study

[0692] Lisenqizumab DP3 was doped with 5 μg / mL PLA2G7 enzyme. The solution was mixed and tested. More information about the study design can be found in Table 21B.

[0693] 5. Results

[0694] Fig. 9 Typical results are shown. The results confirm that PLA2G7 causes PS20 degradation with relatively high activity. However, the PS20 degradation curve caused by PLA2G7 is very different from the PS20 degradation curve observed in DP2 materials. The doping study results indicate that PLA2G7 is unlikely to be the main contributing factor to PS20 degradation in DP2 materials.

[0695] Overview

[0696] The PS20 degradation patterns (curves) in DP3 and DP4 materials doped with six enzymes have been studied. These six enzymes have been detected in DP2 materials (four detected by LC-MS and two detected by ELISA). Three enzymes (PLBL2, PRDX6 and SIAE) show very low activity on PS20 degradation even at very high concentrations. Therefore, these three enzymes are unlikely to be the main root cause of PS20 degradation in DP2 materials. Enzymes CES and PLA2G7 show moderate activity on PS20 degradation. However, the PS20 degradation patterns (curves) caused by CES and PLA2G7 are very different from the patterns (curves) observed in DP2 materials. Therefore, CES and PLA2G7 are also unlikely to be the main root cause of PS20 degradation in DP2 materials. The PS20 degradation pattern (curve) caused by PLA2G15 matches the PS20 degradation pattern in DP2 materials well. This study indicates that PLA2G15 is the key responsible enzyme for PS20 degradation in DP2 drug products.

[0697] Example 8: PLA2G15 inhibition studies confirm that PLA2 is a problematic chaperone

[0698] It has been reported that the small molecule drug fosinopril can inhibit the activity of PLA2G15 (phospholipase A2XV class) with an IC50 of about 0.18uM (Hinkovska-Galcheva et al. (2021) J. Lipid. Res. 62: 100089). Fosinopril is an angiotensin-converting enzyme (ACE) inhibitor used to treat hypertension and some types of chronic heart failure (Murdoch et al. (1992) Drugs 43: 123–140). As a result of the liposomal PLA2G15 co-sedimentation assay, it has been further suggested that fosinopril inhibits PLA2G15 activity by interfering with the binding of PLA2G15 to the liposome surface (Hinkovska-Galcheva et al. (2021) J. Lipid. Res. 62: 100089). To further confirm that PLA2G15 is primarily responsible for the degradation of polysorbate 20 (PS20) in DP2 materials, a series of PLA2G15 inhibition studies were performed by doping DP2 materials with different levels of fosinopril. In this study, DP2 samples were doped with different levels of fosinopril under sterile conditions. All samples (including non-doped samples) were incubated in the dark at room temperature for two weeks. The PS20 degradation levels in these samples were tested using the PS20 subspecies method. The PS20 degradation levels in these samples were compared with the negative control (non-doped samples kept at room temperature in the dark for two weeks) and the positive control (non-doped samples kept at -80°C before testing). Dose-dependent protection (inhibition) of fosinopril against PS20 degradation in DP2 materials has been observed. Protection (inhibition) was observed even at sub-ug / mL doping levels. This result, combined with other studies, further confirms that PLA2G15 is the root cause of PS20 degradation in DP2 materials. The chemical structure of fosinopril is shown below.

[0699]

[0700] Material:

[0701] DP2 material (kept at -80℃)

[0702] Fosinopril Sodium (F13085MG) (from Sigma Aldrich)

[0703] Sample preparation:

[0704] Fosinopril sodium was dissolved in Mill-Q water at a concentration of 0.5 mg / mL. The solution was sterile filtered with a 0.22 syringe filter before use. During filtration, the syringe filter was rinsed with Mill-Q water and then with a 0.5 mg / mL aqueous solution of fosinopril. The lower concentration aqueous solution of fosinopril was aseptically diluted with Mill-Q water. More detailed sample preparation can be found in Table 22. All vials were glass vials, and the solutions were thoroughly mixed before incubation. The samples were incubated in the dark at room temperature for two weeks before testing.

[0705] Table 22. Sample preparation

[0706]

[0707] Test Method

[0708] The PS20 HPLC-CAD subspecies method was used to qualitatively test the PS20 levels and curves in all samples. The solution was transferred to an HPLC bottle and diluted with Milli-Q water (1: 1 dilution). All HPLC bottles were fully mixed and then placed in an HPLC autosampler for testing. Sample description and labeling are shown in Table 23.

[0709] Table 23: Sample information

[0710]

[0711] result:

[0712] Dose (concentration) dependent protection (inhibition) of PS20 degradation by fosinopril in DP2 material solutions was observed during the study period. For example, even at the lowest fosinopril doping level (0.9 μg / mL), a reduction in PS20 degradation was observed compared to the non-doped control sample. Fig.10 Results are shown for a fosinopril doping level of 0.9 μg / mL. Fig.11 Results are shown for a fosinopril doping level of 3.8 μg / mL. Fig.12 Results are shown for a fosinopril doping level of 27.8 μg / mL.

[0713] Since fosinopril co-elutes with PS20 in the chromatogram (peak around retention time 39.5 min), it can be detected at high doping levels (e.g. Fig.12 Further analysis was performed to evaluate potential impacts on the observed PS20 signal. Fig.13 The normalized results for a very high level of fosinopril doping (930 ug / mL) are shown. It shows that the fosinopril peak is around 39.5 minutes. Figure 10-12The relatively low levels of fosinopril adulteration in the samples had negligible impact on retention times > 42 min.

[0714] Overview

[0715] Dose-dependent protection (inhibition) of PS20 degradation by doping fosinopril in DP2 materials has been observed. Fosinopril showed protection (inhibition) even at 0.9 μg / mL doping level. Since fosinopril is a potent PLA2G15 inhibitor, the results of this study combined with other studies further confirm that PLA2G15 should be the root cause of PS20 degradation in DP2 materials.

[0716] Example 9: Risenqizumab drug products DP3 and DP4 have lower levels of associated proteins.

[0717] Residual concomitant proteins in drug substance concentrate samples were determined by enzyme-linked immunosorbent assay (ELISA) as described below.

[0718] PLA2 ELISA

[0719] 1. Principle

[0720] 96-well microtiter plates (Nunc Maxisorp catalog number 439454; VWR catalog number 62409-002) were coated with polyclonal rabbit anti-CHO-PLA2 antibody (1 mg / mL) and then incubated with SuperBlock (Thermo Scientific catalog number 37535) in TBS to block nonspecific sites. Recombinant PLA2 standards ([PLA-2G15 (cg) (34-412)]-6His, 1.05 mg / mL) and bulk drug were then added to the plates. The plates were incubated to allow residual PLA2 present in the standards and samples to bind to the polyclonal anti-PLA2 antibodies. The plates were washed to remove unbound material, and biotinylated rabbit anti-CHO PLA2 polyclonal antibodies (1 mg / mL) were added to the plates. The plates were incubated to allow the biotinylated antibodies to bind to the residual PLA2 antigen bound to the anti-PLA2 antibodies. The plate was washed to remove unbound material and neutravidin-HRP (enzyme-labeled horseradish peroxidase; Thermo Scientific catalog number 31030) was added to the plate. The plate was incubated to allow neutravidin-HRP to bind to the bound biotinylated antibody. The plate was washed to remove unbound material and K-Blue TMB substrate (Neogen catalog number 308177) was added to the plate. The chromogenic substrate was oxidized by the bound enzyme-conjugated antibody to produce a blue color. The reaction was terminated with 4N (2M) sulfuric acid (Ricca catalog number 8322-32) and the color changed to yellow. The color intensity is proportional to the amount of residual PLA2 antigen bound in the well. The plate was read at 450 nanometers using a plate reader.

[0721] 2. Security

[0722] 3. Equipment

[0723] Molecular Devices Plate Reader or equivalent

[0724] BioTek ELx405 96W Microplate Washer or equivalent

[0725] Adjustable pipette with tip Rainin or equivalent

[0726] 8 or 12 channel pipette with tip Rainin or equivalent

[0727] Titer plate shaker, laboratory tubing or equivalent, room temperature

[0728] Incubator / shaker, LabLine Environ Plate Shaker or equivalent, 37°C

[0729] pH Meter

[0730] Balance

[0731] 4. Materials

[0732] 96-well microtiter plate, Nunc Maxisorp Cat. No. 439454 (VWR Cat. No. 62409-002) or equivalent

[0733] ELISA Plate Sealing Tape - Corning Cat. No. 430454 or equivalent

[0734] Polypropylene tube

[0735] Milli-Q water, MPS-66 or equivalent

[0736] Sodium bicarbonate, NaHCO3 FW 84.01 g / mol, JTBaker Cat. No. 3509-01 or equivalent

[0737] 10X Phosphate Buffered Saline with 0.5% Tween-20, Boston BioProducts Catalog No. IBB-171

[0738] 5N Sodium Hydroxide, JT Baker Cat. No. 5671-02 or equivalent

[0739] 5N Hydrochloric acid, JT Baker Cat. No. 5618-02 or equivalent

[0740] SuperBlock in TBS, Thermo Scientific cat. no. 37535 or equivalent

[0741] Sulfuric acid 4N, Ricca catalog number 8322-32 (2N = 1M) or equivalent

[0742] K-Blue TMB Substrate, Neogen Cat. No. 308177 or equivalent

[0743] 0.22 μM CA sterile filter unit, Corning or equivalent

[0744] Abcam sample diluent, Abcam catalog number GR3347356-3; ab193972

[0745] Rabbit anti-CHO-PLA2 coated polyclonal antibody, 1 mg / mL, stored at nominally -80°C

[0746] Biotinylated rabbit anti-CHO-PLA2 polyclonal antibody, 1 mg / mL, stored at nominally -80°C

[0747] Recombinant PLA2 standard, [PLA-2G15(cg)(34-412)]-6His, 1.05 mg / mL, stored at nominally -80°C

[0748] Neutravidin-HRP conjugate, Thermo Scientific cat# 31030 or equivalent, aliquot, store at nominally 4°C

[0749] Lisenqizumab assay control, Lisenqizumab bulk API 145 mg / mL, stored at nominal -80°C

[0750] 5. Preparation of reagents and solutions:

[0751] Note: Use reverse pipetting throughout the assay. Use cold coating buffer and substrate (taken from nominal 4°C immediately before use).

[0752] 5.1 50 mM sodium bicarbonate, pH 9.4 ± 0.1 (coating buffer):

[0753] Add 900 mL of Milli-Q water to a 1 L beaker.

[0754] Add 4.20 g ± 0.01 g sodium bicarbonate.

[0755] Stir until completely dissolved.

[0756] The pH was adjusted to 9.4 ± 0.1 with 5N NaOH and 5N HCl.

[0757] Transfer to a 1 L volumetric flask and adjust volume with Milli-Q water.

[0758] Mix by inversion until smooth.

[0759] Filter through a sterile filter unit (0.22 μm).

[0760] Store at nominally 4 °C for up to 7 days from the date of preparation.

[0761] 5.2 Washing buffer (1X PBS + 0.05% Tween-20):

[0762] In a 1 L graduated cylinder, add 100 mL of 1OX phosphate buffered saline containing 0.5% Tween-20 to 900 mL of Milli-Q water.

[0763] Stir until smooth.

[0764] Filter through a 0.22 μm sterile filter unit.

[0765] Store at room temperature for up to 6 months.

[0766] 5.3 Coating antibody mixture: rabbit anti-CHO PLA2 polyclonal antibody (1 mg / mL), affinity purified:

[0767] Note: Store antibody stocks at nominally -80°C in vials. Prepare aliquots. Remove one aliquot per plate immediately before use. Take 50 mM coating buffer (step 5.1) from nominally 4°C just before use. Apply mixture to plates while cool.

[0768] Immediately before use: dilute coating antibody to a final concentration of 3.0 μg / mL in cold 50 mM sodium bicarbonate as follows.

[0769] For example: Add 36 μL of coating antibody to 11964 μL of cold coating buffer. Mix gently by inversion.

[0770] 5.4 Rabbit anti-CHO PLA2 biotinylated polyclonal antibody (1 mg / mL)

[0771] Note: Stock solution stored in vials at nominally -80 °C. Prepare aliquots. When used, remove one aliquot per plate.

[0772] Immediately before use: dilute the biotinylated antibody to a final concentration of 0.20 μg / mL in SuperBlock in TBS as follows.

[0773] For example: Prepare dilution A by diluting 10 μL of biotinylated antibody in 90 μL of SuperBlock in TBS. Mix gently by inversion. Prepare dilution B by further diluting 24 μL of dilution A in 11976 μL of SuperBlock in TBS for a final dilution factor of 1:5000.

[0774] 5.5 Neutravidin-HRP conjugate (1 mg / mL)

[0775] Note: Stock solutions stored at nominally 4°C. When used, remove one aliquot per plate and warm to room temperature.

[0776] Immediately before use, gently pipette up and down to mix the conjugate. Dilute Neutravidin-HRP conjugate to a final concentration of 0.2 μg / mL. Gently vortex to mix.

[0777] For example: Prepare dilution A by diluting 10 μL of HRP conjugate into 90 μL of SuperBlock in TBS. Mix gently by inversion. Prepare dilution B by further diluting 24 μL of dilution A into 11976 μL of SuperBlock in TBS.

[0778] 6. Preparation of Standards and Adulterants

[0779] NOTE: Stock solution stored in single-use aliquots at nominally -80 °C.

[0780] 6.1 Recombinant PLA2 standard, [PLA-2G15(cg)(34-412)]-6His (1.05 mg / mL)

[0781] 6.1.1 Thaw aliquots at room temperature. Perform serial dilutions in Abcam sample diluent to a concentration of 21 ng / mL. Prepare serial dilutions of the standard curve in polypropylene tubes using Abcam sample diluent as shown in the table below.

[0782] Table 24. Standard dilution scheme for recombinant PLA2

[0783]

[0784] 6.1.2 Gently pipette the standards up and down to mix.

[0785] 6.1.3 Transfer to a polypropylene microtube.

[0786] 6.1.4 Load each standard in triplicate into a 96-well microtiter plate at 100 μL per well.

[0787] 6.2 Preparation of dopants

[0788] In a polypropylene microtube, prepare a high level of PLA2 adulterant 10.5 ng / mL by diluting the 21.000 ng / mL standard (Standard 1 in step 6.1.1) 2X with Abcam sample diluent. Perform a single dilution.

[0789] For example: dilute 400 μL of 21.000 ng / mL (Standard 1) in 400 μL Abcam Sample Diluent for a final concentration of 10.5 ng / mL.

[0790] In a polypropylene microtube, prepare a mid-level PLA2 adulterant of 5.25 ng / mL by diluting the 10.5 ng / mL standard (Standard 2 in step 6.1.1) 2X with Abcam sample diluent. Perform a single dilution.

[0791] In a polypropylene microtube, prepare a mid-level PLA2 adulterant of 1.313 ng / mL by diluting the 2.625 ng / mL standard (Standard 4 in step 6.1.1) 2X with Abcam sample diluent. Perform a single dilution.

[0792] The spikes were loaded in triplicate into 96-well microtiter plates at 100 μL per well.

[0793] 7. Sample Preparation

[0794] 7.1 In a polypropylene tube, dilute the RisenQzumab process 1 BDS to 2.81 mg / mL in Abcam sample diluent (step 4). Pre-dilutions should be made in sufficient volume to allow the serial dilutions to be plated in triplicate at 100 μL / well.

[0795] NOTE: Use the following dilution scheme to prepare adulterated samples (step 8) and the levofloxacin process 1 BDS nominal test dilutions.

[0796] Table 25. Example, pre-dilution:

[0797]

[0798] 7.2 In a polypropylene microtube, further dilute the RisenQizumab Process 1 BDS to 0.18 mg / mL using sample diluent (dilutions 1 to 4 as determined by PLA2 ELISA).

[0799] Table 26. Example:

[0800]

[0801] 7.3 In a polypropylene microtube, dilute the RisenQizumab Process 2BDS 150 mg / mL to 9.38 mg / mL using sample diluent.

[0802] NOTE: Use the following dilution scheme to prepare adulterated samples (step 8) and nominal test dilutions of the levitra process 2 BDS sample.

[0803] Table 27. Example, pre-dilution:

[0804]

[0805] 7.4 In polypropylene microtubes, further dilute the process 2 BDS 150 mg / mL (9.38 mg / mL) solution to 0.59 mg / mL (dilutions 1 to 4 as determined by PLA2 ELISA) using Abcam sample diluent.

[0806] Table 28. Example:

[0807]

[0808] 7.5 In a polypropylene microtube, dilute the 150 mg / mL of Lisenqizumab Process 3 BDS to 37.5 mg / mL.

[0809] NOTE: Use the following dilution scheme to spike samples in step 8 and make a nominal dilution of the Risenzilimab Process 3 BDS sample.

[0810] Table 29. Example, pre-dilution:

[0811]

[0812] 7.6 In a polypropylene microtube, further dilute the 37.5 mg / mL Lisenqizumab Process 3 BDS solution to 2.34 mg / mL using sample diluent (dilutions 1 to 4 for PLA2 ELISA assay)

[0813] Table 30. Example:

[0814]

[0815] 7.7 In a polypropylene microtube, dilute the RisenQizumab Process 4 BDS 150mg / mL to 20mg / mL.

[0816] NOTE: Use the following dilution scheme to prepare adulterated samples (step 8) and nominal test dilutions of the levitra process 4 BDS sample.

[0817] Table 31. Example, pre-dilution:

[0818]

[0819] 7.8 In a polypropylene microtube, further dilute the 20 mg / mL RisenQizumab Process 4 BDS solution to 1.25 mg / mL using sample diluent (dilutions 1 to 4 for PLA2 ELISA assay)

[0820] Table 32. Example:

[0821]

[0822] 7.9 Load triplicate wells of each nominal test solution (dilutions 1-4 from steps 7.2, 7.4, 7.6, 7.8) onto the plate at 100 μL / well.

[0823] 8. Preparation of Doped Samples

[0824] 8.1 In a polypropylene microtube, mix 400 μL of Abcam Sample Diluent with the same volume of 400 μL of pre-diluted Risenqizumab BDS sample (steps 7.1, 7.3, 7.5, and 7.7) as the undoped Risenqizumab BDS sample.

[0825] 8.2 In a polypropylene microtube, mix 400 μL of 21 ng / mL PLA2 standard solution (Standard 1) with the same volume of 400 μL of pre-diluted Risenqizumab BDS samples (steps 7.1, 7.3, 7.5, and 7.7). These were tested as adulterated samples at high levels of PLA2.

[0826] 8.3 In a polypropylene microtube, mix 400 μL of 10.5 ng / mL PLA2 standard solution (Standard 2) with the same volume of 400 μL of pre-diluted Risenqizumab BDS samples (steps 7.1, 7.3, 7.5, and 7.7). These were tested as adulterated samples at medium levels of PLA2.

[0827] 8.4 In a polypropylene microtube, mix 400 μL of 2.625 ng / mL PLA2 standard solution (Standard 4) with the same volume of 400 μL of pre-diluted Risenqizumab BDS samples (steps 7.1, 7.3, 7.5, and 7.7). These were tested as adulterated samples at low levels of PLA2.

[0828] 8.5 The final test concentration of the adulterated samples was the same as the non-adulterated sample preparation (dilutions 1-4) + 5.250 ng / mL (the adulterated samples were diluted 2X from 10.500 ng / mL).

[0829] 8.6 For non-spiked samples and (low, medium and high levels of PLA2) standard spiked samples, load triplicate wells of each spiked sample solution onto the plate at 100 μL / well.

[0830] 9. Preparation of RisenQizumab Sample Control

[0831] 9.1 A control range must be established for each new control stock solution before use in routine testing.

[0832] 9.2 The control was 145 mg / mL of levitra bulk drug substance.

[0833] 9.3 Control Stock Solution: Prepare 50 μL aliquots of a batch of levitra bulk drug substance and store at nominally -80 °C.

[0834] Working Control: Thaw an aliquot of the control at room temperature. In a polypropylene tube, dilute the control to 7.25 mg / mL with Abcam sample diluent. Transfer the 7.25 mg / mL solution to a polypropylene microtube and load 100 μL per well into 3 wells of the plate. A single dilution is appropriate. Record the result in the PA Control Log, rounded to the nearest 0.1 ng / mg.

[0835] Table 33. Example:

[0836]

[0837] 10 Preparation of Assay Controls

[0838] 10.1 Prepare 5.25 ng / mL PLA2 standard assay control by diluting 600 μL of Abcam sample diluent with 200 μL of 21 ng / ml PLA2 standard solution (Standard 1 in step 6.1.1)

[0839] 10.2 Use 5.25 ng / mL PLA2 standard solution as an assay control and load triplicate wells at 100 μL / well.

[0840] 11. Program

[0841] 11.1 Plate washing instructions

[0842] Fill the plate washing bottle with plate washing buffer ((1X PBS + 0.05% Tween-20, refer to step 5.2). Prime the plate washer. Check the following parameters.

[0843] Parameters should be set to: Board Type: 1

[0844] For each cycle (4 cycles total): Volume: 350 μL

[0845] Soaking time: 10 seconds

[0846] Absorption time: 4 seconds

[0847] For the BioTek Elx405 microplate washer, the parameters should be set to:

[0848] Method: 4 cycles

[0849] Soaking / Shaking: Yes

[0850] Soaking duration: 010 seconds

[0851] Shake before soaking: No

[0852] Infusion after soaking: No

[0853] Dispensing: Dispensing volume: 350 μL / well

[0854] Dispensing flow rate: 05

[0855] Dispensing height: 120 (15.240mm)

[0856] - Horizontal DISP POS: 0mm

[0857] First bottom wash: No

[0858] Prime before start: No

[0859] Suction: Suction height: 0.29 (3.683mm)

[0860] Horizontal ASPR POS: -30 (-1.372mm)

[0861] Suction rate: 03 (4 mm / sec)

[0862] Pump delay: 0004 ms

[0863] Horizontal suction: Yes

[0864] Horizontal cross: All

[0865] Horizontal height: 024 (3.048mm)

[0866] Horizontal POS: 30 (1.372mm)

[0867] Final suction: yes

[0868] Final pump delay: 0000 seconds

[0869] After each day of use, fill the washer with at least 4 L of Milli-Q water to remove the plate wash buffer.

[0870] Pour 1 L of warm 1% Tergazyme through the washer, then pour at least 4 L of Milli-Q water to remove the Tergazyme. Store the washer dry.

[0871] 11.2 Measurement Procedure: Use the checklist as a guide by reviewing the steps as they are completed. Also, record all equipment used during the measurement.

[0872] Note: Use reverse pipetting throughout the assay. Use cold coating buffer and substrate. Samples, standards, adulterants, spiked samples, and controls must be diluted in polypropylene tubes (smallest size possible depending on volume) and transferred to polypropylene microtubes for loading onto the plate. Dilutions can also be prepared in polypropylene microtubes if volume permits.

[0873] 11.2.1 Coat the plate with 100 μL / well of coating antibody (step 5.3). Tap the sides of the plate until the coating solution evenly covers the bottom of the wells, cover with sealing tape and incubate on an orbital plate shaker (or equivalent) at 180 rpm at nominally 4°C for 16 hours.

[0874] 11.2.2 After incubation, remove the plate and blocking buffer (SuperBlock in PBS) from the refrigerator and allow to equilibrate to ambient temperature.

[0875] 11.2.3 Aspirate the liquid from the plate and tap the plate firmly on Kimwipes to remove excess buffer.

[0876] 11.2.4 Block the plate by adding 300 μL / well SuperBlock in TBS to each well. Incubate for 1 hour at 37°C with shaking at 200 rpm on an orbital plate shaker.

[0877] 11.2.5 Prepare standards, samples, controls, adulterants, and spiked samples during the blocking incubation (refer to steps 6 to 9). Transfer volumes to polypropylene microtubes. Rearrange tubing to match plate map.

[0878] 11.2.6 Wash the plate for 4 wash cycles using 350 μL / well of plate wash buffer (step 5.2) using a plate washer at the settings listed in step 11.1. Blot the plate firmly on Kimwipes.

[0879] 11.2.7 Using an 8-channel pipette, add 100 μl / well of standards, samples, controls, adulterants, and adulterated samples to triplicate wells of the plate. Add 100 μL / well of Abcam sample diluent to all empty wells of the plate as a blank. Cover with sealing tape and incubate at 37°C for 1 hour while shaking at 200 rpm on an orbital plate shaker (or equivalent). Fill in the template to use as a guide when loading the plate.

[0880] 11.2.8 Wash the plate for 4 wash cycles using 350 μL / well of the plate washer with the plate wash buffer (step 5.2) at the plate washer settings of step 11.1. Blot the plate firmly on Kimwipes.

[0881] 11.2.9 Add 100 μL / well of biotinylated antibody to each well (step 5.4). Cover with sealing tape and incubate the plate at 37°C for 1 hour while shaking at 200 rpm on an orbital plate shaker (or equivalent).

[0882] 11.2.10 Wash the plate for 4 wash cycles using 350 μL / well of plate wash buffer (step 5.2) using a plate washer at the plate washer settings listed in step 11.1. Blot the plate firmly on Kimwipes.

[0883] 11.2.11 Add 100 μL / well of Neutravidin-HRP to each well (step 5.5). Cover with sealing tape and incubate the plate at 37°C for 1 hour while shaking at 200 rpm on an orbital plate shaker (or equivalent).

[0884] 11.2.12 Wash the plate for 4 wash cycles using 350 μL / well of plate wash buffer (step 5.2) using a plate washer at the plate washer settings listed in step 11.1. Blot the plate firmly on Kimwipes.

[0885] 11.2.13 Add 100 μL / well of K-Blue substrate to each well. Cover with sealing tape and incubate at room temperature (25°C ± 2°C) for 10 minutes without shaking.

[0886] 11.2.14 Stop the reaction by adding 100 μL / well of 2M (4N) sulfuric acid to each well.

[0887] 11.2.15 Read the plate using a plate reader at 450 nm.

[0888] 11.2.16 Plate Reader Settings

[0889] Turn on the computer, monitor, and plate reader.

[0890] Log in to your computer. Double-click the Softmax Pro icon and select OPEN from the File drop-down menu.

[0891] Create a new SoftMax file.

[0892] Validation parameters, determination of residual phospholipase A2 concentration by ELISA protocol parameters.

[0893] Set up the template and enter the concentrations of the standards. Do not enter dilution factors for samples, controls, adulterants, or adulterated samples. Designate the wells containing diluent (see step 11.2.7) as blanks to be subtracted from all wells.

[0894] 12. Data Analysis and Calculation

[0895] Note: Only samples, adulterants, adulterated samples, and controls whose optical density falls within the practical limit of quantitation of the standard curve (0.328 ng / mL-21.000 ng / mL standard) and meets the %CV or %Difference criteria described below will be accepted. If the OD of a sample is lower than the 0.328 ng / mL standard, the result should be reported as less than 0.328 ng / mL. This value should be divided by the diluted Risenqizumab sample concentration to report the value in ng / mg. If the sample has a high PLA2 concentration, causing the non-adulterated sample and / or adulterated sample to be above the standard curve, report a value >21.000 ng / mL. This value should be divided by the diluted Risenqizumab sample concentration to report the value in ng / mg.

[0896] Note: If the mean OD value of one sample dilution is ≥ the mean OD value of the 0.328 ng / mL standard and the mean OD value of another dilution is < the mean OD value of the 0.328 ng / mL standard, report the result using the value of the dilution with the mean OD value ≥ the mean OD value of the 0.328 ng / mL standard, as long as the % difference between that ng / mL value and the 0.328 ng / mL value is ≤30%. If the % difference is >30%, repeat the sample.

[0897] 12.1 Standard Curve

[0898] 12.1.1 The concentrations of the standards should be entered into the protocol template. A 4-parameter logistic curve fit should be used.

[0899] 12.1.2 The coefficient of determination must be ≥ 0.99 and the %CV between triplicate wells must be ≤ 20%. If these criteria are not met:

[0900] 12.1.2.1 One standard may be discarded (1 level, 3 wells). If 0.328 ng / mL is discarded, only samples with an optical density falling within the optical density range of 0.656 ng / mL to 21.000 ng / mL (remaining standard curve points) and adulterated samples are acceptable.

[0901] 12.1.2.2 Additionally, for each standard level in triplicate, a single well may be discarded if it is clearly contaminated or shows low binding. If a well is discarded from a standard level, the remaining replicates must have a % difference of ≤ 20%.

[0902] 12.1.2.3 The %CV of the lowest standard that shows an OD value close to the plate background (blank) should be ≤30%. If one well is discarded, the % difference of the remaining replicates must be ≤35%. If the lowest standard is discarded, only samples and adulterated samples whose optical density falls within the optical density range of the remaining standard curve levels are acceptable.

[0903] 12.1.2.4 Calculate the % difference as follows:

[0904] % difference = (absolute (OD1 - OD2) / mean) x 100%.

[0905] If the standard does not meet the above criteria, the assay must be repeated.

[0906] Report %CV and / or %Difference values ​​and standard curve coefficient of determination results.

[0907] 12.2 Samples

[0908] The %CV between triplicate wells should be ≤20%. Report the %CV between triplicate wells. One well may be discarded from each sample dilution. The remaining replicates must have a %Difference of ≤20%. Note: If the unadulterated sample OD is lower than the 0.328 ng / mL standard OD, the %Difference criterion does not apply to the unadulterated results. Refer to calculation in step 12.1.2.4. When one sample dilution is ≥0.328 ng / mL (LOQ) and the second dilution is <0.328 ng / mL, refer to the second note at the beginning of Section 12.

[0909] Report the “Non-Adulterated Sample Result” for each dilution in ng / mL. These values ​​are used in the adulterant recovery calculations.

[0910] Calculate the average "Non-Adulterated Sample Result (ng / mL)" and the % difference between dilutions. Refer to calculation in step 12.1.2.4. The % difference between dilutions must be ≤ 20%. Report the results.

[0911] The PLA2 concentration (ng / mg) was calculated from the mean value (ng / mL) as follows:

[0912]

[0913] Record the results.

[0914] 12.3 Adulterants

[0915] The %CV between triplicate wells should be ≤ 20%. Record the %CV. One well may be discarded from the doping. The remaining wells must have a % difference of ≤ 20%. Refer to calculations in step 12.1.2.4.

[0916] Report PLA2 concentrations in ng / mL. This result is used for adulterant recovery calculations.

[0917] The resulting adulterant concentration (ng / mL) must be ±20% of the theoretical adulterant concentration. Record the result and indicate a pass or fail. If the adulterant result is not within 20% of the theoretical value, the assay must be repeated.

[0918]

[0919] Adulterated samples

[0920] The %CV between triplicate wells should be ≤ 20%. Record the %CV. One well may be discarded from each spiked sample dilution. The remaining replicates must have a % difference of ≤ 20%. Refer to calculations in step 12.1.2.4.

[0921] Report the "Adulterated Sample Result" in ng / mL for each dilution. Record the % difference between the duplicate dilutions (see step 12.1.2.4 for the formula). The % difference between the dilutions should be ≤ 25%. These results are used for the adulterant recovery calculation.

[0922] Calculate the % adulterant recovery for each dilution group using the following formula:

[0923]

[0924] Note: (1) If the OD of the non-adulterated sample value falls below the 0.328 ng / mL standard (LOQ), the value will be considered zero in the % Adulterant Recovery calculation.

[0925] The % adulterant recovery must be 100% ± 50% (50% - 150%) for each dilution of each sample. Record the results and pass / fail.

[0926] 12.5 Comparison

[0927] 12.5.1 The %CV between triplicate wells should be ≤ 20%. Record the %CV results. One well may be discarded from the control. The remaining replicates must have a % difference of ≤ 20%. Refer to calculations in step 12.1.2.4.

[0928] 12.5.2 Record the PLA2 concentration in ng / mL.

[0929] The PLA2 concentration was calculated in ng / mg as follows:

[0930]

[0931] Report the results (ng / mg) appropriately in the control log. If the control is outside the established range, the assay must be repeated.

[0932] 12.6 Blank

[0933] If any blank wells showed significant contamination, the wells were masked.

[0934] 12.7 Measurement range

[0935] The assay range has been determined to be 0.328 ng / mL to 21.000 ng / mL.

[0936] 13. Protocol for Preparation of Recombinant PLA2, Polyclonal Rabbit Anti-CHO PLA2 Antibody and Purified PLA2 Antibody

[0937] 13.1 Production of PLA2G15 Antigen

[0938] A DNA sequence encoding PLA2G15 (UNIPROT G3HK V9; amino acids 1-412) from C. grisea (Chinese hamster) was synthesized and cloned into the pHybE (U.S. Pat. No. 8,187,836 B2) vector followed by an in-frame hexa-histidine tag (SEQ ID NO: 15). The pHybE expression vector utilizes the EF-1α promoter and the OriP origin of replication derived from Epstein-Barr virus (EBV).

[0939] Using transfection reagent polyethyleneimine Max (PEI Max, Polysciences Inc), the plasmid was transfected into CHO-3E7 cells (NRC Canada) grown in BalanCD CHO culture medium (Irvine Scientific) at 3.3x10e6 cells / ml with a PEI:DNA ratio of 8:1. 24 hours after transfection, 4% 1XCHO4 feed (Irvine Scientific), 5% transfection supplement (Irvine Scientific) and 2.5g / L glucose were supplied to the transfected cell culture. On the 7th day after transfection, the transfected cell culture was clarified by centrifugation and then filtered through a Sartopore-2 0.45+0.2mm filter (Sartorius).

[0940] The clarified culture medium is loaded onto the 5ml HisTrap Excel column (Cytiva) balanced with PBS (pH 7.4). The column is washed with 25mm imidazoles in PBS (pH 7.4), and the protein bound by 500mM imidazole elution in PBS (pH 7.4). The protein eluted is concentrated by the Centricon Plus-70 centrifugal filter device (Millipore) with a molecular weight cut-off of 30kDa, and further purified by SEC on the 26 / 60Superdex 200 column (Cytiva) balanced and run with PBS (pH 7.4). The fractions containing PLA2G15 are collected, and the concentration is measured by the absorbance at 280nm, and the samples are analyzed by SEC, SDS-PAGE and mass spectrometry. Purified [PLA2G15 (cg) (34-412)]-6His is stored at -80°C with aliquots.

[0941] 13.2. Generation of polyclonal rabbit anti-CHO PLA2 antibody

[0942] Polyclonal rabbit anti-CHO PLA2 antibodies can be produced by immunizing rabbits (eg, New Zealand White rabbits) with the PLA2G15 antigen described above.

[0943] The antigen can be used with an adjuvant (eg, Freund's adjuvant) to enhance the immune response produced by polyclonal antibodies.

[0944] Antigens can be injected into animals intramuscularly, intradermally or subcutaneously.

[0945] Booster immunizations can be given, for example, 1 to 8 weeks after the initial immunization and continue at 1-4 week intervals.

[0946] Polyclonal antibody production in rabbits can be assessed by collecting serum samples prior to primary immunization and after primary and booster immunizations.

[0947] When antibody titers have reached acceptable levels, polyclonal antibody production can be terminated.

[0948] The animals were bled and serum was collected from the whole blood.

[0949] 13.3. Affinity Purification of Polyclonal Rabbit Anti-CHO PLA2 Antibody

[0950] PLA2-coupled CnBr-Sepharose beads (#17-0430-01) equilibrated in PBS were added to 740 ml of anti-PLA2 serum and incubated with rotation at 4°C for 2 days.

[0951] The beads are drained using an EconoPac column or similar and the flow-through is collected and kept for control purposes.

[0952] The beads were washed with about 200 ml TTBS and about 75 ml mild elution buffer and 200 ml TTBS and eluted with about ~50 ml volume of IgG elution buffer (Pierce #21009).

[0953] The eluate was neutralized with 10% v / v 2M Tris-HCl pH 7.5 (pre-dispensed into collection tubes). Protein peaks were measured with Bradford solution diluted 1:5 in water (200ul Bradford / / well, 20ul eluate added) pre-dispensed into 96-well plates.

[0954] PLA2-coupled CNBr-Sepharose beads were neutralized with 10 bed volumes of PBS and added back to the flow-through from the bead drain step (using EconoPac) for a second round of incubation at 4°C over the weekend.

[0955] The eluate was dialyzed three times on Big Tuna (Unchained Labs) with PBS exchange buffer (50 ml of eluate was exchanged 96% with PBS on Big Tuna). After the first round, 25 ml was removed, mixed, concentration measured, and the library was aliquoted 96 times at 250 ul / aliquot. Another 25 ml was transferred to a new Big Tuna filter plate and subjected to a new round of exchange with PBS 96% to a final concentration of 2x, then biotinylated).

[0956] PLBL2 ELISA

[0957] 1. Principle

[0958] 96-well microtiter plates (Nunc Maxisorp catalog number 439454; VWR catalog number 62409-002) were coated with polyclonal anti-PLBL2 antibodies (2 mg / mL). The plates were then incubated with SuperBlock (Thermo Scientific catalog number 37515) in PBS to block nonspecific sites. Recombinant PLBL2 standards ([PLBL-2 (cg) (38-585)]-6His, 1.27 mg / mL) and bulk drug were then added to the plates. The plates were incubated to allow residual PLBL2 present in the standards and samples to bind to the polyclonal anti-PLBL2 antibodies. The plates were washed to remove unbound material, and biotinylated anti-PLBL2 polyclonal antibodies (1 mg / mL) were added to the plates. The plates were incubated to allow the biotinylated antibodies to bind to the residual PLBL2 antigens bound to the anti-PLBL2 antibodies. The plate was washed to remove unbound material and streptavidin-poly-HRP (enzyme-labeled horseradish peroxidase; Thermo Scientific catalog number 21140) was added to the plate. The plate was incubated to allow streptavidin-poly-HRP to bind to the bound biotinylated antibody. The plate was washed to remove unbound material and K Blue TMB substrate (Neogen catalog number 308177) was added to the plate. The chromogenic substrate was oxidized by the bound enzyme-conjugated antibody to produce a blue color. The reaction was terminated with 4N (2M) sulfuric acid (Ricca catalog number 8310-32) and the color changed to yellow. The color intensity is proportional to the amount of residual PLBL2 antigen bound in the well. The plate was read at 450 nanometers using a plate reader.

[0959] 2. Security

[0960] Standard laboratory safety precautions.

[0961] 3. Equipment

[0962] Molecular Devices Plate Reader or equivalent

[0963] Tecan 96W plate washer or equivalent

[0964] Adjustable pipette with tip Rainin or equivalent

[0965] 8 or 12 channel pipette with tip Rainin or equivalent

[0966] Titer plate shaker, laboratory tubing or equivalent, room temperature

[0967] Incubator / shaker, Lab Line Environ Plate Shaker or equivalent, room temperature

[0968] pH Meter

[0969] Balance

[0970] 4. Materials

[0971] 96-well microtiter plate, Nunc Maxisorp Cat. No. 439454 (VWR Cat. No. 62409-002) or equivalent

[0972] ELISA Plate Sealing Tape - Corning Cat. No. 430454 or equivalent

[0973] Polypropylene tube

[0974] Opaque microtiter plate covers

[0975] Milli-Q water, MPS-66 or equivalent

[0976] Sodium bicarbonate, NaHCO3 FW 84.01 g / mol, JTBaker Cat. No. 3509-01 or equivalent

[0977] Tween-20, JTBaker Catalog No. X251-07 or equivalent

[0978] 5N Sodium Hydroxide, JT Baker Cat. No. 5671-02 or equivalent

[0979] 5N Hydrochloric acid, JT Baker Cat. No. 5618-02 or equivalent

[0980] Sodium chloride, NaCl FW 58.44 g / mol, Sigma catalog number S3014 or equivalent

[0981] Sodium phosphate dibasic, 7-hydrate, crystalline Na2HPO4*7H2O, FW 268.07, JTBaker Cat. No. 3817-01 or equivalent

[0982] SuperBlock in PBS, Thermo Scientific Cat. No. 37515 or equivalent

[0983] Sulfuric acid 4N, Ricca catalog number 8310-32 (2N = 1M), or equivalent

[0984] K-Blue TMB Substrate, Neogen Cat. No. 308177 or equivalent

[0985] 0.22 μM CA sterile filter unit, Corning or equivalent

[0986] Wash buffer, 1X PBS + 0.05% Tween-20, MPS-40, stored at room temperature Anti-id-PLBL2 polyclonal coating antibody, 2 mg / mL, stored at nominally -80°C

[0987] Biotinylated anti-PLBL2 polyclonal antibody, 1 mg / mL, stored at nominally -80°C

[0988] Recombinant PLBL2 standard, [PLBL-2(cg)(38-585)]-6His, 1.27 mg / mL, stored at nominally -80°C

[0989] Streptavidin-poly-HRP conjugate, Thermo Scientific cat# 21140 or equivalent, aliquot, store at nominally 4°C

[0990] PLBL2 Assay Control, Risenzizumab Ultrafiltration / Diafiltration (UF / DF) Retentate 10 mg / mL, Lot No. 91400096, aliquoted, stored at nominally -80°C

[0991] 5. Preparation of reagents and solutions:

[0992] Note: Use reverse pipetting throughout the assay. Use cold coating buffer and substrate (taken from nominal 4°C immediately before use).

[0993] 5.1 50 mM sodium bicarbonate, pH 9.4 ± 0.1 (coating buffer):

[0994] Add 900 mL of Milli-Q water to a 1 L beaker.

[0995] Add 4.20 g ± 0.01 g sodium bicarbonate.

[0996] Stir until completely dissolved.

[0997] The pH was adjusted to 9.4 ± 0.1 with 5N NaOH and 5N HCl.

[0998] Transfer to a 1 L volumetric flask and adjust volume with Milli-Q water.

[0999] Mix by inversion until smooth.

[1000] Filter through a sterile filter unit (0.22 μm).

[1001] Store at nominally 4 °C for up to 7 days from the date of preparation.

[1002] 5.2 10X Phosphate Buffered Saline (PBS), MPS-73:

[1003] Add 800 mL of Milli-Q water to a glass beaker.

[1004] 80.0 g of NaCl was added to achieve a final concentration of 80.0 g / L or 1.37M.

[1005] 2.00 g of KCl was added to achieve a final concentration of 2.00 g / L or 0.0268M.

[1006] 27.88 g of Na2HPO4·7H2O was added to achieve a final concentration of 27.88 g / L or 0.1040 M.

[1007] 2.40 g of KH2PO4 was added to achieve a final concentration of 2.40 g / L or 0.0176 M.

[1008] Make up to 1000 mL with Milli-Q water.

[1009] Stir until smooth.

[1010] Check pH and if necessary adjust to 6.8-6.9 with 5N HCl.

[1011] Stir until smooth.

[1012] Sterilize at 123°C for 30 minutes.

[1013] Store at room temperature for up to 12 months.

[1014] 5.3 Washing buffer / assay diluent / MPS-40 (1X PBS + 0.05% Tween-20):

[1015] Into a 1 L graduated cylinder, add 100 mL of MPS-73 (step 5.2) to 900 mL of Milli-Q water.

[1016] To the solution was added 0.5 mL of Tween-20.

[1017] Stir until smooth.

[1018] Check pH and if necessary adjust to 7.40 ± 0.05 with 5N HCl.

[1019] Stir until smooth.

[1020] Filter through a 0.22 μm sterile filter unit.

[1021] Store at room temperature for up to 6 months.

[1022] 5.4 Coating antibody mixture: anti-id PLBL2 polyclonal antibody (2 mg / mL), affinity purified:

[1023] Note: Store antibody stocks at nominally -80°C in vials. Prepare aliquots. Remove one aliquot per plate immediately before use. Take 50 mM coating buffer (step 5.1) from nominally 4°C just before use. Apply mixture to plates while cool.

[1024] Immediately before use: dilute coating antibody to a final concentration of 1 μg / mL in cold 50 mM sodium bicarbonate as follows.

[1025] For example: Add 6 μL of coating antibody to 11994 μL of cold coating buffer. Mix gently by inversion.

[1026] 5.5 Anti-PLBL2 polyclonal antibody-biotin conjugate (1 mg / mL)

[1027] Note: Stock solution stored in vials at nominally -80 °C. Prepare aliquots. When used, remove one aliquot per plate.

[1028] Immediately before use: dilute the biotinylated antibody to a final concentration of 0.80 μg / mL in MPS-40 (step 5.3) as follows.

[1029] For example: Dilute 10 μL of biotinylated antibody in 12490 μL of MPS-40. Mix gently by inversion.

[1030] 5.6 Streptavidin-poly-HRP conjugate (0.5 mg / mL)

[1031] Note: Stock solutions stored at nominally 4°C. When used, remove one aliquot per plate and warm to room temperature.

[1032] Immediately before use, gently pipette up and down to mix the conjugate. Dilute Streptavidin-Poly-HRP conjugate to a final concentration of 0.083 μg / mL. Gently vortex to mix.

[1033] For example: dilute 10 μL of HRP conjugate into 990 μL of MPS-40 (Diluent A). Then further dilute the HRP conjugate by adding 200 μL of Diluent A to 11800 μL of MPS-40 (Diluent B).

[1034] 6. Preparation of Standards and Adulterants

[1035] NOTE: Stock solution stored in single-use aliquots at nominally -80 °C.

[1036] 6.1 Recombinant PLBL2 standard, [PLBL-2(cg)(38-585)]-6His (1.27 mg / mL)

[1037] 6.1.1 Thaw aliquots at room temperature. Perform serial dilutions in MPS-40 (step 5.3) to a concentration of 4 ng / mL. The serial dilutions for preparing the standard curve using MPS-40 in polypropylene tubes are shown in the table below.

[1038] Table 34. Standard dilution scheme for recombinant PLBL2

[1039]

[1040]

[1041] 6.1.2 Gently pipette the standards up and down to mix.

[1042] 6.1.3 Transfer to a polypropylene microtube.

[1043] 6.1.4 Load each standard in triplicate into a 96-well microtiter plate at 100 μL per well.

[1044] 6.2 Preparation of dopants

[1045] 6.2.1 In a polypropylene microtube, prepare 0.320 ng / mL recombinant PLBL2 spike from the 0.640 ng / mL standard (Standard 3) prepared above (Step 6.1.1) by diluting it 2X with MPS-40 (Step 5.3). Perform a single dilution.

[1046] 6.2.2 For example: dilute 300 μL of 0.640 ng / mL (Standard 3) in 300 μL MPS-40 to a final concentration of 0.320 ng / mL.

[1047] 6.2.3 Load the spiked substances in triplicate into a 96-well microtiter plate at 100 μL per well.

[1048] 6.2.4 Spike the samples using the 0.640 ng / mL standard (Standard 3) from step 6.1.1.

[1049] 7. Sample Preparation

[1050] 7.1 In a polypropylene tube, dilute BDS to 0.045 mg / mL in MPS-40 (step 5.3). Make a pre-dilution in sufficient volume to plate the serial dilutions in triplicate at 100 μL / well.

[1051] NOTE: Use the following dilution scheme to prepare adulterated samples (step 8) and BDS nominal test dilutions.

[1052] Table 35. Example, pre-dilution:

[1053]

[1054] 7.2 In a polypropylene microtube, further dilute the 0.1788 mg / mL solution to 0.01117 mg / mL in MPS-40 (step 5.3).

[1055] Table 36. Example:

[1056]

[1057] 7.3 Risenqizumab Clarified Harvest followed a similar dilution scheme as Risenqizumab BDS, where the final inoculated serial dilutions were 1000X, 2000X, 4000X and 8000X.

[1058] NOTE: Use the following dilution scheme to prepare nominal test dilutions of adulterated samples (step 8) and clarified harvest.

[1059] Table 37. Example, pre-dilution:

[1060]

[1061] 7.4 In a polypropylene microtube, further dilute the 0.00098 mg / mL (0.98 μg / mL) solution to 0.061 μg / mL in MPS-40 (step 5.3).

[1062] Table 38. Example:

[1063]

[1064] 7.5 Risenqizumab Protein A eluate followed a similar dilution scheme as Risenqizumab BDS, where the final serial dilutions inoculated were 1000X, 2000X, 4000X and 8000X.

[1065] NOTE: Use the following dilution scheme in step 8 to spike samples and prepare nominal test dilutions of the Protein A eluate.

[1066] Table 39. Example, pre-dilution:

[1067]

[1068] 7.6 In a polypropylene microtube, further dilute the 0.0165 mg / mL solution to 0.00103 mg / mL in MPS-40 (step 5.3).

[1069] Table 40. Example:

[1070]

[1071]

[1072] 7.7 Load triplicate wells of each nominal test solution onto the plate at 100 μL / well for a total of 36 wells.

[1073] 8. Preparation of Doped Samples

[1074] 8.1 In polypropylene microtubes, add 400 μL of 0.640 ng / mL standard (Standard 3) to a fresh number of microtubes equal to the number of non-adulterated sample test dilutions.

[1075] 8.2 From steps 7.1, 7.3, and 7.5, transfer 400 μL of the 500X pre-dilutions (i.e., 0.1788 mg / mL BDS, 0.00098 mg / mL clarified harvest, and 0.0165 mg / mL Protein A eluate, respectively) to a fresh microtube containing 400 μL of 0.640 ng / mL spike. These are tested as spike dilution 1.

[1076] 8.3 Dilute dilutions 1, 2, and 3 from each non-adulterated sample preparation (steps 7.2, 7.4, and 7.6) 2X by adding 400 μL of sample to a fresh microtube containing 400 μL of 0.640 ng / mL adulterant. These are tested as adulterated dilutions 2-4.

[1077] For example: RisenQizumab BDS 89.38 mg / mL was adulterated into final test dilutions 1000X, 2000X, 4000X, 8000X.

[1078] Table 41.

[1079]

[1080]

[1081] 8.4 The final test concentration of the adulterated samples was the same as the non-adulterated sample preparation (dilutions 1-4) 0.320 ng / mL (the adulterated samples were diluted 2X from 0.640 ng / mL).

[1082] 8.5 Load triplicate wells of each spiked sample solution onto the plate at 100 μL / well for a total of 36 wells.

[1083] 9. Preparation of Controls

[1084] 9.1 A control range must be established for each new control stock solution before use in routine testing.

[1085] The control was RisenQzumab UF / DF retentate 10 mg / mL.

[1086] 9.2 Control Stock Solution: Prepare a batch of 500 μL aliquots of the RisenQzumab UF / DF retentate and store at nominally -80 °C.

[1087] 9.3 Working Control: Thaw an aliquot of the control at room temperature. In a polypropylene tube, dilute the control to 1 mg / mL with MPS-40 (step 5.3). Transfer the 1 mg / mL solution to a polypropylene microtube and load 100 μL per well into 3 wells of the plate. A single dilution is appropriate. Record the result in the PA Control Log, rounded to the nearest 0.1 pg / mg.

[1088] Table 42. Example:

[1089]

[1090] 10. Procedure

[1091] 10.1 Plate washing instructions

[1092] Fill the plate washer bottle with wash buffer (refer to step 5.3, MPS-40). Prime the plate washer. Check the following parameters.

[1093] Parameters should be set to: Board Type: 1

[1094] For each cycle (4 cycles total): Volume: 300 μL

[1095] Soaking time: 10 seconds

[1096] Absorption time: 4 seconds

[1097] For the BioTek Elx405 microplate washer, the parameters should be set to:

[1098] Method: 4 cycles

[1099] Soaking / Shaking: Yes

[1100] Soaking duration: 010 seconds

[1101] Shake before soaking: No

[1102] Infusion after soaking: No

[1103] Dispensing: Dispensing volume: 300 μL / well

[1104] Dispensing flow rate: 05

[1105] Dispensing height: 120 (15.240mm)

[1106] - Horizontal DISP POS: 0mm

[1107] First bottom wash: No

[1108] Prime before start: No

[1109] Suction: Suction height: 0.29 (3.683mm)

[1110] Horizontal ASPR POS: -30 (-1.372mm)

[1111] Suction rate: 03 (4 mm / sec)

[1112] Pump delay: 0004 ms

[1113] Horizontal suction: Yes

[1114] Horizontal cross: All

[1115] Horizontal height: 024 (3.048mm)

[1116] Horizontal POS: 30 (1.372mm)

[1117] Final suction: yes

[1118] Final pump delay: 0000 seconds

[1119] After each day of use, fill the washer with at least 4 L of Milli-Q water to remove the plate wash buffer.

[1120] Pour 1 L of warm 1% Tergazyme through the washer, then pour at least 4 L of Milli-Q water to remove the Tergazyme. Store the washer dry.

[1121] 10.2 Measurement Procedure: Use the checklist as a guide by reviewing the steps as they are completed. Also, record all equipment used during the measurement.

[1122] Note: Use reverse pipetting throughout the assay. Use cold coating buffer and substrate. Samples, standards, adulterants, spiked samples, and controls must be diluted in polypropylene tubes (smallest size possible depending on volume) and transferred to polypropylene microtubes for loading onto the plate. Dilutions can also be prepared in polypropylene microtubes if volume permits.

[1123] 10.2.1 Coat the plate with 100 μL / well of coating antibody (step 5.4). Tap the sides of the plate until the coating solution evenly covers the bottom of the wells, cover with sealing tape and incubate on an orbital plate shaker (or equivalent) at 180 rpm at nominally 4°C for 18 ± 1 hours.

[1124] 10.2.2 After incubation, remove the plate and blocking buffer (SuperBlock in PBS) from the refrigerator and allow to equilibrate to ambient temperature.

[1125] 10.2.3 Evacuate the liquid from the plate into a reservoir and tap the plate firmly on Kimwipes to remove excess buffer.

[1126] 10.2.4 Block the plate by adding 300 μL / well SuperBlock in PBS to each well. Incubate at room temperature for 1 hour without shaking.

[1127] 10.2.5 Prepare standards, samples, controls, adulterants, and spiked samples during the blocking incubation (refer to steps 6 to 9). Transfer volumes to polypropylene microtubes. Rearrange tubing to match plate map.

[1128] 10.2.6 Wash the plate using a plate washer MPS-40 (step 5.3) for 4 wash cycles using 300 μL / well. Blot the plate firmly on Kimwipes.

[1129] 10.2.7 Using an 8-channel pipette, add 100 μL / well of standards, samples, controls, adulterants, and adulterated samples to triplicate wells of the plate. Add 100 μL / well of MPS-40 (step 5.3) to all empty wells of the plate as a blank. Cover with sealing tape and incubate at room temperature (25°C ± 2°C) for 2 hours while shaking at 400 rpm on an orbital plate shaker (or equivalent). Fill in the template to use as a guide when loading the plate.

[1130] 10.2.8 Wash the plate using a plate washer MPS-40 (step 5.3) for 4 wash cycles using 300 μL / well. Blot the plate firmly on Kimwipes.

[1131] 10.2.9 Add 100 μL / well of biotinylated antibody (step 5.5) to each well. Cover with sealing tape and opaque plate cover to protect the reaction from light. Incubate the plate at room temperature (25°C ± 2°C) for 45 minutes while shaking at 400 rpm on an orbital plate shaker (or equivalent).

[1132] 10.2.10 Wash the plate with 300 μL / well for 4 wash cycles using MPS-40 (step 5.3) using a plate washer. Blot the plate firmly on Kimwipes.

[1133] 10.2.11 Add 100 μL / well of Streptavidin-Poly-HRP (step 5.6) to each well. Cover with sealing tape and opaque plate cover to protect the reaction from light. Incubate the plate at room temperature (25°C ± 2°C) for 30 minutes while shaking at 400 rpm on an orbital plate shaker (or equivalent).

[1134] 10.2.12 Wash the plate using a plate washer MPS-40 (step 5.3) for 4 wash cycles using 300 μL / well. Blot the plate firmly on Kimwipes.

[1135] 10.2.13 Add 100 μL / well of K-Blue substrate to each well. Cover with sealing tape and opaque plate cover to protect the reaction from light. Incubate at room temperature (25°C ± 2°C) for 10 minutes while shaking at 400 rpm on an orbital plate shaker (or equivalent).

[1136] 10.2.14 Stop the reaction by adding 100 μL / well of 2M (4N) sulfuric acid to each well.

[1137] 10.2.15 Read the plate using a plate reader at 450 nm.

[1138] 10.2.16 Plate Reader Settings

[1139] Turn on the computer, monitor, and plate reader.

[1140] Log in to your computer. Double-click the Softmax Pro icon and select OPEN from the File drop-down menu.

[1141] Create a new SoftMax file and set the parameters and plate map.

[1142] Validation parameters, determination of residual phospholipase B-like protein 2 concentration by ELISA protocol parameters.

[1143] Set up the template and enter the concentrations of the standards. Do not enter dilution factors for samples, controls, adulterants, or adulterated samples. Designate the wells containing diluent (see step 10.2.7) as blanks to be subtracted from all wells.

[1144] 11 Data Analysis and Calculation

[1145] Note: Only samples, adulterants, adulterated samples, and controls whose optical density falls within the practical limit of quantitation of the standard curve (0.041 ng / mL - 4 ng / mL standard) and meets the %CV or %Difference criteria described below were accepted. If the OD of a sample is below the 0.041 ng / mL standard, the result should be reported as less than 0.041 ng / mL. This value should be divided by the diluted sample concentration and multiplied by 1000 to report the value in pg / mg. If the sample has a high PLBL2 concentration, resulting in non-adulterated and / or adulterated samples above the standard curve, report a value >4 ng / mL. This value should be divided by the diluted sample concentration and multiplied by 1000 to report the value in pg / mg. When a sample is below the 0.041 ng / mL standard, the sample value is considered zero for adulterant recovery calculations.

[1146] Note: If the mean OD value of one sample dilution is ≥ the mean OD value of the 0.041 ng / mL standard and the mean OD value of another dilution is < the mean OD value of the 0.041 ng / mL standard, report the result using the value of the dilution with the mean OD value ≥ the mean OD value of the 0.041 ng / mL standard, as long as the % difference between that ng / mL value and the 0.041 ng / mL value is ≤ 30%. If the % difference is > 30%, repeat the sample.

[1147] 11.1 Standard Curve

[1148] 11.1.1 The concentrations of the standards should be entered into the protocol template. A 4-parameter logistic curve fit was used.

[1149] 11.1.2 The coefficient of determination must be ≥ 0.99 and the %CV between triplicate wells must be ≤ 20%. If these criteria are not met:

[1150] 11.1.2.1 One standard may be discarded (1 level, 3 wells). If 0.016 ng / mL is discarded, only samples with optical densities falling within the optical density range of 0.041 ng / mL to 4 ng / mL (remaining standard curve points) and adulterated samples are acceptable.

[1151] 11.1.2.2 Additionally, for each standard level in triplicate, a single well may be discarded if it is clearly contaminated or shows low binding. If a well is discarded from a standard level, the remaining replicates must have a % difference of ≤ 20%.

[1152] 11.1.2.3 The %CV of the lowest standard that shows an OD value close to the plate background (blank) should be ≤30%. If one well is discarded, the % difference of the remaining replicates must be ≤35%. If the lowest standard is discarded, only samples and adulterated samples whose optical density falls within the optical density range of the remaining standard curve levels are acceptable.

[1153] 11.1.2.4 Calculate the % difference as follows:

[1154] % difference = (absolute (OD1 - OD2) / mean) x 100%.

[1155] 11.1.3 If the standard does not meet the above criteria, the assay must be repeated.

[1156] 11.1.4 Report the %CV and / or %Difference values ​​and the coefficient of determination of the standard curve results.

[1157] 11.2 Samples

[1158] The %CV between triplicate wells should be ≤20%. Report the %CV between triplicate wells. One well may be discarded from each sample dilution. The remaining replicates must have a %Difference of ≤20%. Note: If the unadulterated sample OD is lower than the 0.041 ng / mL standard OD, the %Difference criterion does not apply to the unadulterated results. Refer to calculation in step 11.1.2.4. When one sample dilution is ≥0.041 ng / mL (LOQ) and the second dilution is <0.041 ng / mL, refer to the second note at the beginning of Section 11.

[1159] Report the “Non-Adulterated Sample Result” for each dilution in ng / mL. These values ​​are used in the adulterant recovery calculations.

[1160] Calculate the average "Non-Adulterated Sample Result (ng / mL)" and the % difference between dilutions. Refer to calculation in step 11.1.2.4. The % difference between dilutions must be ≤ 25%. Report the results.

[1161] The PLBL2 concentration (pg / mg) was calculated from the mean value (ng / mL) as follows:

[1162]

[1163] Record the results.

[1164] 11.3 Adulterants

[1165] The %CV between triplicate wells should be ≤ 20%. Record the %CV. One well may be discarded from the doping. The remaining wells must have a % difference of ≤ 20%. Refer to calculations in step 11.1.2.4.

[1166] Report PLBL2 concentrations in ng / mL. This result is used for adulterant recovery calculations.

[1167] The resulting adulterant concentration (ng / mL) must be ±20% of the theoretical adulterant concentration. Record the result and indicate a pass or fail. If the adulterant result is not within 20% of the theoretical value, the assay must be repeated.

[1168]

[1169] 11.4 Adulterated Samples

[1170] The %CV between triplicate wells should be ≤ 20%. Record the %CV. One well may be discarded from each spiked sample dilution. The remaining replicates must have a % difference of ≤ 20%. Refer to calculations in step 11.1.2.4.

[1171] Report the "Adulterated Sample Result" in ng / mL for each dilution. Record the % difference between the duplicate dilutions (see step 11.1.2.4 for the formula). The % difference between the dilutions should be ≤ 25%. These results are used for the adulterant recovery calculation.

[1172] Calculate the % adulterant recovery for each dilution group using the following formula:

[1173]

[1174] Note: (1) If the OD of the non-adulterated sample value falls below the 0.041 ng / mL standard (LOQ), the value will be considered zero in the % Adulterant Recovery calculation.

[1175] The % adulterant recovery must be 100% ± 50% (50% - 150%) for each dilution of each sample. Record the results and pass / fail.

[1176] 11.5 Comparison

[1177] The %CV between triplicate wells should be ≤20%. Record the %CV results. One well may be discarded from the control. The remaining replicates must have a % difference of ≤20%. Refer to calculations in step 11.1.2.4.

[1178] PLBL2 concentrations were recorded in ng / mL.

[1179] Calculate PLBL2 concentration in pg / mg as follows:

[1180]

[1181] Report the results appropriately (pg / mg) in the control log. If the control is outside the established range, the assay must be repeated.

[1182] 11.6 Blank

[1183] If any blank wells showed significant contamination, the wells were masked.

[1184] 11.7 Measurement range

[1185] The assay range has been determined to be 0.041 ng / mL to 4.000 ng / mL.

[1186] LPL ELISA

[1187] 1. Principle

[1188] The assay was performed using a commercial kit: Mouse LPL / lipoprotein lipase ELISA kit (Catalog No. LS-F11957; Lifespan Bioscience (LSBio)). The 96-well plate in the purchased kit was pre-coated with anti-LPL antibody and blocked with a blocking agent. Mouse LPL standards (provided with the kit) and lisinopril monoclonal antibody samples (serial dilutions were performed using the sample diluent provided in the kit) were added to the 96-well plate. The 96-well plate was incubated to allow the LPL present in the standards and samples to bind to the polyclonal anti-LPL antibody bound to the plate. The 96-well plate was washed to remove unbound material, and biotinylated anti-LPL antibody (detection reagent A) was added to the plate. The plate was incubated to allow the biotinylated antibody to bind to the LPL antigen bound to the anti-LPL antibody. The plate was washed to remove unbound material and streptavidin-HRP conjugate (enzyme-labeled horseradish peroxidase; detection reagent B) was added to the plate. The plate was incubated to allow streptavidin-HRP to bind to the bound biotinylated antibody. The plate is washed to remove unbound material and TMB substrate is added to the plate. The chromogenic substrate TMB is oxidized by the bound enzyme (HRP) conjugated antibody to produce blue. The colorimetric reaction is stopped with the stop solution provided and the color becomes yellow. The optical density of each well is directly proportional to the amount of CHO LPL antigen bound in the well. The plate is read at 450nm within 2 minutes after adding the stop solution. Blank deduction is performed.

[1189] 2. Security

[1190] Standard laboratory safety precautions.

[1191] 3. Equipment

[1192] Molecular Devices Plate Reader or equivalent

[1193] Relay pipette, Eppendorf or equivalent

[1194] Sterile filtration device (0.2μm)

[1195] Adjustable pipette with tip Rainin or equivalent

[1196] 8 or 12 channel pipette with tip Rainin or equivalent

[1197] Titer plate shaker, Wallac Delfia Cat. No. 1296-004 (1.5 mm shaking orbit) or equivalent, room temperature speed approximately 350 RPM

[1198] pH Meter

[1199] Balance

[1200] 4. Materials

[1201] ELISA Sealing Tape - Corning Cat. No. 430454 or equivalent

[1202] Polypropylene microtube

[1203] Immunoware microtubes with holder, 1.1 ml or equivalent

[1204] Low protein binding tubes, Eppendorf catalog numbers 022431064 (0.5 mL), 022431081 (1.5 mL), 022431102 (2.0 mL), and 0030108302 (5.0 mL)

[1205] MilliQ water

[1206] 0.22 μM CA sterile filter unit, Corning or equivalent

[1207] Mouse LPL / Lipoprotein Lipase ELISA Kit, Lifespan Bioscience Cat. No. LS-F11957, each kit contains:

[1208] 96-well ELISA plate (pre-coated with anti-LPL antibody and then blocked with blocking buffer by the supplier)

[1209] Mouse LPL Standard (lyophilized), 2 vials (depending on the lot number, lots from 2019 have 2 vials of 4 ng / vial lyophilized stock, lots from 2021 have 2 vials of 1 ng / vial lyophilized stock)

[1210] Sample diluent (20mL)

[1211] Biotinylated anti-LPL antibody (capture antibody) - detection reagent A (120 μL)

[1212] Determination diluent A (> 10mL)

[1213] Streptavidin-HRP conjugate-detection reagent B (120 μL)

[1214] Determination diluent B (> 10mL)

[1215] Wash buffer (25X concentration), 30 mL

[1216] TMB Substrate, 10ML

[1217] Stop solution, 10 mL

[1218] Adhesive Board Sealant

[1219] 5. Preparation of reagents and solutions:

[1220] NOTE: Reverse pipetting was used throughout the assay unless otherwise stated. All buffers were used at room temperature.

[1221] 5.1 1X plate washing buffer

[1222] In a 1 L graduated cylinder, add 30 mL of 25X Wash Buffer Concentrate to 720 mL of MilliQ water. Mix until smooth.

[1223] Once prepared, store the wash buffer at 4°C.

[1224] 5.2 Assay buffer (LSbio exclusive component)

[1225] Use the sample diluent and assay diluent for the biotinylated antibody and neutravidin-HRP conjugate provided with the kit (Assay Diluent A and B).

[1226] 5.3 Substrate

[1227] The TMB substrate provided by the kit was used.

[1228] 5.4 Stop solution

[1229] Use the stop solution provided by the kit.

[1230] 5.5 Detection Reagent A Working Solution

[1231] Immediately before use: Dilute Detection Reagent A 1:100 with Assay Diluent A.

[1232] For example: If 11 mL of Detection Reagent A working solution must be prepared, add 110 μL of Detection Reagent A to 10,890 μL of Assay Diluent A.

[1233] 5.6 Detection Reagent B Working Solution

[1234] Immediately before use: Dilute Detection Reagent B 1:100 with Assay Diluent B.

[1235] For example: If 11 mL of Detection Reagent B working solution must be prepared, add 110 μL of Detection Reagent B to 10,890 μL of Assay Diluent B.

[1236] 5.7 Assay control

[1237] A control was prepared with an LPL level of 62.5 pg / mL which fell within the assay range. Single-use aliquots were prepared and stored at nominally -80°C.

[1238] For example: Use 200 μL of sample diluent and mix with 200 μL of 125 pg / mL LPL standard;

[1239] 6 Preparation of standards and adulterants

[1240] 6.1 Preparation of standard curve solution

[1241] 6.1.1 For historical batches (2019-2020), reconstitute the lyophilized standard with 2 mL of sample diluent to prepare a LPL stock concentrate at a concentration of 2000 pg / mL. Incubate the LPL stock solution at room temperature for 10 minutes without vortexing or vigorous mixing.

[1242] 6.1.2 For the latest batch of the kit (from 2021), reconstitute the lyophilized standard with 1 mL of Sample Diluent to prepare a 1000 pg / mL LPL stock concentrate and incubate at room temperature for 10 minutes while vortexing or mixing vigorously.

[1243] 6.1.3 Prepare standard curve dilutions by serially diluting LPL stock concentrate (2000 pg / mL) using sample diluent to the following concentrations: 1000, 500, 250, 125, 62.5, and 31.25 pg / mL from step 6.1.1. See the table below for examples:

[1244] Table 43. Example of dilution scheme for LPL standards

[1245]

[1246] 6.1.4 Prepare standard curve dilutions by serially diluting LPL stock concentrate 1000 pg / mL using sample diluent to the following concentrations: 500, 250, 125, 62.5 and 31.25 pg / mL from step 6.1.2. See the table below for examples.

[1247] Table 44.

[1248]

[1249] NOTE: Depending on the batch size of the commercial kit used for this assay, then dilute the standard curve with concentrations ranging from 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, and 31.25 pg / mL using steps 6.1.3 or 6.1.4.

[1250] 6.2 Preparation for determination of adulterants

[1251] Prepare a high level LPL assay adulterant 250 pg / mL standard by diluting 1000 pg / mL (Standard 1 in step 6.1) 4X with sample diluent.

[1252] For example: dilute 200 μL of 1000 pg / mL (Standard 1) in 600 μL of Sample Diluent for a final concentration of 250 pg / mL.

[1253] A mid-level LPL assay adulterant of 125 pg / mL was prepared by diluting 500 pg / mL (Standard 2 in step 6.1) 4X with sample diluent.

[1254] Prepare a low level LPL assay adulterant at 31.25 pg / mL by diluting it 4X with sample diluent, 125 pg / mL (Standard 4 in step 6.1).

[1255] Spikes were loaded in triplicate into 96-well microtiter plates at 100 μL per well.

[1256] 6.3 Sample preparation

[1257] 6.3.1 Dilute the Risenqizumab process 4 BDS (150 mg / mL) to a concentration of 0.075 mg / mL for the preparation of adulterated samples and the nominal dilution of the Risenqizumab BDS samples

[1258] Table 45. Example of pre-dilution

[1259]

[1260] 6.4 Preparation of doped samples

[1261] 6.4.1 In a polypropylene microtube, mix 200 μL of Abcam sample diluent with the same volume of 200 μL of pre-diluted levitra BDS sample (step 6.3.1) as the undoped levitra BDS sample

[1262] In a polypropylene microtube, mix 200 μL (Standard 2 from step 6.1) 500 pg / mL standard solution with 200 μL pre-diluted liscencizumab BDS sample (step 6.3.1) as a high-level PLA2 doped sample.

[1263] In a polypropylene microtube, mix 200 μL (Standard 3 from step 6.1) 250 pg / mL standard solution with 200 μL pre-diluted liscencizumab BDS sample (step 6.3.1) as a medium-level PLA2-doped sample.

[1264] In a polypropylene microtube, mix 200 μL (standard 4 from step 6.1) 125 pg / mL standard solution with 200 μL pre-diluted liscencizumab BDS sample (step 6.3.1) as a medium-level PLA2-doped sample.

[1265] 7. Procedure

[1266] 7.1 Assay Procedure: The checklist can be used as a guide by checking the steps as they are completed. Reverse pipetting is always used unless otherwise specified.

[1267] 7.1.1 Place all contents of the kit at room temperature (RT) for at least 2 hours.

[1268] 7.1.2 Prepare standards, unadulterated samples, adulterated samples and controls.

[1269] 7.1.3 Using a multichannel pipette, pipette 100 μL / well of standards, non-adulterated samples, adulterated samples (if applicable), and controls into triplicate wells of the plate, and pipette 100 μL / well of assay buffer into all empty wells as blanks. Cover with sealing tape and incubate at 37°C while shaking at approximately 100 rpm for two hours.

[1270] 7.1.4 Aspirate the liquid from each well and discard into a trough. Tap the plate gently on a paper towel or kimwipe to remove liquid residue and then do not wash.

[1271] 7.1.5 Add 100 μL / well of Detection Reagent A working solution (step 5.5). Cover with sealing tape and incubate at 37°C in a room while shaking at approximately 100 rpm for one hour.

[1272] 7.1.6 Aspirate liquid from each well and discard into a trough. Gently tap the plate on a paper towel or kimwipe to remove liquid residue. Wash the plate 3 times with 350 μL / well of wash buffer in each well. For each wash, incubate the plate for 2 minutes and then aspirate the wash buffer. Blot the plate dry on a paper towel.

[1273] 7.1.7 Add 100 μL / well of Detection Reagent B working solution (step 5.6). Cover with sealing tape and incubate at 37°C while shaking at approximately 100 rpm for one hour.

[1274] 7.1.8 Aspirate liquid from each well and discard into a trough. Gently tap the plate on a paper towel or kimwipe to remove liquid residue. Wash the plate 5 times with 350 μL / well of wash buffer in each well. For each wash, incubate the plate for 2 minutes and then aspirate the wash buffer. Blot the plate dry on a paper towel.

[1275] 7.1.9 Add 90 μL / well of TMB substrate (step 5.3), cover with sealing tape, protect the plate from light and incubate at 37°C without shaking for 10 minutes (start the timer once the substrate is added to the first column).

[1276] 7.1.10 Stop the reaction (step 5.4) by adding 50 μL / well of stop solution.

[1277] 7.1.11 Read the plate at 450 nm within 2 minutes after adding the stop solution. Perform blank subtraction.

[1278] 8 Data analysis and calculation

[1279] Note: Only sample and control dilutions with OD values ​​that fall within the range of the 31.25 pg / mL standard (assay LOQ) and 1000 pg / mL (highest standard) and pass the adulterant recovery standard (if applicable) will be accepted. If a sample falls below the 31.25 pg / mL standard (<31.25 pg / mL) or the LOQ, the result should be reported as less than 31.25 pg / mL or less than LOQ. This result should then be multiplied by the dilution factor and divided by the initial sample concentration (mg / mL) to report the result in pg / mg. If the sample has a high LPL concentration that causes the sample to be above the standard curve (1000 pg / mL), repeat at a dilution high enough to be within the standard curve. If the sample does not pass adulterant recovery, repeat with more dilutions.

[1280] Table 46. Assay acceptance criteria:

[1281]

[1282]

[1283] 8.1 Standard curve

[1284] 8.1.1 The concentrations of the standards should be entered into the protocol template.

[1285] 8.1.2 Use 4-parameter curve fitting (4P) to draw a standard curve using each response value (OD).

[1286] Note: The above 4-parameter fitting equation is equivalent to:

[1287]

[1288] 8.1.3 If the reference standard does not meet the acceptance criteria, the assay must be repeated.

[1289] 8.2 Doped samples (if applicable)

[1290] 8.2.1 For doped results within the assay range (evaluate each well): Multiply the doped sample result by the dilution factor. Calculate the mean and %CV of the dilution factor-corrected doped sample results. The %CV between dilutions must be ≤ 20%. Use the following equation to calculate the % dopant recovery.

[1291] % dopant recovery = ((doped sample result (pg / mL) - undoped sample result (pg / mL)) ÷ (theoretical amount of doping (S) in pg / mL)) × 100

[1292] 8.2.2 The dopant recovery based on the mean result must be within 50% and 150%.

[1293] 8.2.3 If this criterion is not met, the sample must be repeated.

[1294] 8.3 Undoped samples

[1295] 8.3.1 For sample dilutions within the assay range (evaluate each well): Calculate the mean and %CV of the obtained dilution factor-corrected results. The %CV between dilutions must be ≤ 20%. If this criterion is not met, the sample must be repeated.

[1296] 8.3.2 Using the acceptable results, calculate the LPL concentration (ng / mg) by dividing the mean result corrected by the final dilution factor by the initial sample stock solution concentration.

[1297]

[1298] 8.3.3 Record the final result as an integer.

[1299] 8.4 Controls

[1300] 8.4.1 For control dilutions within the assay range (evaluate each well): Calculate the mean and %CV of the obtained dilution factor-corrected results. The %CV between dilutions must be ≤ 20%. If this criterion is not met, the assay must be repeated. Record the final result to 1 decimal place in ng / mL. If the control is outside the established range, the assay must be repeated.

[1301] 8.5 Measurement range: The measurement range is 31.25pg / mL to 1000pg / mL.

[1302] result

[1303] PLA2, PLBL2 and LPL levels were determined in different batches of DP1, DP2, DP3 and DP4 and are shown in Table 47 below.

[1304] Table 47 Associated protein levels

[1305] Technology PLA2(ng / mg) PLBL2 (ng / mg) LPL (ng / mg) DP1 0.29-1.09 5.7-16.43 0.004-0.019 DP2 0.25-1.03 0.51-1.34 0.019-0.291 DP3 0.07-0.27 0.003-0.020 0.008-0.658 DP4 <0.009 <0.0006 0.015-0.098

[1306] As shown in Table 47, the level of PLBL2 in DP3 and DP4 was greatly reduced compared to DP1 and DP2. For example, the level of PLBL2 in DP4 was reduced by almost 1000-fold compared to DP1 and DP2.

[1307] Similarly, the levels of PLA2 in DP3 and DP4 were greatly reduced compared to DP1 and DP2. DP1 and DP2 had at least 0.25 ng / mg (250 pg / mg) of PLA2.

[1308] Example 10: Increased polysorbate stability in Risenqizumab drug products DP3 and DP4

[1309] The stability of polysorbate 20 (PS20) and polysorbate 80 (PS80) was determined by examining the levels of PS20 or PS80 and free fatty acids (FFA) in samples stored at different temperatures (ie, 5°C, 25°C, and 40°C) for 6 months.

[1310] Determination of PS20 / PS80 using Charged Aerosol Detector (CAD)

[1311] The following describes a method for measuring the levels of PS20 in various drug products. The same method was used to determine the levels of PS80 in various drug products.

[1312] 1. Principle

[1313] The polysorbate 20 content in the 150 mg / mL formulation test sample was determined using a charged aerosol detector (CAD). Detection was based on the atomization of the analyte in a continuous nitrogen stream. After the mobile phase was removed, a second positively charged nitrogen stream caused the formation of charged particles. The charge released was proportional to the amount of polysorbate 20.

[1314] 2 Equipment

[1315] HPLC system with gradient elution, temperature controlled autosampler, column oven and degasser. Also, stainless steel capillaries (to connect the HPLC column and to connect the column to the detector).

[1316] Corona Veo RS with Charged Aerosol Detector (CAD), Thermo Fisher Scientific part number 5081.0020

[1317] Chromatography data system (e.g., Empower)

[1318] Column: Waters Oasis MAX column (2.1×20 mm, 30 μm), part number 186002052

[1319] Amber HPLC vials, screw cap, Agilent, catalog number 5182-0716 or equivalent Blue screw cap, Agilent, catalog number 5182-0717 or equivalent

[1320] Analytical Balance

[1321] pH Meter

[1322] Stir Plates, Stir Bars, and Vortex Mixers

[1323] Volumetric flask

[1324] 3 Materials

[1325] Ammonium formate, Fisher, LC-MS grade, Catalog No. A115-50 or equivalent Methanol, EMD, LC-MS grade, Catalog No. MX0486-6 or equivalent Isopropanol, Fisher, LC-MS grade, Catalog No. A461-212 or equivalent Acetonitrile, Fisher, Optima LC-MS grade, Catalog No. A955-212 or equivalent Formic acid, ThermoScientific, Catalog No. 28905 or equivalent Polysorbate 20 (PS20), JT Baker, Catalog No. 4116-02 or equivalent Project GB Reference Standards

[1326] Purified water (Type 1 rating, e.g., Milli-Q, WFI, or equivalent)

[1327] 4. Solution Preparation

[1328] 4.1 Dilute formic acid solution (1:1) for pH adjustment of mobile phase A

[1329] 4.1.1 In a glass beaker, add 7.5 mL of formic acid and 7.5 mL of purified water.

[1330] 4.1.2 Mix the solution until uniform.

[1331] 4.2 Mobile phase A (10 mM ammonium formate pH 3.0 / 20% isopropanol)

[1332] 4.2.1 Add 1400 mL of purified water to a 2 L beaker.

[1333] 4.2.2 Weigh 1.26 g ± 0.01 g of ammonium formate and add it to the beaker.

[1334] 4.2.3 Using the dilute formic acid solution from step 4.1, adjust the pH of the solution to 3.0 ± 0.1.

[1335] 4.2.4 Add 400 mL of isopropanol.

[1336] 4.2.5 Transfer the solution into a 2L volumetric flask.

[1337] 4.2.6 Adjust the volume with purified water.

[1338] 4.2.7 Transfer the solution into a suitable container.

[1339] 4.2.8 Mix the solution for approximately 15 minutes.

[1340] 4.2.9 Check the homogeneity of the solution by visual inspection.

[1341] 4.2.10 Store at room temperature for up to 1 week.

[1342] 4.3 Mobile phase B (50% isopropanol / 50% acetonitrile)

[1343] 4.3.1 Add 500 mL of isopropanol and 500 mL of acetonitrile to a 1 L glass bottle.

[1344] 4.3.2 Mix the solution for approximately 15 minutes.

[1345] 4.3.3 Check the homogeneity of the solution by visual inspection.

[1346] 4.3.4 Store at room temperature for up to 1 month.

[1347] 4.4 Autosampler rinsing solution (20% methanol)

[1348] 4.4.1 Add 200 mL of methanol and 800 mL of purified water to a 1 L glass bottle.

[1349] 4.4.2 Mix the solution until uniform.

[1350] 4.4.3 Store at room temperature for up to 1 week.

[1351] 5. Preparation of standards, samples and blanks

[1352] 5.1 Recommendations for preparation of standards and samples

[1353] 5.1.1 Accurate pipetting of polysorbate 20

[1354] Care should be taken when pipetting samples, standards and dilutions containing PS20. It is recommended to balance the pipette tip by pipetting up and down 5 times. Pipetting should be done slowly. It is important to wait for highly viscous solutions to be completely aspirated and for the thin film of solution to completely leave the pipette tip after pipetting. For downward pipetting, it is equally important to wait for the solution to completely clear from the pipette tip. It may be necessary to wait for approximately 15 seconds in each direction (up / down).

[1355] 5.1.2 Volumes of standard and sample solutions

[1356] The volumes of the calibration standards and diluted sample solutions may deviate from 500 μL. However, the volumes of the standards and diluted sample solutions must be the same.

[1357] 5.2 10 mg / mL Polysorbate 20 Stock Solution I

[1358] 5.2.1 Weigh 1.00 g ± 0.01 g of polysorbate 20 into a 100 mL tared volumetric flask.

[1359] 5.2.2 Adjust the volume with purified water.

[1360] 5.2.3 Carefully add a...

Claims

1. A liquid composition comprising: (1) lisenqizumab; and (2) phospholipase A2 (PLA2) in an amount less than about 250 pg / mg lisenqizumab.

2. The liquid composition of claim 1, comprising about 60 mg / ml to about 150 mg / ml of Risenqizumab.

3. The liquid composition of claim 1 or 2, wherein the PLA2 is PLA2G15.

4. The liquid composition of any one of claims 1-3, wherein the level of PLA2 is less than about 240 pg, less than about 220 pg, less than about 200 pg, less than about 180 pg, less than about 160 pg, less than about 140 pg, less than about 120 pg, less than about 100 pg, less than about 90 pg, less than about 80 pg, less than about 70 pg, less than about 60 pg, less than about 50 pg, less than about 40 pg , less than about 30pg, less than about 25pg, less than about 20pg, less than about 15pg, less than about 10pg, less than about 9pg, less than about 8pg, less than about 7pg, less than about 6pg, less than about 5pg, less than about 4.4pg, less than about 3pg, less than about 2pg, less than about 1pg, less than about 0.5pg, less than about 0.1pg, less than about 0.05pg or less than about 0.01pg / mg of lisenqizumab.

5. The liquid composition of any one of claims 1-3, wherein the level of PLA2 is greater than about 240 pg, greater than about 220 pg, greater than about 200 pg, greater than about 180 pg, greater than about 160 pg, greater than about 140 pg, greater than about 120 pg, greater than about 100 pg, greater than about 90 pg, greater than about 80 pg, greater than about 70 pg, greater than about 60 pg, greater than about 50 pg, greater than about 40 pg. g, greater than about 30pg, greater than about 25pg, greater than about 20pg, greater than about 15pg, greater than about 10pg, greater than about 9pg, greater than about 8pg, greater than about 7pg, greater than about 6pg, greater than about 5pg, greater than about 4pg, greater than about 3pg, greater than about 2pg, greater than about 1pg, greater than about 0.5pg, greater than about 0.1pg, greater than about 0.05pg or greater than about 0.01pg / mg of lisenqizumab.

6. The liquid composition of any one of claims 1 to 3, wherein the level of PLA2 is from about 200 pg to about 249 pg, from about 160 pg to about 200 pg, from about 120 pg to about 160 pg, from about 100 pg to about 120 pg, from about 80 pg to about 100 pg, from about 60 pg to about 80 pg, from about 40 to about 60 pg, from about 25 pg to about 40 pg, from about 10 pg to about 20 pg. 5pg, about 5pg to about 10pg, about 4pg to about 10pg, about 1pg to about 5pg, about 1pg to about 4pg, about 1pg to about 3pg, about 1pg to about 2pg, about 0.5pg to about 1pg, about 0.1pg to about 0.5pg, about 0.05pg to about 0.1pg, about 0.01pg to about 0.5pg or about 70pg to about 240pg / mg of lisenqizumab.

7. The liquid composition of any one of claims 1-3, wherein the level of PLA2 is about 240, about 220, about 200, about 180, about 160, about 140, about 120, about 100, about 90, about 80, about 70, about 60, about 50, about 40, about 30, about 25, about 20, about 15, about 10, about 9, about 8, about 7, about 6, about 5, about 4.4, about 3, about 2, about 1, about 0.5, about 0.1, about 0.05 or about 0.01 pg / mg of lisenqizumab.

8. The liquid composition according to any one of claims 1 to 7, wherein the level of PLA2 is determined by ELISA.

9. The liquid composition of any one of claims 1-8, wherein the risankizumab has been produced in a CHO cell line.

10. The liquid composition according to any one of claims 1 to 9, further comprising one or more of a surfactant, a polyol and a buffer.

11. The liquid composition of claim 10, wherein the polyol is selected from the group consisting of trehalose, mannitol, sucrose and sorbitol.

12. The liquid composition of claim 11, wherein the polyol is trehalose.

13. The liquid composition of claim 12, wherein the amount of trehalose is about 150 to about 220 mM.

14. The liquid composition of claim 13, wherein the amount of trehalose is about 185 mM.

15. The liquid composition of any one of claims 10-14, wherein the buffer is selected from the group consisting of acetate buffer, histidine buffer, citrate buffer, phosphate buffer, glycine buffer and arginine buffer.

16. The liquid composition of claim 15, wherein the buffer is acetate buffer.

17. The liquid composition of claim 16, wherein the amount of acetate buffer is about 5 to about 50 mM.

18. The liquid composition of claim 17, wherein the amount of acetate buffer is about 10 mM.

19. The liquid composition of any one of claims 10-18, wherein the surfactant is selected from the group consisting of polysorbate 20 (PS20), polysorbate 80 (PS80), polysorbate 40 (PS40), polysorbate 60 (PS60), polysorbate 65 (PS65) and poloxamer 188.

20. The liquid composition of claim 19, wherein the surfactant is PS20.

21. The liquid composition of claim 20, wherein the amount of PS20 is about 0.2 mg / mL.

22. The liquid composition of claim 21, comprising: 150mg / ml lisenqizumab; 185 mM trehalose; 10 mM acetate; and 0.20mg / mL polysorbate 20, The liquid composition has a pH of about 5.

7.

23. The liquid composition of claim 21, comprising: 150mg / ml lisenqizumab; 0.054 mg / mL acetic acid; 1.24 mg / mL sodium acetate trihydrate; 70 mg / mL trehalose dihydrate; 0.20 mg / mL polysorbate 20; and Water for injection, USP; wherein the liquid composition has a pH of about 5.

7.

24. The liquid composition of any one of claims 20-23, wherein after storage at 5°C for 6 months, at least 80% of the initial concentration of PS20 is present in the composition.

25. The liquid composition of any one of claims 20-23, wherein after storage at 5°C for 24 months, at least 70% of the initial concentration of PS20 is present in the composition.

26. The liquid composition of any one of claims 20-23, wherein after storage at 25°C for 6 months, at least 60% of the initial concentration of PS20 is present in the composition.

27. The liquid composition of any one of claims 20-23, wherein after storage at 40°C for 6 months, at least 40% of the initial concentration of PS20 is present in the composition.

28. The liquid composition of any one of claims 20-23, wherein the total concentration of free fatty acids (FFA) present in the composition does not increase by more than 1.5 times after storage at 5°C for 6 months.

29. The liquid composition of any one of claims 20-23, wherein the total concentration of FFA present in the composition does not exceed 20 nmol / ml after storage at 5°C for 6 months.

30. The liquid composition of any one of claims 20-23, wherein the total concentration of FFAs present in the composition does not exceed 3.2 times after storage at 25°C for 6 months.

31. The liquid composition of any one of claims 20-23, wherein the total concentration of FFA present in the composition does not exceed 25 nmol / ml after storage at 25°C for 6 months.

32. The liquid composition of any one of claims 20-23, wherein the total concentration of FFAs present in the composition does not exceed 3-fold after storage at 40°C for 6 months.

33. The liquid composition of any one of claims 20-23, wherein the total concentration of FFA present in the composition does not exceed 35 nmol / ml after storage at 40°C for 6 months.

34. The liquid composition of claim 19, wherein the surfactant is PS80.

35. The liquid composition of claim 34, wherein after storage at 5°C for 6 months, at least 80% of the initial concentration of PS80 is present in the composition.

36. The liquid composition of claim 34, wherein after storage at 25°C for 6 months, at least 60% of the initial concentration of PS80 is present in the composition.

37. The liquid composition of claim 34, wherein after storage at 40°C for 6 months, at least 60% of the initial concentration of PS80 is present in the composition.

38. The liquid composition of claim 34, wherein the total concentration of FFAs present in the composition does not increase more than 8-fold after storage at 5°C for 6 months.

39. The liquid composition of claim 34, wherein the total concentration of FFA present in the composition does not exceed 40 nmol / ml after storage at 5°C for 6 months.

40. The liquid composition of claim 34, wherein the total concentration of FFAs present in the composition does not increase by more than 12-fold after storage at 25°C for 6 months.

41. The liquid composition of claim 34, wherein the total concentration of FFA present in the composition does not exceed 60 nmol / ml after storage at 25°C for 6 months.

42. The liquid composition of claim 34, wherein the total concentration of FFAs present in the composition does not increase by more than 2.5-fold after storage at 40°C for 6 months.

43. The liquid composition of claim 34, wherein the total concentration of FFA present in the composition does not exceed 15 nmol / ml after storage at 40°C for 6 months.

44. The liquid composition of any one of claims 24-43, wherein the PS20 or PS80 is measured using HPLC-CAD.

45. The liquid composition of any one of claims 24-43, wherein the FFA is measured using a LC-FFA assay.

46. ​​The liquid composition of any one of claims 1-45, wherein no visible or shiny particles are observed within 24 months at 4°C.

47. The liquid composition of any one of claims 1-46, wherein the liquid composition is packaged in a vial, a pre-filled syringe, or an on-body device.

48. The liquid composition of any one of claims 1-47, wherein the liquid composition is a pharmaceutical composition and is suitable for subcutaneous injection.

49. The liquid composition of any one of claims 1-47, wherein the liquid composition is a pharmaceutical composition and is suitable for intravenous injection.

50. A method of treating an immune disease using the composition of any one of claims 1-49.

51. A composition comprising risankizumab, wherein the composition has one or more of the following characteristics: (a) less than about 5.4% of the total levitra species having N-glycosylation have high mannose N-glycans; and / or (b) After a single subcutaneous 150 mg dose of the composition is administered to humans, the incidence of anti-drug antibodies (ADA) during treatment is less than about 4.7%.

52. The composition of claim 51, wherein the composition is a pharmaceutical composition.

53. A composition as described in claim 51 or 52, wherein the composition is a liquid composition.

54. The composition of any one of claims 51-53, wherein the composition comprises about 60 mg / ml to about 150 mg / ml risankizumab.

55. The composition of any one of claims 51-54, wherein the composition has at least the following characteristic (a): less than about 5.4% of the total risankizumab species having N-glycosylation have high mannose N-glycans.

56. The composition of claim 55, wherein the high mannose N-glycans comprise one or more high mannose N-glycans selected from mannose 5 N-glycans (M5), mannose 6 N-glycans (M6), and mannose 7 N-glycans (M7).

57. The composition of claim 56, wherein the high mannose N-glycans are M5, M6 and M7.

58. The composition of any of claims 51-57, wherein the level of lisenqizumab having the high mannose N-glycans is less than 5.3%, less than about 5.2%, less than about 5.1%, less than about 5.0%, less than about 4.9%, less than about 4.8%, less than about 4.7%, less than about 4.6%, less than about 4.5%, less than about 4.4%, less than about 4.3%, less than about 4.2%, less than about 4.1%, less than about 4.0%, less than about 3.9%, less than about 3.8% or less than about 3.7% of the total lisenqizumab material having N-glycosylation.

59. The composition of any of claims 51-58, wherein the level of lisenqizumab having said high mannose N-glycans is greater than about 5.3%, greater than about 5.2%, greater than about 5.1%, greater than about 5.0%, greater than about 4.9%, greater than about 4.8%, greater than about 4.7%, greater than about 4.6%, greater than about 4.5%, greater than about 4.4%, greater than about 4.3%, greater than about 4.2%, greater than about 4.1%, greater than about 4.0%, greater than about 3.9%, greater than about 3.8%, greater than about 3.7% or greater than about 3.6% of the total lisenqizumab material having N-glycosylation.

60. The composition of any of claims 51-59, wherein the level of lisenqizumab having the high mannose N-glycans is about 3.6% to about 5.3%, about 3.6% to about 5.0%, about 3.6% to about 4.8%, about 3.6% to about 4.5%, about 3.6% to about 4.1%, about 3.6% to about 3.8%, about 3.8% to about 5.3%, about 4.1% to about 5.3%, about 4.5% to about 5.3%, about 4.8% to about 5.3%, about 5.0% to about 5.3%, about 4.3% to about 4.9%, or about 3.6% to about 4.9% of the total lisenqizumab material having N-glycosylation.

61. The composition of any of claims 51-60, wherein the level of lisenqizumab having the high mannose N-glycans is about 5.3%, about 5.2%, about 5.1%, about 5.0%, about 4.9%, about 4.8%, about 4.7%, about 4.6%, about 4.5%, about 4.4%, about 4.3%, about 4.2%, about 4.1%, about 4.0%, about 3.9%, about 3.8%, about 3.7% or about 3.6% of the total lisenqizumab material having N-glycosylation.

62. The composition of claim 56, wherein the high mannose N-glycan is M5.

63. The composition of claim 62, wherein the level of lisenqizumab with M5 is less than 5.3%, less than about 5.2%, less than about 5.1%, less than about 5.0%, less than about 4.9%, less than about 4.8%, less than about 4.7%, less than about 4.6%, less than about 4.5%, less than about 4.4%, less than about 4.3%, less than about 4.2%, less than about 4.1%, less than about 4.0%, less than about 3.9%, less than about 3.8%, less than about 3.7%, less than about 3.6%, less than about 3.5%, less than about 3.4%, less than about 3.3%, less than about 3.2%, less than about 3.1%, less than about 3.0%, less than about 2.9% or less than about 2.8% of the total lisenqizumab material with N-glycosylation.

64. The pharmaceutical composition of claim 62 or 63, wherein the level of lisenqizumab with M5 is greater than about 5.2%, greater than about 5.1%, greater than about 5.0%, greater than about 4.9%, greater than about 4.8%, greater than about 4.7%, greater than about 4.6%, greater than about 4.5%, greater than about 4.4%, greater than about 4.3%, greater than about 4.2%, greater than about 4.1%, greater than about 4.0%, greater than about 3.9%, greater than about 3.8%, greater than about 3.7%, greater than about 3.6%, greater than about 3.5%, greater than about 3.4%, greater than about 3.3%, greater than about 3.2%, greater than about 3.1%, greater than about 3.0%, greater than about 2.9%, greater than about 2.8%, or greater than about 2.7% of the total lisenqizumab material with N-glycosylation.

65. The composition of any of claims 62-64, wherein the level of lisenqizumab with M5 is about 2.7% to about 5.2%, about 3.1% to about 5.2%, about 3.5% to about 5.2%, about 4.0% to about 5.2%, about 4.5% to about 5.2%, about 5% to about 5.2%, about 2.7% to about 5.0%, about 2.7% to about 4.5%, about 2.7% to about 4.0%, about 2.7% to about 3.5%, about 2.7% to about 3.1%, about 3.2% to about 3.7%, or about 2.7% to about 3.7% of the total lisenqizumab material with N-glycosylation.

66. The composition of any of claims 62-65, wherein the level of lisenqizumab with M5 is about 2.7%, about 2.8%, about 2.9%, about 3.0%, about 3.1%, about 3.2%, about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%, about 3.9%, about 4.0%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, about 5.0%, about 5.1% or about 5.2% of the total lisenqizumab material with N-glycosylation.

67. The composition of claim 56, wherein the high mannose polysaccharide is M6.

68. The composition of claim 67, wherein the level of lisenqizumab with M6 is less than about 2.6%, less than about 2.5%, less than about 2.4%, less than about 2.3%, less than about 2.2%, less than about 2.1%, less than about 2.0%, less than about 1.9%, less than about 1.8%, less than about 1.7%, less than about 1.6%, less than about 1.5%, less than about 1.4%, less than about 1.3%, less than about 1.2%, less than about 1.1%, less than about 1.0%, less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6% or less than about 0.5% of the total lisenqizumab material with N-glycosylation.

69. The composition of claim 67 or 68, wherein the level of lisenqizumab with M6 is greater than about 2.5%, greater than about 2.4%, greater than about 2.3%, greater than about 2.2%, greater than about 2.1%, greater than about 2.0%, greater than about 1.9%, greater than about 1.8%, greater than about 1.7%, greater than about 1.6%, greater than about 1.5%, greater than about 1.4%, greater than about 1.3%, greater than about 1.2%, greater than about 1.1%, greater than about 1.0%, greater than about 0.9%, greater than about 0.8%, greater than about 0.7%, greater than about 0.6%, greater than about 0.5% or greater than about 0.4% of the total lisenqizumab material with N-glycosylation.

70. The composition of any of claims 67-69, wherein the level of lisenqizumab with M6 is about 0.4% to about 2.5%, about 0.4% to about 2.4%, about 0.4% to about 2.2%, about 0.4% to about 2.0%, about 0.4% to about 1.8%, about 0.4% to about 1.6%, about 0.4% to about 1.4%, about 0.4% to about 1.2%, about 0.4% to about 1.0%, about 0.4% to about 0.9%, about 0.4% to about 0.8%, about 0.4% to about 0.7%, about 0.4% to about 0.6%, about 0.4% to about 0.5%, or about 0.6% to about 0.7% of the total lisenqizumab material with N-glycosylation.

71. The composition of any of claims 67-70, wherein the level of lisenqizumab with M6 is about 2.5%, about 2.4%, about 2.3%, about 2.2%, about 2.1%, about 2.0%, about 1.9%, about 1.8%, about 1.7%, about 1.6%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1.0%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5% or about 0.4% of the total lisenqizumab material with N-glycosylation.

72. The composition of claim 56, wherein the high mannose polysaccharide is M7.

73. The composition of claim 72, wherein the level of lisenqizumab with M7 is less than 2.0%, less than about 1.9%, less than about 1.8%, less than about 1.7%, less than about 1.6%, less than about 1.5%, less than about 1.4%, less than about 1.3%, less than about 1.2%, less than about 1.1%, less than about 1.0%, less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, less than about 0.5% or greater than about 0.4% of the total lisenqizumab material with N-glycosylation.

74. The composition of claim 72 or 73, wherein the level of lisenqizumab with M7 is greater than about 1.9%, greater than about 1.8%, greater than about 1.7%, greater than about 1.6%, greater than about 1.5%, greater than about 1.4%, greater than about 1.3%, greater than about 1.2%, greater than about 1.1%, greater than about 1.0%, greater than about 0.9%, greater than about 0.8%, greater than about 0.7%, greater than about 0.6%, greater than about 0.5% or greater than about 0.4% of the total lisenqizumab material with N-glycosylation.

75. The composition of any of claims 72-74, wherein the level of lisenqizumab with M7 is about 0.4% to about 1.9%, about 0.4% to about 1.8%, about 0.4% to about 1.6%, about 0.4% to about 1.4%, about 0.4% to about 1.2%, about 0.4% to about 1.0%, about 0.4% to about 0.9%, about 0.4% to about 0.8%, about 0.4% to about 0.7%, about 0.4% to about 0.6%, about 0.4% to about 0.5%, or about 0.5% to about 0.6% of the total lisenqizumab material with N-glycosylation.

76. The composition of any of claims 72-75, wherein the level of lisenqizumab with M7 is about 1.9%, about 1.8%, about 1.7%, about 1.6%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1.0%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5% or about 0.4% of the total lisenqizumab material with N-glycosylation.

77. The composition of any one of claims 51-76, wherein the level of risankizumab with the high mannose N-glycans is determined by 2-AB and HILIC-FL chromatography.

78. The composition of any one of claims 51-76, wherein the level of risankizumab with the high mannose N-glycans is determined by RapiFluor HILIC-FL chromatography.

79. The composition of any one of claims 51-78, wherein greater than about 84.4% of the total risankizumab species having N-glycosylation have fucosylated complex oligosaccharides.

80. The composition of claim 79, wherein about 88.0% to about 90.9% of the total risankizumab species having N-glycosylation have fucosylated complex oligosaccharides.

81. The composition of claim 79 or 80, wherein the level of risankizumab with fucosylated complex oligosaccharides is determined by 2-AB and HILIC-FL chromatography.

82. The composition of claim 79 or 80, wherein the level of risankizumab with said high mannose N-glycans is determined by RapiFluor HILIC-FL chromatography.

83. The composition of any one of claims 51-82, wherein the pharmaceutical composition comprises about 0.8% to about 1.4% aglycosylated risankizumab.

84. The composition of claim 83, wherein the aglycosylated risankizumab is determined by tryptic peptide mapping.

85. The composition of any one of claims 51-53, wherein the composition has at least the following characteristic (d): following administration of a single subcutaneous dose of the composition to a human, the incidence of anti-drug antibodies (ADA) that emerge during treatment in the human is less than about 4.7%.

86. The composition of claim 85, wherein the incidence of ADA occurring during treatment is less than about 4.5%, less than about 4.0%, less than about 3.5%, less than about 3.0%, less than about 2.5%, less than about 2.0%, less than about 1.5%, less than about 1.0%, less than about 0.5%, less than about 0.4%, less than about 0.3%, less than about 0.2%, less than about 0.1%, less than about 0.01%, less than about 0.001% or less than about 0.0001%.

87. The composition of claim 85 or 86, wherein the incidence of ADA occurring during treatment is less than about 4.7%, less than about 4.5%, less than about 4.0%, less than about 3.5%, less than about 3.0%, less than about 2.5%, less than about 2.0%, less than about 1.5%, less than about 1.0%, less than about 0.5%, less than about 0.4%, less than about 0.3%, less than about 0.2%, less than about 0.1%, less than about 0.01%, less than about 0.001%, less than about 0.0001%, or about 0.0%.

88. A composition as described in any one of claims 85 to 87, wherein the incidence of ADA occurring during treatment is measured after a single subcutaneous injection of a 150 mg dose of the composition is administered to the human being.

89. The composition of any one of claims 85-88, wherein the presence of ADA is determined using a validated titer-based bridging electrochemiluminescent immunoassay.

90. The composition of any one of claims 51-89, wherein the risenizumab is produced in a CHO cell line.

91. The composition of any one of claims 52-90, further comprising a pharmaceutically acceptable excipient.

92. A composition comprising: (1) risankizumab; and (2) poloxamer 188 (P188).

93. The composition of claim 92, comprising about 60 mg / ml to about 150 mg / ml of lisenqizumab.

94. The composition of claim 92 or 93, further comprising phospholipase A2 (PLA2).

95. The composition of claim 94, wherein the PLA2 is PLA2G15.

96. The composition of claim 94 or 95, wherein the level of PLA2 is greater than about 250 pg, greater than about 260 pg, greater than about 270 pg, greater than about 280 pg, greater than about 290 pg, greater than about 300 pg, greater than about 310 pg, greater than about 320 pg, greater than about 330 pg, greater than about 340 pg, greater than about 350 pg, greater than about 360 pg, greater than about 380 pg, greater than about 400 pg, greater than about 450 pg, greater than about 500 pg, greater than about 550 pg, greater than about 600 pg, greater than about 650 pg, greater than about 700 pg, greater than about 750 pg, greater than about 800 pg, greater than about 900 pg, or greater than about 1000 pg / mg of lisenqizumab.

97. The composition of any of claims 92-95, wherein the level of PLA2 is about 250 pg to about 1100 pg, about 260 pg to about 1100 pg, about 270 pg to about 1100 pg, about 280 pg to about 1100 pg, about 290 pg to about 1100 pg, about 300 pg to about 1100 pg, about 310 pg to about 1100 pg, about 320 pg to about 1100 pg, about 340 pg to about 1100 pg, about 360 pg to about 1100 pg. To about 1100pg, about 250pg to about 1000pg, about 250pg to about 900pg, about 250pg to about 800pg, about 250pg to about 700pg, about 250pg to about 600pg, about 250pg to about 500pg, about 250pg to about 400pg, about 250pg to about 1030pg, about 290pg to about 1090pg, about 360pg to about 450pg or about 310pg to about 920pg / mg of lisenqizumab.

98. The composition of any one of claims 92-95, wherein the level of PLA2 is about 260 pg, about 270 pg, about 280 pg, about 290 pg, about 300 pg, about 310 pg, about 320 pg, about 330 pg, about 340 pg, about 350 pg, about 360 pg, about 380 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about 750 pg, about 800 pg, about 900 pg, about 1000 pg or about 1100 pg / mg of lisenqizumab.

99. A composition as described in any one of claims 94-98, wherein the level of PLA2 is determined by ELISA.

100. The composition of any one of claims 92-99, wherein after storage at 5°C for 6 months, at least 85% of the initial amount of P188 is retained.

101. The composition of any one of claims 92-99, wherein after storage at 5°C for 6 months, at least 80% of the initial amount of P188 is retained.

102. The composition of any one of claims 92-99, wherein after storage for 3 months at 25°C, at least 65% of the initial amount of P188 is retained.

103. The composition of any one of claims 92-99, wherein after storage for 6 months at 25°C, at least 60% of the initial amount of P188 is retained.

104. The composition of any one of claims 92-99, wherein after storage at 40°C for 3 months, at least 60% of the initial amount of P188 is retained.

105. The composition of any one of claims 92-99, wherein after storage at 40°C for 6 months, at least 60% of the initial amount of P188 is retained.

106. The composition of any one of claims 100-105, wherein the P188 is measured using the Pluronic F-68 colorimetric assay.

Images