High-concentration risankizumab formulation

Abstract

The present disclosure relates to a novel antibody formulation containing a high concentration of risankizumab. The present disclosure further relates to a method of using such formulation to treat diseases (e.g., psoriasis and Crohn's disease).

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 612,905, filed December 20, 2023 and U.S. Provisional Application No. 63 / 612,928, filed December 20, 2023, the contents of each of which are incorporated herein by reference in their entirety.

[0003] Sequence list

[0004] The present application includes a list of electronic sequences submitted in XML file format with the present application, the entire contents of which are incorporated herein by reference. The XML file of the sequence list submitted with the present application is titled '13371-301-228_SEQLISTING.xml', was created on December 19, 2024, and is 17,798 bytes in size.

[0005] 1. Field

[0006] The present disclosure relates to a novel antibody formulation containing a high concentration of risankizumab. The present disclosure further relates to a method of using such formulation to treat diseases (e.g., psoriasis and Crohn's disease). Background Technology

[0007] 2. Background Technology

[0008] Risankizumab is a humanized immunoglobulin G1 (IgG1) monoclonal antibody that inhibits the activation of the IL-23 signaling cascade by binding with high affinity to the p19 subunit of IL-23 (IL-23p19). Risankizumab is approved to treat inflammatory diseases such as psoriasis and Crohn's disease.

[0009] Most approved antibody drugs are administered intravenously. However, for drugs intended to treat chronic diseases where outpatient or home administration is preferable, subcutaneous injection has been preferred. Compared to intravenous infusion, subcutaneous injection beneficially enables drug administration outside the hospital setting, reduces drug delivery-related healthcare costs, saves time, and alleviates the discomfort associated with intravenous infusion.

[0010] Subcutaneous injections are limited to small injection volumes because large volumes are often associated with injection pain, side effects at the injection site, and leakage. As such, high-concentration antibody formulations are desirable and advantageous for the subcutaneous delivery of large volumes of antibody therapeutics.

[0011] A key challenge in developing high-concentration antibody formulations is the increase in formulation viscosity associated with higher antibody concentrations. High viscosity of antibody formulations can affect their injectability, potentially rendering subcutaneous delivery routes completely impossible. High viscosity increases the force and time required for subcutaneous injection, leading to increased pain during administration. Furthermore, high viscosity may render manufacturing processes, such as pumping or tangential flow filtration for antibody concentration, impossible. Additionally, high-concentration antibody formulations can increase the likelihood of antibody aggregation and particle formation, which reduces the efficacy and increases immunogenicity of the formulation and correlates with filter clogging that occurs during the manufacturing process.

[0012] Another challenge in developing antibody formulations is the presence of hitchhiker proteins within the formulation. Even trace amounts of hitchhiker protein contaminants can induce polysorbate 20 hydrolysis, leading to particle formation (therapeutic protein and / or free fatty acid aggregates) and consequently shortening shelf life (Reference [Khan et al. (2015) European Journal of Pharmaceutics and Biopharmaceutics[97:60-67]). Various commercial risankizumab 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. These commercial formulations contain the surfactant polysorbate 20 (PS20). One significant factor contributing to the presence of hitchhiker protein impurities in recombinant therapeutic monoclonal antibody formulations is the association of the product monoclonal antibody [mAb] with these impurities (Nogal et al. (2012) Biotechnol. Prog. It has been reported that mAbs can preferentially bind to hitchhiker proteins (HPs) for selection, and that the degree of interaction and / or the type of associated HP may vary depending on the mAb (Nogal et al. (2012) Biotechnol. Prog. [28:454-458]). In addition, the Hitchhiker protein content of mAbs in protein A [protein A, PrA] eluent is specific to specific antibodies (reference [Zhang et al. (2016) Biotechnol. Prog. [32:708-717]), the primary sequence of the mAb was shown to be responsible for the binding of specific hitchhiker proteins and the subsequent co-purification (Reference [Bee et al. (2016) Biotechnol. Prog. 00: 1-6]). Another factor is the similar physicochemical properties of a specific hitchhiker protein to a specific mAb to be purified, which leads to co-purification with the mAb. Because different hitchhiker proteins are co-purified with different recombinant antibodies, it is impossible to predict before the experiment whether a hitchhiker problem will occur during the production of the novel antibody, as well as which hitchhiker protein will be problematic.

[0013] Furthermore, a major problem with protein-based therapeutics is their immunogenicity, that is, their tendency to trigger an unwanted immune response against themselves, generating so-called 'anti-drug antibodies' or 'ADAs'.

[0014] Therefore, there remains a need for high-concentration risankizumab formulations that exhibit viscosity and stability suitable for subcutaneous injection and antibody manufacturing processes. Additionally, there remains a need for the development of risankizumab formulations with high levels of purity (e.g., having a higher percentage of antibody monomers), low levels of Hitchhiker protein impurities, and low immunogenicity. means of solving the problem

[0015] 3. Summary

[0016] The present disclosure provides a novel formulation comprising risankizumab at a high concentration of about 180 mg / ml. The formulation disclosed herein advantageously provides a high concentration of risankizumab having suitable viscosity (e.g., less than about 20.0 mPa·s) and stability, which makes subcutaneous injection of the formulation feasible.

[0017] The viscosity profile of each antibody is unpredictable, even for very similar monoclonal antibodies. For example, the literature [Liu et al , J. Pharm. Sci. [94 (9) (2005) 1928-1940] showed that monoclonal antibodies that share essentially the same constant region and differ only in the complementarity determining region exhibit very different viscosity profiles in nearly identical formulations. Liu observed that while all antibodies generally showed an increase in viscosity as protein concentration increased, significant differences were observed in the absolute viscosity values ​​and specific viscosity-antibody concentration relationships of the different antibodies.

[0018] Determining the relationship between antibody concentration and viscosity in formulations can be challenging. Several factors contribute to this difficulty. For example, the relationship between antibody concentration and viscosity is often nonlinear. Small changes in concentration can lead to disproportionate changes in viscosity, and the nature of this relationship can be influenced by various factors, including protein-protein interactions and structural changes. Antibodies are complex proteins capable of interacting with one another within a formulation. At higher concentrations, the likelihood of protein-protein interactions increases, leading to the formation of aggregates or complexes. The impact of these interactions on viscosity can be unpredictable and difficult to model. Similarly, changes in antibody structure, particularly at higher concentrations, can affect how antibodies interact with each other and with the solvent. These structural changes can have complex effects on viscosity, and accurately predicting these changes is difficult.

[0019] Solutions with a viscosity of up to 20 m / Pas have been reported to exhibit good tolerability for subcutaneous injection (e.g., literature [Berteau et al , Med Devices [Refer to 8(2015) 473-484]). For any specific antibody, due to the unpredictability of the relationship between antibody concentration and viscosity, empirical studies and careful formulation development are required to identify a high antibody concentration or maximum antibody concentration cut-off that allows the formulation to maintain its subcutaneous injectability and other desired properties.

[0020] The present disclosure is based in part on the surprising discovery regarding the unique viscosity profile of risankizumab. In determining the maximum acceptable antibody concentration of a subcutaneous formulation of risankizumab, it was discovered that a surprising cut-off effect exists at a concentration of about 180 mg / ml. In particular, the viscosity of the risankizumab formulation increased with protein concentration; it was found to be consistently less than about 20.0 mPa·s at concentrations of about 180 mg / ml or less, but increased exponentially at concentrations of about 180 mg / ml or more. Formulations containing about 180 mg / ml of risankizumab exhibited clinically acceptable glide force and ejection time when tested with a subcutaneous syringe. Furthermore, the present disclosure found that formulations containing about 180 mg / ml of risankizumab are stable under freeze / thaw and mechanical stress.

[0021] Furthermore, it has been well recognized in the technical field that identifying optimized parameters within the manufacturing process to reduce SVP in high-concentration antibody formulations is complex due to the intricate interactions between process variables, individual antibody characteristics, and stability requirements. Since antibodies share structural similarities but can exhibit unique behaviors due to differences in specific amino acid sequences, structures, and properties, the need arises for different manufacturing parameters for each antibody.

[0022] The present disclosure is also based, in part, on the surprising finding that adjusting specific parameters of the manufacturing process can significantly reduce the level of subvisible particles (SVP) in formulations containing high concentrations of risankizumab (e.g., 10,000 or fewer SVPs per ml, where SVPs have a diameter greater than 2 μm), and thus reduce the risk of filter clogging.

[0023] For example, as demonstrated in Section 6 below, changing the freezing and thawing speeds from low to high reduced the level of SVP in the risankizumab formulation. Additionally, controlling the mechanical power input of the mixing process and the mixing time within a specific range also significantly reduced the level of SVP in the risankizumab formulation. Accordingly, the present disclosure also provides a high-concentration risankizumab formulation having not only a low level of SVP but also desirable filtration for the manufacturing process.

[0024] The present disclosure further discovered that a specific hitchhiker protein phospholipase A2 (PLA2) co-purified with risankizumab negatively affects the stability of polysorbate (e.g., polysorbate 20) in risankizumab liquid pharmaceutical formulations. Reducing the level of PLA2 within the formulation beneficially increased the long-term stability of the formulation (e.g., reduced particle formation, extended shelf life of the risankizumab medicine, etc.). Without being bound by any single theory, the present disclosure describes the development of novel risankizumab formulations having reduced levels of PLA2. These risankizumab formulations exhibited increased post-storage stability over time, which may be a result of the improved risankizumab active pharmaceutical ingredient manufacturing process disclosed herein (e.g., see Section 6).

[0025] Certain novel risankizumab formulations disclosed herein having one or more additional or alternative beneficial properties may be derived from the improved risankizumab active pharmaceutical ingredient manufacturing process exemplified herein, e.g., in Section 6. In particular, some of the risankizumab formulations disclosed herein have reduced immunogenicity. Additionally, certain risankizumab formulations disclosed herein have a unique and favorable glycosylation profile for risankizumab, e.g., low levels of gomannose N-glycans, high levels of fucosylated biantenary oligosaccharides, high levels of sialylated glycans, and / or overall low levels of non-glycosylated risankizumab. While not intended to be confined to any single theory, the glycosylation profile of risankizumab may affect the immunogenicity, stability, protein folding, structural integrity, aggregation, and other properties of risankizumab within the formulation.

[0026] The present disclosure also describes the finding that poloxamer 188 (P188) has improved stability over time compared to polysorbate 20 and polysorbate 80 in liquid risankizumab formulations prepared using a traditional process, and has similar stability over time compared to polysorbate 20 and polysorbate 80 in liquid risankizumab formulations prepared by the improved process exemplified herein.

[0027] Accordingly, in one embodiment, the present disclosure provides a liquid formulation comprising (i) risankizumab, (ii) water, and (iii) a surfactant, wherein the liquid formulation has a pH of 5.0 to 6.5. In a specific embodiment, the liquid formulation is a liquid aqueous pharmaceutical formulation.

[0028] In another aspect, the present disclosure provides a liquid formulation comprising risankizumab, wherein the formulation comprises 10,000 or fewer invisible particles (SVP) per ml, wherein the SVP have a diameter greater than 2 μm. In a specific embodiment, the liquid formulation is a liquid aqueous pharmaceutical formulation.

[0029] In another aspect, the present disclosure provides a liquid formulation comprising risankizumab at a concentration of about 180 mg / ml and phospholipase A2 (PLA2) in an amount of less than 250 pg per mg of risankizumab.

[0030] In another aspect, the present disclosure provides a liquid formulation comprising risankizumab at a concentration of about 180 mg / ml, 10,000 or fewer invisible particles (SVP) per ml, and phospholipase A2 (PLA2) in an amount of less than 250 pg per mg of risankizumab, wherein the SVP has a diameter greater than 2 μm.

[0031] In another aspect, the present disclosure provides a liquid formulation comprising risankizumab at a concentration of about 180 mg / ml, wherein a smaller percentage of the antibody in the formulation has a gomannose N-glycan compared to the antibody in a previously disclosed commercial formulation. Such gomannose N-glycans include mannose 5 N-glycan (M5), mannose 6 N-glycan (M6), and mannose 7 N-glycan (M7).

[0032] In another aspect, the present disclosure provides a liquid formulation comprising risankizumab at a concentration of about 180 mg / ml, and a higher percentage of the antibody in the formulation has fucosylated bianteneric oligosaccharides compared to previously disclosed commercial formulations.

[0033] In another aspect, the present disclosure provides a liquid formulation comprising risankizumab at a concentration of about 180 mg / ml, wherein a higher percentage of the antibody in the formulation has sialylated glycan compared to previously disclosed commercial formulations.

[0034] In another aspect, the present disclosure provides a liquid formulation comprising risankizumab at a concentration of about 180 mg / ml, wherein a smaller percentage of the antibody in the formulation is non-glycosylated compared to previously disclosed commercial formulations.

[0035] In another aspect, the present disclosure provides a liquid formulation comprising risankizumab at a concentration of about 180 mg / ml, and the incidence of treatment-emergent anti-drug antibodies (ADA) after administration of a single subcutaneous 150 mg dose of the liquid formulation to human subjects is less than 4.7%.

[0036] In another aspect, the present disclosure provides a liquid formulation comprising risankizumab at a concentration of about 180 mg / ml, wherein at least 99.1% of the antibody species in the formulation are present as monomers. In certain embodiments, a smaller percentage of the antibody in the formulation is a high molecular weight or low molecular weight species compared to those in previously disclosed commercial formulations.

[0037] In another aspect, the present disclosure provides a liquid formulation comprising risankizumab and poloxamer 188 (P188) at a concentration of about 180 mg / ml.

[0038] In a specific embodiment, the viscosity of the liquid formulation is less than about 20.0 mPa.s at 20°C. In a specific embodiment, the viscosity of the liquid formulation is 14.0 mPa.s to 20.0 mPa.s at 20°C. In a specific embodiment, the viscosity of the liquid formulation is about 15 mPa.s, about 17 mPa.s, or about 19 mPa.s at 20°C. In a specific embodiment, the viscosity of the liquid formulation is measured by the ALP method or a falling ball viscometer.

[0039] In a specific embodiment, the surfactant is polysorbate 20 (PS20). In a specific embodiment, PS20 is at a concentration of about 0.2 mg / ml.

[0040] In specific embodiments, the liquid formulations disclosed herein further comprise (iii) an isotonic agent, (iv) a buffer, and / or (v) a solubilizing agent.

[0041] In certain embodiments, the liquid formulation comprises an isotonic agent. In certain embodiments, the isotonic agent is a polyol. In certain embodiments, the isotonic agent is trehalose or mannitol. In certain embodiments, the isotonic agent is a salt.

[0042] In a specific embodiment, the liquid formulation comprises a buffer. In a specific embodiment, the buffer is an acetate buffer.

[0043] In certain embodiments, the liquid formulation does not contain a buffer.

[0044] In a specific embodiment, the liquid formulation includes a solubilizing agent. In a specific embodiment, the solubilizing agent is an amino acid. In a specific embodiment, the solubilizing agent is proline or glycine.

[0045] In a specific embodiment, the liquid formulation has an osmotic concentration of 320 mOsmol / kg to 350 mOsmol / kg.

[0046] In a specific embodiment, the liquid formulation has a pH of 5.0 to 6.5. In a specific embodiment, the liquid formulation has a pH of about 5.5.

[0047] In a specific embodiment, the formulation underwent a freeze-thaw process (F / T), wherein F / T comprises freezing the formulation to about -70°C and then thawing the formulation to about 18°C. In a specific embodiment, F / T comprises freezing the formulation from about 18°C ​​to about -70°C for 10 hours or less and then thawing the formulation from about -70°C to about 18°C ​​for 15 hours or less.

[0048] In a specific embodiment, the formulation underwent a mixing process, wherein the power per unit volume (P / V) of the mixing process is 1000 W / m² 3 The mixing time of the mixing process is 30 minutes or less.

[0049] In another aspect, the present disclosure provides a method for reducing the formation of SVP in a liquid aqueous pharmaceutical formulation comprising risankizumab at a concentration of about 180 mg / ml. In a specific embodiment, the method comprises F / T, wherein F / T comprises freezing the formulation from about 18 °C to about -70 °C for 10 hours or less, and then thawing the formulation from about -70 °C to about 18 °C for 15 hours or less. In a specific embodiment, the method comprises a mixing process, wherein the power per volume (P / V) of the mixing process is 1000 W / m² 3 The mixing time of the mixing process is 30 minutes or less.

[0050] In another aspect, the present disclosure also provides a liquid aqueous pharmaceutical formulation produced by a method for reducing the formation of SVP disclosed herein. Brief explanation of the drawing

[0051] 4. Brief description of the drawing Figures 1a and 1b represents the viscosity of formulations with four different concentrations of risankizumab (i.e., 150 mg / ml, 162 mg / ml, 180 mg / ml, and 198 mg / ml) and three different pH values ​​(i.e., 5.2, 5.7, and 6.2). Viscosity was not applied to any stress test and was measured by ALP at 20°C after preparation. Fig. 1a represents the viscosity of each formulation at different pH levels. Fig. 1b represents the fitted curve of the viscosity profile of risankizumab as a function of pH (left panel) or antibody concentration (right panel). Fig. 2 Figure 1 shows a comparison of viscosities measured at 20°C using both the falling ball method and the ALP method. For each formulation, the left bar represents the viscosity measured by the ALP method, and the right bar represents the viscosity measured by the falling ball method. FIGS. 3a to 3eFigure 1 represents the size-exclusion ultra-high performance liquid chromatography (SE-UHPLC) evaluation of formulations containing risankizumab at a concentration of 180 mg / ml and three different pH values ​​(i.e., 5.2, 5.7, and 6.2). Measurements were performed after 1) preparations not subjected to any stress test (no stress), 2) four freeze / thaw (ft) stress cycles in which each cycle was +20 °C / -40 °C and maintained for 30 minutes at each temperature, or 3) shaking a 100 μl sample of the formulation at 2200 rpm for 1 day at 20 °C to induce mechanical stress (mech). Fig. 3a This represents the percentage of monomers (monomer %) determined by SE-UHPLC in the unstressed formulation (left panel), the sample subjected to freeze / thaw stress (middle panel), and the formulation subjected to mechanical stress (right panel). Fig. 3b This represents the percentage (HMW %) of high molecular weight [HMW] species determined by SE-UHPLC in the unstressed formulation (left panel), the samples subjected to freeze / thaw stress (middle panel), and the formulation subjected to mechanical stress (right panel). Fig. 3c This represents the percentage (LMW %) of low molecular weight [LMW] species determined by SE-UHPLC in the unstressed formulation (left panel), the samples subjected to freeze / thaw stress (middle panel), and the formulation subjected to mechanical stress (right panel). Fig. 3d It shows the main effect plot for monomer % as a function of pH (left panel) or stress (right panel). Fig. 3e It shows the main effect plot for HMW % as a function of pH (left panel) or stress (right panel). FIGS. 4a to 4fThis represents the SE-UHPLC evaluation of formulations containing 180 mg / ml risankizumab in four different base formulations (i.e., mannitol formulation, trehalose / acetate formulation, glycine formulation, and proline formulation) and a benchmark formulation containing 150 mg / ml risankizumab in a trehalose / acetate formulation. Measurements were performed after applying the 180 mg / ml risankizumab formulation and the benchmark formulation at 40 °C for 7 or 21 days (full points (days)). Fig. 4a represents the monomer % at each pool point determined by SE-UHPLC. Fig. 4b represents the HMW % at each pool point determined by SE-UHPLC. Fig. 4c represents the LMW % at each pool point determined by SE-UHPLC. FIGS. 4d to 4f is the monomer % for the formulation over time at 40 ℃ determined by SE-UHPLC ( Fig. 4d ), HMW %( Fig. 4e ) and LMW %( Fig. 4f Represents the change (delta) of ). Delta was calculated as D = Tx - T0, where Tx is the measurement at day 7 or 21, and T0 is the measurement at day 0. Method variability ± 0.4%. FIGS. 5a to 5f It comes in four different basic formulations (i.e., mannitol formulation, trehalose / acetate formulation, glycine formulation, proline). This shows the CEX-UHPLC evaluation of a formulation containing 180 mg / ml risankizumab in a formulation and a benchmark formulation containing 150 mg / ml risankizumab in a trehalose / acetate formulation. Measurements were performed after applying the 180 mg / ml risankizumab formulation and the benchmark formulation at 40 ℃ for 7 or 21 days (full points [days]). Major isoform %( Fig. 5a ), acid species (acidic substance) content ( Fig. 5b ), basic species (basic substances) content ( Fig. 5c ), Delta major isoform content ( Fig. 5d), delta acid species content ( Fig. 5e ) and delta basic species content ( Fig. 5f ) was determined by CEX-UHPLC at each of the indicated pool points. Method variability ± 2%. FIGS. 6a to 6d represents the temperature profile for the FT scale-down model [SDM] and transport simulation. Fig. 6a represents the low-speed FT for upright freezer freezing and benchtop thawing. Fig. 6b represents the high-speed FT for rapid freezer freezing and forced-air convection chamber (FACC) thawing. Fig. 6c represents a full-scale transportation study. Fig. 6d Is It represents the worst transportation conditions in the past. Figures 7a and 7b represents the pH (right y-axis) and temperature (left y-axis) of Risa DS during the FT process. Fig. 7a Is Risa 180 UHC shows low-temperature pH changes. Fig. 7b Is Risa 150 shows the pH change at low temperature in the control group. FIGS. 8a to 8c The density and viscosity of risankizumab (Risa) drug substance (DS) as a function of temperature and antibody concentration represents. Fig. 8a It represents the change in density as a function of temperature for batches of Risa DS at different concentrations. Fig. 8b Is It shows the temperature dependence of viscosity at various concentrations. The experimental data were fitted to an Arrhenius model for viscosity to predict the viscosity-temperature profile. Fig. 8c represents the antibody concentration dependence of viscosity at 4 ℃, 20 ℃, and 25 ℃. Experimental data were fitted to an exponential model to predict the viscosity-concentration profile. FIGS. 9a to 9drepresents the number of SVPs of Risa bulk drug substance [BDS] at different FT rates and cycles by MFI. Cycle 0 is the initial time point prior to FT. Fig. 9a represents Risa 180 UHC stressed by low-speed FT. Fig. 9b represents a Risa 150 control group stressed by low-speed FT. Fig. 9c represents Risa 180 UHC stressed by high-speed FT. Fig. 9d represents the Risa 150 control group stressed by high-speed FT. For each cycle group, the bars from left to right represent particle sizes greater than 2 μM, greater than 5 μM, greater than 10 μM, and greater than 25 μM, respectively. FIGS. 10a to 10d represents the clarity of 180 mg / mL risankizumab (Risa 180) ultra-high concentration (UHC) and 150 mg / mL risankizumab (Risa 150) control in simulations of different FT rates, cycles, and transport based on turbidity. Cycle 0 is the starting point before FT or transport. Fig. 10a represents a Risa BDS stressed by a low-speed FT. Fig. 10b represents Risa BDS stressed by high-speed FT. Fig. 10c represents the Risa BDS full-scale transport study and SDM validation. Fig. 10d This represents the historical worst-case transport simulation of Risa BDS. For each cycle group, the bars from left to right represent Risa 180 UHC and Risa 150 control, respectively. FIGS. 11a to 11d represents the product quality [PQ] data of Risa BDS at different FT rates and cycles by SEC, CEX, and CE-SDS-NR. Cycle 0 is the initial point in time prior to FT. Fig. 11a Is This represents Risa 180 UHC stressed by low-speed FT. Fig. 11b Is This represents the Risa 150 control group stressed by low-speed FT. Fig. 11c represents Risa 180 UHC stressed by high-speed FT. Fig. 11d represents a Risa 150 control group stressed by high-speed FT. *This condition was not performed due to the limited availability of control materials. FIGS. 12a to 12c Figure 1 represents the PQ data of Risa BDS in -20°C transport simulations by SEC, CEX, and CE-SDS-NR. Cycle 0 is the initial point in time before transport. Fig. 12a This represents the Risa 180 UHC full-scale study. For each condition group, the bars from left to right represent % LMV, % HMV, and % monomer, respectively. Fig. 12b It represents Risa 180 UHC under stress during up to two worst-case transport cycles. Fig. 12c represents a Risa 150 control group subjected to stress during up to two worst-case transport cycles. FIGS. 13a to 13c Is PQ data of Risa 180 UHC from storage time studies by SEC, CEX, and CE-SDS-NR is shown. Fig. 13a indicates storage at 4 ℃. Fig. 13b indicates storage at room temperature (approx. 21.5 ℃). Fig. 13c indicates storage at -20 ℃. Fig. 14 Figure 1 represents the correlation between Risa final BDS filtration efficiency and SVP. 150 L / m² 2 Low filtration rates of less than this likely clogged full-scale sterile filters in the DP manufacturing process. Figures 15a and 15brepresents the PQ data of Risa BDS mixed SDM by SEC, CEX, and CE-SDS-NR. N=0 rpm, 0 min was the initial sample before mixing. Fig. 15a Is Risa 180 UHC is stressed by mechanical mixing. Fig. 15b Is This represents the Risa 150 control group stressed by mechanical mixing. Figures 15a and 15b For each group of the upper panels, the bars from left to right represent % LMV and % HMW, respectively. Figures 15a and 15b For each group of the middle panels, the bars from left to right represent % basic and % acidic, respectively. Figures 15a and 15b For each group of the lower panels, the bars from left to right represent % purity, % LMV, and % HMW, respectively. Figures 16a and 16b represents the mathematical modeling of the Risa final BDS mixture for shear rate, mixing rate, and filling volume. Fig. 16a Is Risa represents an 180 UHC 200 L SS BDS tank. Fig. 16b Is Risa 180 UHC XDM-50 L BDS SUM represents. FIGS. 17a to 17d represents the number of SVPs of Risa BDS in the -20 ℃ transport simulation. Cycle 0 is the initial point before transport. Fig. 17a Is Risa 180 UHC represents full-scale transport study and SDM verification. Fig. 17b Is Risa 150 represents the control group's full-scale transport SDM. Fig. 17c Is Risa 180 UHC represents a simulation of past worst-case transport conditions. Fig. 17d Is This represents the past worst-case transport simulation of the Risa 150 control group. For each condition group, the bars from left to right represent particle sizes greater than 2 μM, greater than 5 μM, greater than 10 μM, and greater than 25 μM, respectively. FIGS. 18a to 18c represents particle identification of Risa BDS mixed SDM. N=0 rpm, 0 min is the initial sample before mixing. Fig. 18a Is Shows the MFI reading for Risa 180 UHC. Fig. 18b Is Shows the MFI readings for the Risa 150 control group. Fig. 18c Is Shows the turbidity measurements for Risa 180 UHC and Risa 150 control group. Figures 18a and 18b For each condition group, the bars from left to right represent particle sizes greater than 2 μM, greater than 5 μM, greater than 10 μM, and greater than 25 μM, respectively. Fig. 18c For each condition group, the bars from left to right represent Risa 180 UHC and Risa 150 control, respectively. Figures 19a and 19b represents the mathematical modeling of the Risa final BDS mixture for P / V, mixing rate, and filling volume. Fig. 19a Is Risa represents an 180 UHC 200 L SS BDS tank. Fig. 19b Is Risa 180 UHC XDM-50 L BDS SUM represents. Fig. 20 Figure 1 represents the mathematical modeling of the Risa final BDS mixture for P / V, mixing rate, and filling volume. The model was based on Risa 150 BDS tank mixing data to validate the model for predicting the risk level of particle formation. Fig. 21 ... represents the number of low molecular weight (LMW) hitchhiker proteins (HP), total HP, and LMW HP and total HP in DP1 and DP2 by affinity purification. Fig. 22 This represents a general overview of a newly developed purification process for risankizumab active pharmaceutical ingredient (referred to herein as the development of Process 4). Fig. 23 represents Western blots probed with anti-PLA2G15 antibody. 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. FIGS. 24a to 24d Is It demonstrates the stability of PS20 at different temperatures. Fig. 24a represents the stability of PS20 at 5°C measured by CAD assay in samples prepared from various control cell lines and knockout cell lines. Fig. 24b represents the stability of PS20 at 25°C as measured by the CAD assay in samples prepared from various control and knockout cell lines. #5 Placebo: PS20 control. #6: BDS control: DP4 BDS. Fig. 24c represents the stability of PS20 at 5°C as measured by the FFA assay in samples prepared from various control and knockout cell lines. #5 Placebo: PS20 control. #6: BDS control: DP4 BDS. Fig. 24d represents the stability of PS20 at 25°C as measured by the FFA assay in samples prepared from various control and knockout cell lines. #5 Placebo: PS20 control. #6: BDS control: DP4 BDS. FIGS. 25a and 25b is the PS20 subspecies chromatogram represents. Fig. 25ashows the chromatogram overlay of PS20 subspecies of sample C1 injection 1 (DP2 after 30 months at 2 to 8 ℃), sample A1 injection 2 (DP3 spiked with 1 μg / mL PLA2G15 after about 9 hours of incubation at room temperature and 25 ℃), and sample D1 injection 1 (unspiketed DP3 material, no significant PS20 degradation). Fig. 25b represents the chromatogram overlay of PS20 subspecies degradation in DP4 solution at different PLA2G15 spiking levels after 4 days of incubation at 25°C. Arm 8 was the DP2 control sample. The small difference around 42 minutes for Arm 8 may have been caused by the leaching from the syringe filter used for this laboratory filling of this arm, as this peak was not observed in past data. Fig. 26 shows the chromatogram overlays of PS20 subspecies of sample E3 injection 2 (DP3; no significant PS20 degradation), sample A3 injection 6 (DP3 spiked with 5 μg / mL PLBL2 after incubation at 25°C for about 30 hours; total incubation time after spiked was 26 hours at 25°C plus about 20 hours at 2 to 8°C), and sample D3 injection 2 (PS20 in DP2 after storage at 2 to 8°C for about 30 months). FIGS. 27a and 27b is the PS20 subspecies chromatogram represents. Fig. 27a shows the chromatogram overlay of PS20 subspecies of sample E3 injection 2 (DP3; no significant PS20 degradation) and sample B3 injection 6 (DP3 spiked with 5 μg / mL CES 1 after incubation for about 30 hours at 25 ℃; total incubation time after spiked was 27 hours at 25 ℃ plus about 20 hours at 2 to 8 ℃). Fig. 27bshows the chromatogram overlay of PS20 subspecies of sample D3 injection 2 (PS20 degradation in DP2, after storage at 2 to 8 °C for about 30 months) and sample B3 injection 6 (DP3 spiked with 5 μg / mL CES 1 after incubation at 25 °C for about 30 hours; total incubation time after spiked was 27 hours at 25 °C in addition to about 20 hours at 2 to 8 °C). Fig. 28 shows the chromatogram overlay of PS20 subspecies of sample E3 injection 2 (DP3, no significant PS20 degradation) and sample C3 injection 6 (DP3 spiked with 5 μg / mL SIAE after incubation for about 30 hours at 25°C; total incubation time after spiked was 28 hours at 25°C in addition to about 20 hours at 2 to 8°C). Fig. 29 shows the chromatogram overlays of PS20 subspecies of sample D4 injection 2 (DP3, no significant PS20 degradation), sample A4 injection 7 (DP3 spiked with 5 μg / mL PRDX6 after incubation at 25°C for about 30 hours; total incubation time after spiked was about 27 hours at 25°C), and sample C4 injection 2 (PS20 in DP2 after storage at 2 to 8°C for about 30 months). Fig. 30 represents the chromatogram overlay of PS20 subspecies of sample D4 injection 2 (DP3, no significant 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 to 8°C for about 30 months). Fig. 31Figure 1 shows the chromatogram overlays of the PS20 subspecies of samples H7-9, H7-8, and H7-7. H7-9: DP2 (maintained at -80 °C), H7-8: DP2 (maintained at room temperature for 2 weeks), H7-7: DP2 spiked with 0.9 µg / mL posinopril (maintained at room temperature for 2 weeks). The signal was normalized to correct for concentration changes due to spiking (normalized to the peak at 37 min, which was stable in the DP2 substance based on historical data). Fig. 32 Figure 7 shows the chromatogram overlays of the PS20 subspecies of samples H7-9, H7-8, and H7-6. H7-9: DP2 (maintained at -80 °C), H7-8: DP2 (maintained at room temperature for 2 weeks), H7-6: DP2 spiked with 3.8 µg / mL fosinopril (maintained at room temperature for 2 weeks). The signal was normalized to correct for concentration changes due to spiking (normalized to the peak at 37 min, which was stable in the DP2 substance based on historical data). Fig. 33 Figure 7 shows the chromatogram overlays of the PS20 subspecies of samples H7-9, H7-8, and H7-3. H7-9: DP2 (maintained at -80 °C), H7-8: DP2 (maintained at room temperature for 2 weeks), H7-3: DP2 spiked with 27.8 µg / mL posinopril (maintained at room temperature for 2 weeks). The signal was normalized to correct for concentration changes due to spikes (normalized to the peak at 37 min, which was stable in the DP2 material based on historical data). The higher background in the spiked samples at approximately 39 to 42 min must be due to the co-elution of posinopril. Fig. 34 represents the chromatogram overlay of the PS20 subspecies of samples A6 and C6. A6: DP2 substance (maintained at -80 °C, diluted 1:1 with water prior to testing), C6: DP2 substance (spiked with 930 µg / mL fosinopril, maintained at room temperature for 2 weeks, diluted 1:1 with water prior to testing). The signal from C6 was normalized for better comparison with A5. FIGS. 35a to 35c Is It demonstrates the stability of PS20 at different temperatures. Fig. 35a This represents the stability of PS20 in DP2(PS20) and DP3(PS20) measured by CAD calibration at 5 ℃. Fig. 35b This represents the stability of PS20 in DP2(PS20) and DP3(PS20) measured by CAD calibration at 25 ℃. Fig. 35c This represents the stability of PS20 in DP2(PS20) and DP3(PS20) measured by CAD calibration at 40 ℃. Figures 36a and 36b Is It demonstrates the stability of PS20 at different temperatures. Fig. 36a represents the stability of PS20 in DP2(PS20) and DP3(PS20) as measured by FFA assay at 5 ℃. Fig. 36b This represents the stability of PS20 in DP2(PS20) and DP3(PS20) as measured by FFA testing at 25 ℃. FIGS. 37a to 37c Is It demonstrates the stability of PS20 at different temperatures. Fig. 37a This represents the stability of PS20 in DP2(PS20) and DP4(PS20) measured by CAD calibration at 5 ℃. Fig. 37b This represents the stability of PS20 in DP2(PS20) and DP4(PS20) measured by CAD calibration at 25 ℃. Fig. 37c This represents the stability of PS20 in DP2(PS20) and DP4(PS20) measured by CAD calibration at 40 ℃. FIGS. 38a to 38d Is It demonstrates the stability of PS20 at different temperatures. . Figs. 38a and 38b represents the stability of PS20 in DP2(PS20) and DP4(PS20) as measured by FFA assay at 5 ℃. FIGS. 38c and 38d This represents the stability of PS20 in DP2(PS20) and DP4(PS20) as measured by FFA testing at 25 ℃. Figures 39a and 39bFigure 4 shows the 2-AB and HILIC-FL chromatograms of the Process 4 drug substance (DS) batch and the Process 1 reference standard DS1-RS2. Process 4 batch DS4-001 and the Process 1 reference standard DS1-RS2 were analyzed side-by-side. Results for the reference standards accompanying the other three Process 4 DS batches are not shown. Differences in retention times from different analytical runs were observed as expected. The assay performance (relative peak quantification) was unaffected. Fig. 39b Is Fig. 39a It is an enlarged view of. Fig. 40 Figure 1 shows the RapiFluor and HILIC-FL chromatograms of process 1, 2, and 4 DS batches. FIGS. 41a to 41d Is Shows the UP-SEC results. Fig. 41a represents the relative distribution of UP-SEC monomer results for risankizumab process 1, 2, and 4 DS batches. Fig. 41b Figure 1 shows the relative distribution of UP-SEC HMW results for risankizumab process batches 1, 2, and 4 DS. Fig. 41c This shows the UP-SEC results of risankizumab process 4 DS batch DS4-005 and process 1 reference standard DS1-RS2. Fig. 41d Is Fig. 41c It is an enlarged view of. FIGS. 42a to 42d Is Shows the CGE-NR results. Fig. 42a represents the relative distribution of CGE-NR major peak results for risankizumab process batches 1, 2, and 4 DS. Fig. 42b represents the relative distribution of CGE-NR LMW results for risankizumab process batches 1, 2, and 4 DS. Fig. 42c This shows the CGE-NR results of risankizumab process 4 DS batch DS4-005 and process 1 reference standard DS1-RS2. Fig. 42d Is Fig. 42c It indicates the magnification of. Fig. 43This is a summary of the total particle concentrations (≥ 0.5 μm) of formulations 1 to 4 of Example 2 compared with a benchmark formulation within the range of short-term stability studies (40 ℃, 21 days) at full points t0, t7, and t21. FIGS. 44a to 44c is 5 ℃( Fig. 44a ), 25 ℃( Fig. 44b ) and 40 ℃( Fig. 44c This shows the stability of PS80 in DP2 (PS80), DP3 (PS80), and DP4 (PS80) as measured by CAD testing. FIGS. 45a to 45f is 5 ℃( Figs. 45a and 45b ), 25 ℃( Figs. 45c and 45d ) and 40 ℃( Figs. 45e and 45f This shows the stability of PS80 in DP2 (PS80), DP3 (PS80), and DP4 (PS80) as measured by FFA testing. Fig. 46 represents the P188 and PS20 levels in DP2 DS. Fig. 47 represents the P188 and PS20 levels in DP3 DS. Fig. 48 This is a table showing P188, PS20, HMW, LMW, APG, and BPG data generated from Example 19. Specific details for implementing the invention

[0052] 5. Detailed explanation

[0053] The present disclosure relates to a novel antibody formulation that advantageously provides a high concentration of risankizumab having suitable viscosity and stability, which enables subcutaneous injection of the formulation.

[0054] In one aspect, the present disclosure provides a liquid formulation containing risankizumab at a high concentration of about 180 mg / ml (e.g., the liquid formulation of Section 5.2). In certain embodiments, the liquid formulation is a liquid aqueous pharmaceutical formulation. The present disclosure further provides a method of using the formulation disclosed herein to treat a disease (e.g., psoriasis and Crohn's disease) (e.g., the method of Section 5.3).

[0055] The viscosity profile of each antibody is unpredictable, even for very similar monoclonal antibodies. It can be difficult to determine the relationship between antibody concentration and viscosity in a formulation. A solution with a viscosity of up to 20 m·Pas It has been reported to show good tolerability for subcutaneous injection (e.g., literature [Berteau et al , Med Devices [Refer to 8(2015) 473-484]). For any specific antibody, due to the unpredictability of the relationship between antibody concentration and viscosity, empirical studies and prudent formulation development are required to identify high antibody concentrations or maximum antibody concentration cut-offs at which the formulation maintains its subcutaneous injectability and other desired properties. This disclosure is based in part on the surprising discovery that a surprising cut-off effect exists at a concentration of about 180 mg / ml. In particular, the viscosity of the risankizumab formulation increased with protein concentration and was found to be consistently less than about 20.0 mPa·s at concentrations of about 180 mg / ml or less, but increased exponentially at concentrations of about 180 mg / ml or more. Additionally, formulations containing about 180 mg / ml of risankizumab exhibited clinically acceptable glide and ejection times when tested with a subcutaneous syringe. In addition, the present disclosure found that a formulation containing about 180 mg / ml of risankizumab is stable under freezing / thawing and mechanical stress.

[0056] The present disclosure also relates to a novel method for preparing a liquid aqueous pharmaceutical formulation comprising a high concentration of risankizumab (e.g., about 180 mg / ml of risankizumab) that results in a reduction in the level of SVP (Section 5.4). A resulting high concentration risankizumab formulation having a low level of SVP is also provided herein.

[0057] The present disclosure also relates to an improved process for manufacturing a risankizumab active pharmaceutical ingredient that produces a risankizumab formulation further comprising low levels of PLA2, reduced immunogenicity, a unique and favorable glycosylation profile, and / or increased purity (Section 5.5). This is based, in part, on the discovery of certain hitchhiker proteins, PLA2, whose presence negatively affects the stability of polysorbates (e.g., PS20 and / or PS80) in risankizumab liquid pharmaceutical formulations, and the discovery that reducing PLA2 from the formulation beneficially increases the stability of the formulation (e.g., reduced particle formation, increased shelf life of the risankizumab finished product).

[0058] The initial pharmaceutical formulation developed for risankizumab had a concentration of 90 mg / ml. A 150 mg / ml formulation was subsequently approved by the U.S. FDA to enable a single subcutaneous injection of a total therapeutic dose of 150 mg. Both the commercial 75 mg / 0.83 ml (90 mg / mL) and 150 mg / ml risankizumab formulations are disclosed in the FDA-approved drug labels and the 'Full Prescribing Information' for SKYRIZI® (risankizumab-rzaa) as revised in December 2022, the contents of which are incorporated herein by reference in their entirety. Both FDA-approved risankizumab formulations contain a highly purified, recombinantly produced risankizumab active pharmaceutical ingredient (API). However, when the 150 mg / ml risankizumab formulation was diluted to investigate the feasibility of development when used with a specific product form, e.g., a body-worn device, unacceptable levels of particles consisting of risankizumab and / or free fatty acid aggregates were formed under specific storage conditions.

[0059] This unexpected problem is believed to be caused at least partially by residual trace levels of hitchhiker protein co-purified with a highly pure risankizumab API, purified using a state-of-the-art orthogonal column chromatography process. Since the type of hitchhiker protein co-purified with a monoclonal antibody (mAb) varies depending on the mAb, it is impossible to predict prior to the experiment whether a hitchhiker protein issue will occur during the production of the novel antibody, as well as which hitchhiker protein will be problematic.

[0060] The present disclosure identifies PLA2 as a specific problematic hitchhiker protein co-purified with risankizumab. It is demonstrated herein that PLA2 co-purified with risankizumab causes degradation of the surfactant polysorbate 20 (PS20), leading to particle formation in risankizumab products. An optimized purification process has been developed specifically targeting a reduction in the levels of PLA2 co-purified with risankizumab. Accordingly, in some embodiments, the present disclosure provides a risankizumab liquid formulation having reduced levels of PLA2 and improved stability and shelf life.

[0061] In some embodiments, the present disclosure relates to an improved risankizumab composition having a unique antibody glycosylation profile that may confer or contribute to advantageous properties, such as increased stability, reduced aggregation, and reduced immunogenicity, as a result of the novel manufacturing process described herein. For example, in certain embodiments, the present disclosure relates to an improved risankizumab composition having a reduced level of risankizumab species modified with highmannose N-glycans (e.g., M5, M6, and / or M7) that have reduced immunogenicity. Reduced immunogenicity (e.g., a low incidence of anti-drug antibodies occurring after treatment following administration of a single 150 mg subcutaneous liquid formulation to humans) also indicates improved product quality of the risankizumab composition described herein. In certain embodiments, the present disclosure relates to an improved risankizumab composition having a higher level of risankizumab species modified with fucosylated biantenary oligosaccharides. In certain embodiments, the present disclosure relates to an improved risankizumab composition having a higher level of risankizumab species modified with sialylated glycans. In certain embodiments, the present disclosure relates to an improved risankizumab composition having a lower level of non-glycosylated risankizumab species.

[0062] In some embodiments, the present disclosure relates to novel risankizumab compositions having a higher level of purity or homogeneity of monomers having a reduced level of high molecular weight or low molecular weight antibody species.

[0063] In some aspects, the present disclosure relates to a novel risankizumab composition comprising poloxamer 188 (P188). The present disclosure describes the finding that P188 has improved stability over time compared to polysorbate 20 and polysorbate 80 in liquid risankizumab formulations prepared using a traditional process, and has similar stability over time compared to polysorbate 20 and polysorbate 80 in liquid risankizumab formulations prepared by the improved process exemplified herein.

[0064] 5.1 Definition

[0065] The singular forms 'one', 'one', and 'above' include multiple referents unless the context clearly indicates otherwise.

[0066] In this document, the term 'and / or' as used in phrases such as 'A and / or B' is intended to mean 'A and B', 'A or B', or 'A' or 'B'.

[0067] The term 'between' used in phrases such as 'between A and B' or 'between A and B' refers to a range that includes both A and B.

[0068] The term 'optional' or 'optional' means that the situation described subsequently may or may not occur, and thus the description includes cases where the situation occurs and cases where the situation does not occur.

[0069] The term “about” generally refers to a range of numbers that a person skilled in the art would consider equivalent to (i.e. having the same function or result) the cited value, including within 5%, 4%, 3%, 2%, 1%, or lesser variation of the given value or range. In many cases, the term “about” may include numbers rounded to the nearest significant figure.

[0070] Unless the context requires otherwise, the terms 'comprise', 'comprises', and 'comprise-ing' are used based on the clear understanding that they indicate the inclusion of the cited features but should be interpreted inclusively rather than exclusively so as not to exclude one or more of these features.

[0071] Terms such as 'patient', 'object', and 'individual' refer to humans.

[0072] As used herein, the terms 'treat,' 'treatment,' 'treating,' 'to treat,' 'alleviate,' 'alleviation,' 'alleviating,' or 'to alleviate' refer to therapeutic measures aimed at curing, slowing down, reducing / and / or the symptoms of, or halting the progression of pathological conditions or disorders. Accordingly, subjects requiring treatment include those already suffering from a disease.

[0073] 5.2 Liquid formulation

[0074] The present disclosure provides a liquid formulation comprising risankizumab at a concentration of about 180 mg / ml. The formulation disclosed herein advantageously provides a high concentration of risankizumab having suitable viscosity (e.g., less than about 20.0 mPa·s at 20°C) and stability, which makes subcutaneous injection of the formulation feasible.

[0075] In a specific embodiment, the liquid formulation comprises risankizumab at a concentration of about 180 mg / ml and a surfactant (e.g., the surfactant of Section 5.2.1), wherein the liquid formulation has a pH of 5.0 to 6.5. In a specific embodiment, the liquid formulation further comprises an isotonic agent (e.g., the isotonic agent of Section 5.2.2), a solubilizer (e.g., the solubilizer of Section 5.2.3), and / or a buffer (e.g., the buffer of Section 5.2.4). In a specific embodiment, the viscosity of the liquid formulation is less than about 20.0 mPa·s at 20 °C. In a specific embodiment, the liquid formulation has an osmotic concentration of 300 mOsmol / kg to 400 mOsmol / kg.

[0076] In a specific embodiment, the liquid formulation disclosed herein comprises risankizumab at a concentration of about 180 mg / ml and comprises 10,000 or fewer invisible particles (SVP) per ml (see Section 5.2.8). In a specific embodiment, the SVP has a diameter of at least 2 μm.

[0077] In a specific embodiment, the liquid formulation is produced by the method disclosed in Section 5.4.

[0078] In a specific embodiment, the liquid formulation disclosed herein comprises risankizumab at a concentration of about 180 mg / ml and less than 250 pg of phospholipase A2 (PLA2) per mg of risankizumab (see Section 5.2.9).

[0079] The liquid formulations covered by the present disclosure may include added water, such as USP grade water. In certain embodiments, the liquid formulations described herein further include pharmaceutically acceptable excipients. In certain embodiments, the liquid formulations are liquid aqueous pharmaceutical formulations. In certain embodiments, the liquid formulations described herein are packaged in vials, pre-filled syringes, or body-worn devices. In certain embodiments, the liquid formulations described herein are suitable for parenteral administration. In certain embodiments, the liquid formulations disclosed herein are suitable for subcutaneous or intravenous injection.

[0080] 5.2.1 Surfactants

[0081] In certain embodiments, the liquid formulation disclosed herein comprises a surfactant. In certain embodiments, the surfactant disclosed herein may inhibit protein aggregation and / or minimize surface adsorption at the air-water interface, in the packaging container, and / or when diluted with an intravenous solution.

[0082] Any suitable surfactant known in the art may be used with the formulations disclosed herein. In certain embodiments, the surfactant is polysorbate 20 (PS20), polysorbate 80 (PS80), polysorbate 40 (PS40), polysorbate 60 (PS60), polysorbate 65 (PS65), or poloxamer 188. In certain embodiments, the surfactant is polysorbate. In certain embodiments, the surfactant is PS20. In certain embodiments, the surfactant is PS80.

[0083] In a specific embodiment, the liquid formulation contains a surfactant at a concentration of 0.02 mg / ml to 2 mg / ml. In a specific embodiment, the liquid formulation contains a surfactant at a concentration of at least 0.1 mg / ml, at least 0.2 mg / ml, at least 0.3 mg / ml, at least 0.4 mg / ml, up to 0.5 mg / ml, up to 0.4 mg / ml, up to 0.3 mg / ml, or up to 0.2 mg / ml. In a specific embodiment, the liquid formulation contains a surfactant at a concentration of 0.1 mg / ml to 0.3 mg / ml. In a specific embodiment, the liquid formulation contains a surfactant at a concentration of about 0.2 mg / ml.

[0084] In a specific embodiment, the surfactant is PS20. In a specific embodiment, the liquid formulation contains PS20 at a concentration of 0.02 mg / ml to 2 mg / ml. In a specific embodiment, the liquid formulation contains PS20 at a concentration of at least 0.1 mg / ml, at least 0.2 mg / ml, at least 0.3 mg / ml, at least 0.4 mg / ml, up to 0.5 mg / ml, up to 0.4 mg / ml, up to 0.3 mg / ml, or up to 0.2 mg / ml. In a specific embodiment, the liquid formulation contains PS20 at a concentration of 0.1 mg / ml to 0.3 mg / ml. In a specific embodiment, the liquid formulation contains PS20 at a concentration of about 0.2 mg / ml.

[0085] In a specific embodiment, the surfactant is PS80, and the amount of PS80 is up to 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).

[0086] 5.2.2 Topics of Appearance

[0087] In certain embodiments, the liquid formulation disclosed herein further comprises an isotonic agent. In certain embodiments, the isotonic agent disclosed herein may adjust the isotonicity of the formulation.

[0088] Any suitable isotonic agent known in the art may be used with the formulations disclosed herein. In certain embodiments, the isotonic agent is a salt. In certain embodiments, the isotonic agent is a polyol. In certain embodiments, the polyol is a sugar or a sugar alcohol. In certain embodiments, the polyol is a sugar. In certain embodiments, the sugar is trehalose. In certain embodiments, the polyol is a sugar alcohol. In certain embodiments, the sugar alcohol is mannitol.

[0089] In a specific embodiment, the liquid formulation contains a polyol at a concentration of 100 mM to 500 mM. In a specific embodiment, the liquid formulation contains a polyol at a concentration of at least 170 mM, at least 180 mM, at least 190 mM, up to 200 mM, up to 190 mM, or up to 180 mM. In a specific embodiment, the liquid formulation contains a polyol at a concentration of 180 mM to 190 mM. In a specific embodiment, the liquid formulation contains a polyol at a concentration of about 185 mM. In a specific embodiment, the liquid formulation contains a polyol at a concentration of about 190 mM.

[0090] In a specific embodiment, the isotonic agent is trehalose. In a specific embodiment, the liquid formulation contains trehalose at a concentration of 100 mM to 500 mM. In a specific embodiment, the liquid formulation contains trehalose at a concentration of at least 170 mM, at least 180 mM, at least 190 mM, up to 200 mM, up to 190 mM, or up to 180 mM. In a specific embodiment, the liquid formulation contains trehalose at a concentration of 180 mM to 190 mM. In a specific embodiment, the liquid formulation contains trehalose at a concentration of about 185 mM.

[0091] In a specific embodiment, the isotonic agent is mannitol. In a specific embodiment, the liquid formulation contains mannitol at a concentration of 100 mM to 500 mM. In a specific embodiment, the liquid formulation contains mannitol at a concentration of at least 170 mM, at least 180 mM, at least 190 mM, up to 200 mM, up to 190 mM, or up to 180 mM. In a specific embodiment, the liquid formulation contains mannitol at a concentration of 180 mM to 190 mM. In a specific embodiment, the liquid formulation contains mannitol at a concentration of about 190 mM.

[0092] In certain embodiments, the formulations disclosed herein do not contain isotonic agents. In certain embodiments, the formulations disclosed herein do not contain polyols (e.g., sugars or sugar alcohols).

[0093] 5.2.3 Solubilizers

[0094] In certain embodiments, the liquid formulation disclosed herein comprises a solubilizing agent. In certain embodiments, the solubilizing agent disclosed herein may improve the bioavailability of the antibody and / or reduce antibody aggregation.

[0095] Any suitable solubilizing agent known in the art may be used with the formulations disclosed herein. In certain embodiments, the solubilizing agent is an amino acid. In certain embodiments, the solubilizing agent is proline. In certain embodiments, the solubilizing agent is glycine.

[0096] In a specific embodiment, the liquid formulation contains a solubilizing agent at a concentration of 100 mM to 500 mM. In a specific embodiment, the liquid formulation contains a solubilizing agent at a concentration of at least 100 mM, at least 200 mM, at least 300 mM, at least 400 mM, up to 500 mM, up to 400 mM, up to 300 mM, or up to 200 mM. In a specific embodiment, the liquid formulation contains a solubilizing agent at a concentration of 200 mM to 250 mM. In a specific embodiment, the liquid formulation contains a solubilizing agent at a concentration of about 225 mM.

[0097] In a specific embodiment, the solubilizing agent is proline. In a specific embodiment, the liquid formulation contains proline at a concentration of 100 mM to 500 mM. In a specific embodiment, the liquid formulation contains proline at a concentration of at least 100 mM, at least 200 mM, at least 300 mM, at least 400 mM, up to 500 mM, up to 400 mM, up to 300 mM, or up to 200 mM. In a specific embodiment, the liquid formulation contains proline at a concentration of 200 mM to 250 mM. In a specific embodiment, the liquid formulation contains proline at a concentration of about 225 mM.

[0098] In a specific embodiment, the solubilizing agent is glycine. In a specific embodiment, the liquid formulation contains glycine at a concentration of 100 mM to 500 mM. In a specific embodiment, the liquid formulation contains glycine at a concentration of at least 100 mM, at least 200 mM, at least 300 mM, at least 400 mM, up to 500 mM, up to 400 mM, up to 300 mM, or up to 200 mM. In a specific embodiment, the liquid formulation contains glycine at a concentration of 200 mM to 250 mM. In a specific embodiment, the liquid formulation contains glycine at a concentration of about 225 mM.

[0099] In certain embodiments, the liquid formulation disclosed herein does not contain a solubilizing agent. In certain embodiments, the liquid formulation disclosed herein does not contain glycine or proline.

[0100] 5.2.4 Buffer

[0101] In certain embodiments, the liquid formulation disclosed herein further comprises a buffer. Any suitable buffer known in the art may be used with the formulation disclosed herein. In certain embodiments, the buffer is an acetate buffer.

[0102] In a specific embodiment, the liquid formulation contains a buffer at a concentration of 1 mM to 100 mM. In a specific embodiment, the liquid formulation contains a buffer at a concentration of at least 1 mM, at least 5 mM, at least 10 mM, at least 15 mM, up to 20 mM, up to 15 mM, up to 10 mM, or up to 5 mM. In a specific embodiment, the liquid formulation contains a buffer at a concentration of 5 mM to 15 mM. In a specific embodiment, the liquid formulation contains a buffer at a concentration of about 9.5 mM. In a specific embodiment, the liquid formulation contains a buffer at a concentration of about 10 mM.

[0103] In a specific embodiment, the buffer is an acetate buffer. In a specific embodiment, the liquid formulation contains the acetate buffer at a concentration of 1 mM to 100 mM. In a specific embodiment, the liquid formulation contains the acetate buffer at a concentration of at least 1 mM, at least 5 mM, at least 10 mM, at least 15 mM, up to 20 mM, up to 15 mM, up to 10 mM, or up to 5 mM. In a specific embodiment, the liquid formulation contains the acetate buffer at a concentration of 5 mM to 15 mM. In a specific embodiment, the liquid formulation contains the acetate buffer at a concentration of about 9.5 mM. In a specific embodiment, the liquid formulation contains the acetate buffer at a concentration of about 10 mM.

[0104] In a specific embodiment, the acetate buffer comprises sodium acetate and acetic acid. In a specific embodiment, the liquid formulation comprises sodium acetate at a concentration of 1 mM to 100 mM and acetic acid at a concentration of 0.1 mM to 20 mM. In a specific embodiment, the liquid formulation comprises sodium acetate at a concentration of 1 mM to 10 mM and acetic acid at a concentration of 0.1 mM to 10 mM. In a specific embodiment, the liquid formulation comprises sodium acetate at a concentration of about 9 mM and acetic acid at a concentration of about 0.5 mM. In a specific embodiment, the liquid formulation comprises sodium acetate at a concentration of about 9 mM and acetic acid at a concentration of about 1 mM. In a specific embodiment, the liquid formulation comprises sodium acetate at a concentration of about 9.1 mM and acetic acid at a concentration of about 0.4 mM. In a specific embodiment, the liquid formulation comprises sodium acetate at a concentration of about 9.1 mM and acetic acid at a concentration of about 0.9 mM. In a specific embodiment, the liquid formulation comprises sodium acetate at a concentration of about 8.7 mM and acetic acid at a concentration of about 1.3 mM.

[0105] In certain embodiments, the liquid formulations disclosed herein do not contain a buffer (e.g., acetate buffer).

[0106] 5.2.5 Viscosity

[0107] The formulation disclosed herein advantageously provides a high concentration of risankizumab of 180 mg / ml having an acceptable viscosity, which makes subcutaneous injection of the formulation feasible.

[0108] In specific embodiments, the viscosity of the formulation disclosed herein is 20.0 mPa.s or less at 20°C or less. In specific embodiments, the viscosity of the formulation disclosed herein is 19.0 mPa.s or less at 20°C or less, 18.0 mPa.s or less at 20°C or less, 17.0 mPa.s or less at 20°C or less, 16.0 mPa.s or less at 20°C or less, 15.0 mPa.s or less at 20°C or less at 20°C or less at 20°C or 14.0 mPa.s or less at 20°C or less at 20°C. In specific embodiments, the viscosity of the formulation disclosed herein is 15.0 mPa.s to 20.0 mPa.s at 20°C. In specific embodiments, the viscosity of the formulation disclosed herein is 14.0 mPa.s to 20.0 mPa.s at 20°C.

[0109] In a specific embodiment, the viscosity of the formulation disclosed herein is about 20.0 mPa.s at 20°C. In a specific embodiment, the viscosity of the formulation disclosed herein is about 19.0 mPa.s at 20°C. In a specific embodiment, the viscosity of the formulation disclosed herein is about 18.0 mPa.s at 20°C. In a specific embodiment, the viscosity of the formulation disclosed herein is about 17.0 mPa.s at 20°C. In a specific embodiment, the viscosity of the formulation disclosed herein is about 16.0 mPa.s at 20°C. In a specific embodiment, the viscosity of the formulation disclosed herein is about 15.0 mPa.s at 20°C. In a specific embodiment, the viscosity of the formulation disclosed herein is about 14.0 mPa.s at 20°C.

[0110] In a specific embodiment, the viscosity of the formulation disclosed herein is about 17.6 mPa.s at 20°C. In a specific embodiment, the viscosity of the formulation disclosed herein is about 18.7 mPa.s at 20°C. In a specific embodiment, the viscosity of the formulation disclosed herein is about 18.9 mPa.s at 20°C. In a specific embodiment, the viscosity of the formulation disclosed herein is about 19.8 mPa.s at 20°C. In a specific embodiment, the viscosity of the formulation disclosed herein is about 14.7 mPa.s at 20°C. In a specific embodiment, the viscosity of the formulation disclosed herein is about 18.6 mPa.s at 20°C.

[0111] Methods for measuring viscosity are known in the technical field and may utilize, for example, rotational rheometers, capillary viscometers (e.g., Ostwald-viscosometer, Ubbelohde viscometer, Cannon-Fenske-viscosometer), falling ball viscometers (e.g., Gilmont falling ball viscometer, Hakke falling ball viscometer, Brookfield falling ball viscometer), bubble viscometers (e.g., Cole Parmer), cone and plate viscometers (Brookfield), and liquid handling systems [e.g., ALP (Liquid Characterization) Hamilton system, Opentrons (OT-2) system], but are not limited thereto.

[0112] In certain embodiments, the viscosity of the formulation disclosed herein is measured by an Alternating Liquid Profiling (ALP) method. In certain embodiments, the viscosity of the formulation disclosed herein is measured by a falling ball viscometer.

[0113] The ALP method can be performed on a liquid handling system acting as a high-throughput viscometer. Viscosity measurements can be evaluated using the liquid handling system by analyzing the mass flow behavior and pressure profile through a pipette tip during transient flow, and by comparing either the dispensed weight or pressure reading with a calibration curve. An exemplary protocol for using the ALP method to measure viscosity is described in the literature [Deshmukh et al. , ACS Comb Sci. (2016) Jul 11;18(7):405-14] and [Soh et al. , Digital Discovery

[2023] 2, 481-488] is disclosed, and the contents of each of these are incorporated by reference in their entirety.

[0114] A falling ball viscometer can measure the time required for a spherical ball to fall a defined distance under gravity through a tube filled with a tested formulation. Measurements are collected under temperature-controlled conditions, and the results are provided as dynamic viscosity using milli-Pascal seconds [mPa·s], an internationally standardized absolute unit. An exemplary protocol for using a falling ball viscometer to measure viscosity is described in the literature [Uribe and Sampedro, Biol. Proced. Online (2003) 5, 108-115] and [Allmendinger et al. , CHALLENGES IN PROTEIN PRODUCT DEVELOPMENT. AAPS ADVANCES IN THE PHARMACEUTICAL SCIENCES SERIES, (2018) vol 38. Springer, Cham] is disclosed, and the entire contents of each of these are incorporated by reference.

[0115] 5.2.6 Osmotic Concentration and pH

[0116] In specific embodiments, the liquid formulation disclosed herein has an osmolarity of 300 mOsm / kg to 400 mOsm / kg. In specific embodiments, the liquid formulation disclosed herein has an osmolarity of at least 300 mOsm / kg, at least 325 mOsm / kg, at least 350 mOsm / kg, at least 375 mOsm / kg, up to 400 mOsm / kg, up to 375 mOsm / kg, up to 350 mOsm / kg, or up to 325 mOsm / kg. In specific embodiments, the liquid formulation disclosed herein has an osmolarity of 320 mOsm / kg to 350 mOsm / kg. In specific embodiments, the liquid formulations disclosed herein have an osmotic concentration of about 325 mOsm / kg, about 329 mOsm / kg, about 339 mOsm / kg, about 338 mOsm / kg, about 346 mOsm / kg, or about 350 mOsm / kg.

[0117] In certain embodiments, the liquid formulation disclosed herein has a pH of 5.0 to 7.0. In certain embodiments, the liquid formulation disclosed herein has a pH of 5.0 to 6.5.

[0118] In specific embodiments, the liquid formulation disclosed herein has a pH of at least 6.5, at least 6.4, at least 6.3, at least 6.2, at least 6.1, at least 6.0, at least 5.9, at least 5.8, at least 5.7, at least 5.6, at least 5.5, at least 6.0, at least 5.9, at least 5.8, at least 5.7, at least 5.6, at least 5.5, at least 5.4, at least 5.3, at least 5.2, at least 5.1, or at least 5.0.

[0119] In specific embodiments, the liquid formulation disclosed herein has a pH of 5.0 to 6.5, 5.0 to 6.5, 5.0 to 6.1, 5.0 to 5.8, 5.0 to 5.3, 5.4 to 6.5, 5.4 to 6.1, 5.4 to 5.8, 5.8 to 6.5, 5.8 to 6.3, or 6.0 to 6.4.

[0120] In certain embodiments, the liquid formulation disclosed herein has a pH of 5.1 to 5.3. In certain embodiments, the liquid formulation disclosed herein has a pH of about 5.2.

[0121] In certain embodiments, the liquid formulation disclosed herein has a pH of 5.4 to 5.6. In certain embodiments, the liquid formulation disclosed herein has a pH of about 5.5.

[0122] In certain embodiments, the liquid formulation disclosed herein has a pH of 5.6 to 5.8. In certain embodiments, the liquid formulation disclosed herein has a pH of about 5.7.

[0123] In certain embodiments, the liquid formulation disclosed herein has a pH of 6.1 to 6.3. In certain embodiments, the liquid formulation disclosed herein has a pH of about 6.2.

[0124] 5.2.7 Exemplary liquid formulations

[0125] In a specific embodiment, the liquid formulation disclosed herein comprises risankizumab at a concentration of about 180 mg / ml, (b) a surfactant (e.g., the surfactant of Section 5.2.1), (c) a buffer (e.g., the buffer of Section 5.2.4), and (d) an isotonic agent (e.g., the isotonic agent of Section 5.2.2), wherein the liquid formulation has a pH of about 5.5, about 5.7, about 5.2, or about 6.2. In a specific embodiment, the liquid formulation is a liquid aqueous pharmaceutical formulation.

[0126] In a specific embodiment, the liquid formulation disclosed herein comprises risankizumab at a concentration of about 180 mg / ml, (b) PS20, (c) acetate buffer, and (d) trehalose, wherein the liquid formulation has a pH of about 5.5. In a specific embodiment, the liquid formulation is a liquid aqueous pharmaceutical formulation.

[0127] In a specific embodiment, the liquid formulation disclosed herein comprises (a) risankizumab at a concentration of about 180 mg / ml, (b) PS20, (c) acetate buffer, and (d) trehalose, wherein the liquid formulation has a pH of about 5.7. In a specific embodiment, the liquid formulation is a liquid aqueous pharmaceutical formulation.

[0128] In a specific embodiment, the liquid formulation disclosed herein comprises (a) risankizumab at a concentration of about 180 mg / ml, (b) PS20, (c) acetate buffer, and (d) trehalose, wherein the liquid formulation has a pH of about 5.2. In a specific embodiment, the liquid formulation is a liquid aqueous pharmaceutical formulation.

[0129] In a specific embodiment, the liquid formulation disclosed herein comprises (a) risankizumab at a concentration of about 180 mg / ml, (b) PS20, (c) acetate buffer, and (d) trehalose, wherein the liquid formulation has a pH of about 6.2. In a specific embodiment, the liquid formulation is a liquid aqueous pharmaceutical formulation.

[0130] In certain embodiments, the liquid formulation disclosed herein comprises (a) risankizumab at a concentration of about 180 mg / ml, (b) PS20 at a concentration of about 0.2 mg / ml, (c) acetate buffer at a concentration of about 9.5 mM, and (d) trehalose at a concentration of about 185 mM, wherein the liquid formulation has a pH of 5.2 to 6.2, optionally a pH of about 5.2, about 5.5, about 5.7, or about 6.2. In certain embodiments, the liquid formulation is a liquid aqueous pharmaceutical formulation.

[0131] In certain embodiments, the liquid formulation disclosed herein comprises (a) risankizumab at a concentration of about 180 mg / ml, (b) PS20 at a concentration of about 0.2 mg / ml, (c) sodium acetate at a concentration of about 9.1 mM and acetic acid at a concentration of about 0.4 mM, and (d) trehalose at a concentration of about 185 mM, wherein the liquid formulation has a pH of 5.2 to 6.2, optionally a pH of about 5.2, about 5.5, about 5.7, or about 6.2. In certain embodiments, the liquid formulation is a liquid aqueous pharmaceutical formulation.

[0132] In a specific embodiment, the liquid formulation disclosed herein comprises (a) risankizumab at a concentration of about 180 mg / ml, (b) PS20 at a concentration of about 0.2 mg / ml, (c) acetate buffer at a concentration of about 9.5 mM, and (d) trehalose at a concentration of about 185 mM, wherein the liquid formulation has a pH of about 5.7. In a specific embodiment, the liquid formulation is a liquid aqueous pharmaceutical formulation.

[0133] In a specific embodiment, the liquid formulation disclosed herein comprises (a) risankizumab at a concentration of about 180 mg / ml, (b) PS20 at a concentration of about 0.2 mg / ml, (c) sodium acetate at a concentration of about 9.1 mM and acetic acid at a concentration of about 0.4 mM, and (d) trehalose at a concentration of about 185 mM, wherein the liquid formulation has a pH of about 5.7. In a specific embodiment, the liquid formulation is a liquid aqueous pharmaceutical formulation.

[0134] In a specific embodiment, the liquid formulation disclosed herein comprises (a) risankizumab at a concentration of about 180 mg / ml, (b) PS20 at a concentration of about 0.2 mg / ml, (c) acetate buffer at a concentration of about 9.5 mM, and (d) trehalose at a concentration of about 185 mM, wherein the liquid formulation has a pH of about 5.5. In a specific embodiment, the liquid formulation is a liquid aqueous pharmaceutical formulation.

[0135] In a specific embodiment, the liquid formulation disclosed herein comprises (a) risankizumab at a concentration of about 180 mg / ml, (b) PS20 at a concentration of about 0.2 mg / ml, (c) sodium acetate at a concentration of about 9.1 mM and acetic acid at a concentration of about 0.4 mM, and (d) trehalose at a concentration of about 185 mM, wherein the liquid formulation has a pH of about 5.5. In a specific embodiment, the liquid formulation is a liquid aqueous pharmaceutical formulation.

[0136] In certain embodiments, the liquid formulation disclosed herein comprises (a) risankizumab at a concentration of about 180 mg / ml, (b) PS20 at a concentration of about 0.2 mg / ml, (c) acetate buffer at a concentration of about 10 mM, and (d) trehalose at a concentration of about 185 mM, wherein the liquid formulation has a pH of 5.2 to 6.2, optionally a pH of about 5.2, about 5.5, about 5.7, or about 6.2. In certain embodiments, the liquid formulation is a liquid aqueous pharmaceutical formulation.

[0137] In certain embodiments, the liquid formulation disclosed herein comprises (a) risankizumab at a concentration of about 180 mg / ml, (b) PS20 at a concentration of about 0.2 mg / ml, (c) sodium acetate at a concentration of about 9.1 mM and acetic acid at a concentration of about 0.9 mM, and (d) trehalose at a concentration of about 185 mM, wherein the liquid formulation has a pH of 5.2 to 6.2, optionally a pH of about 5.2, about 5.5, about 5.7, or about 6.2. In certain embodiments, the liquid formulation is a liquid aqueous pharmaceutical formulation.

[0138] In certain embodiments, the liquid formulation disclosed herein comprises (a) risankizumab at a concentration of about 180 mg / ml, (b) PS20 at a concentration of about 0.2 mg / ml, (c) sodium acetate at a concentration of about 9.1 mM and acetic acid at a concentration of about 0.9 mM, and (d) trehalose at a concentration of about 185 mM, wherein the liquid formulation has a pH of 5.2 to 6.2, optionally a pH of about 5.2, about 5.5, about 5.7, or about 6.2. In certain embodiments, the liquid formulation is a liquid aqueous pharmaceutical formulation.

[0139] In certain embodiments, the liquid formulation disclosed herein comprises (a) risankizumab at a concentration of about 180 mg / ml, (b) PS20 at a concentration of about 0.2 mg / ml, (c) sodium acetate at a concentration of about 8.7 mM and acetic acid at a concentration of about 1.3 mM, and (d) trehalose at a concentration of about 185 mM, wherein the liquid formulation has a pH of 5.2 to 6.2, optionally a pH of about 5.2, about 5.5, about 5.7, or about 6.2. In certain embodiments, the liquid formulation is a liquid aqueous pharmaceutical formulation.

[0140] In a specific embodiment, the liquid formulation disclosed herein comprises (a) risankizumab at a concentration of about 180 mg / ml, (b) PS20 at a concentration of about 0.2 mg / ml, (c) acetate buffer at a concentration of about 10 mM, and (d) trehalose at a concentration of about 185 mM, wherein the liquid formulation has a pH of about 5.5. In a specific embodiment, the liquid formulation is a liquid aqueous pharmaceutical formulation.

[0141] In a specific embodiment, the liquid formulation disclosed herein comprises (a) risankizumab at a concentration of about 180 mg / ml, (b) PS20 at a concentration of about 0.2 mg / ml, (c) sodium acetate at a concentration of about 8.7 mM and acetic acid at a concentration of about 1.3 mM, and (d) trehalose at a concentration of about 185 mM, wherein the liquid formulation has a pH of about 5.5. In a specific embodiment, the liquid formulation is a liquid aqueous pharmaceutical formulation.

[0142] In a specific embodiment, the liquid formulation disclosed herein comprises (a) risankizumab at a concentration of about 180 mg / ml, (b) PS20 at a concentration of about 0.2 mg / ml, (c) acetate buffer at a concentration of about 10 mM, and (d) trehalose at a concentration of about 185 mM, wherein the liquid formulation has a pH of about 5.7. In a specific embodiment, the liquid formulation is a liquid aqueous pharmaceutical formulation.

[0143] In a specific embodiment, the liquid formulation disclosed herein comprises (a) risankizumab at a concentration of about 180 mg / ml, (b) PS20 at a concentration of about 0.2 mg / ml, (c) sodium acetate at a concentration of about 9.1 mM and acetic acid at a concentration of about 0.9 mM, and (d) trehalose at a concentration of about 185 mM, wherein the liquid formulation has a pH of about 5.7. In a specific embodiment, the liquid formulation is a liquid aqueous pharmaceutical formulation.

[0144] In a specific embodiment, the liquid formulation disclosed herein comprises (a) risankizumab at a concentration of about 180 mg / ml, (b) PS20 at a concentration of about 0.2 mg / ml, (c) acetate buffer at a concentration of about 10 mM, and (d) trehalose at a concentration of about 185 mM, wherein the liquid formulation has a pH of about 5.2. In a specific embodiment, the liquid formulation is a liquid aqueous pharmaceutical formulation.

[0145] In a specific embodiment, the liquid formulation disclosed herein comprises (a) risankizumab at a concentration of about 180 mg / ml, (b) PS20 at a concentration of about 0.2 mg / ml, (c) sodium acetate at a concentration of about 9.1 mM and acetic acid at a concentration of about 0.9 mM, and (d) trehalose at a concentration of about 185 mM, wherein the liquid formulation has a pH of about 5.2. In a specific embodiment, the liquid formulation is a liquid aqueous pharmaceutical formulation.

[0146] In a specific embodiment, the liquid formulation disclosed herein comprises (a) risankizumab at a concentration of about 180 mg / ml, (b) PS20 at a concentration of about 0.2 mg / ml, (c) acetate buffer at a concentration of about 10 mM, and (d) trehalose at a concentration of about 185 mM, wherein the liquid formulation has a pH of about 6.2. In a specific embodiment, the liquid formulation is a liquid aqueous pharmaceutical formulation.

[0147] In a specific embodiment, the liquid formulation disclosed herein comprises (a) risankizumab at a concentration of about 180 mg / ml, (b) PS20 at a concentration of about 0.2 mg / ml, (c) sodium acetate at a concentration of about 9.1 mM and acetic acid at a concentration of about 0.9 mM, and (d) trehalose at a concentration of about 185 mM, wherein the liquid formulation has a pH of about 6.2. In a specific embodiment, the liquid formulation is a liquid aqueous pharmaceutical formulation.

[0148] In a specific embodiment, the liquid formulation disclosed herein comprises (a) risankizumab at a concentration of about 180 mg / ml, (b) a surfactant (e.g., the surfactant of Section 5.2.1), and (c) an isotonic agent (e.g., the isotonic agent of Section 5.2.2), wherein the liquid formulation has a pH of about 5.7. In a specific embodiment, the liquid formulation is a liquid aqueous pharmaceutical formulation.

[0149] In a specific embodiment, the liquid formulation disclosed herein comprises (a) risankizumab at a concentration of about 180 mg / ml, (b) PS20, and (c) mannitol, wherein the liquid formulation has a pH of about 5.7. In a specific embodiment, the liquid formulation is a liquid aqueous pharmaceutical formulation.

[0150] In a specific embodiment, the liquid formulation disclosed herein comprises (a) risankizumab at a concentration of about 180 mg / ml, (b) PS20 at a concentration of about 0.2 mg / ml, and (c) mannitol at a concentration of about 190 mM, wherein the liquid formulation has a pH of about 5.7. In a specific embodiment, the liquid formulation is a liquid aqueous pharmaceutical formulation.

[0151] In a specific embodiment, the liquid formulation disclosed herein comprises (a) risankizumab at a concentration of about 180 mg / ml, (b) a surfactant (e.g., the surfactant of Section 5.2.1), and (c) a solubilizer (e.g., the solubilizer of Section 5.2.3), wherein the liquid formulation has a pH of about 5.7. In a specific embodiment, the liquid formulation is a liquid aqueous pharmaceutical formulation.

[0152] In a specific embodiment, the liquid formulation disclosed herein comprises (a) risankizumab at a concentration of about 180 mg / ml, (b) PS20, and (c) glycine, wherein the liquid formulation has a pH of about 5.7. In a specific embodiment, the liquid formulation is a liquid aqueous pharmaceutical formulation.

[0153] In a specific embodiment, the liquid formulation disclosed herein comprises (a) risankizumab at a concentration of about 180 mg / ml, (b) PS20 at a concentration of about 0.2 mg / ml, and (c) glycine at a concentration of about 225 mM, wherein the liquid formulation has a pH of about 5.7. In a specific embodiment, the liquid formulation is a liquid aqueous pharmaceutical formulation.

[0154] In a specific embodiment, the liquid formulation disclosed herein comprises (a) risankizumab at a concentration of about 180 mg / ml, (b) PS20, and (c) proline, wherein the liquid formulation has a pH of about 5.7. In a specific embodiment, the liquid formulation is a liquid aqueous pharmaceutical formulation.

[0155] In a specific embodiment, the liquid formulation disclosed herein comprises (a) risankizumab at a concentration of about 180 mg / ml, (b) PS20 at a concentration of about 0.2 mg / ml, and (c) proline at a concentration of about 225 mM, wherein the liquid formulation has a pH of about 5.7. In a specific embodiment, the liquid formulation is a liquid aqueous pharmaceutical formulation.

[0156] 5.2.8 Particles invisible to the naked eye

[0157] In certain embodiments, the liquid formulation disclosed herein contains risankizumab at a concentration of about 180 mg / ml and low levels of invisible particles (SVP). Controlling the formulation to such low levels of SVP is advantageously increased in the filterability of the antibody formulation and prevents the occurrence of filter-clogging events during the antibody manufacturing process. In certain embodiments, the liquid formulation contains 10,000 or fewer SVPs per ml to ensure a safe manufacturing process.

[0158] SVP is typically a particle with a diameter ranging from 0.1 μm to 100 μm. As used herein, SVP effectively refers to a proteinaceous aggregate. In certain embodiments, SVP has a diameter greater than 2 μm. In certain embodiments, SVP has a diameter greater than 5 μm. In certain embodiments, SVP has a diameter greater than 10 μm. In certain embodiments, SVP has a diameter greater than 25 μm.

[0159] In a specific embodiment, the liquid formulation contains 10,000 or fewer SVPs per ml. In a specific embodiment, the liquid formulation contains 9,000 or fewer SVPs per ml. In a specific embodiment, the liquid formulation contains 8,000 or fewer SVPs per ml. In a specific embodiment, the liquid formulation contains 7,000 or fewer SVPs per ml. In a specific embodiment, the liquid formulation contains 6,000 or fewer SVPs per ml. In a specific embodiment, the liquid formulation contains 5,000 or fewer SVPs per ml. In a specific embodiment, the liquid formulation contains 4,000 or fewer SVPs per ml. In a specific embodiment, the liquid formulation contains 3,000 or fewer SVPs per ml. In a specific embodiment, the liquid formulation contains 2,000 or fewer SVPs per ml. In a specific embodiment, the liquid formulation contains 1,000 or fewer SVPs per ml. In a specific embodiment, the liquid formulation contains 800 or fewer SVPs per ml. In a specific embodiment, the liquid formulation contains 600 or fewer SVPs per ml. In a specific embodiment, the liquid formulation contains 400 or fewer SVPs per ml or 200 or fewer SVPs per ml.

[0160] In a specific embodiment, the liquid formulation contains 10,000 or fewer SVPs per ml, wherein the SVPs have a diameter greater than 2 μm. In a specific embodiment, the liquid formulation contains 9,000 or fewer SVPs per ml, wherein the SVPs have a diameter greater than 2 μm. In a specific embodiment, the liquid formulation contains 8,000 or fewer SVPs per ml, wherein the SVPs have a diameter greater than 2 μm. In a specific embodiment, the liquid formulation contains 7,000 or fewer SVPs per ml, wherein the SVPs have a diameter greater than 2 μm. In a specific embodiment, the liquid formulation contains 6,000 or fewer SVPs per ml, wherein the SVPs have a diameter greater than 2 μm. In a specific embodiment, the liquid formulation contains 5,000 or fewer SVPs per ml, wherein the SVPs have a diameter greater than 2 μm. In a specific embodiment, the formulation contains 4,000 or fewer SVPs per ml, wherein the SVPs have a diameter greater than 2 μm. In a specific embodiment, the liquid formulation contains 3,000 or fewer SVPs per ml, wherein the SVPs have a diameter greater than 2 μm. In a specific embodiment, the liquid formulation contains 2,000 or fewer SVPs per ml, wherein the SVPs have a diameter greater than 2 μm. In a specific embodiment, the liquid formulation contains 1,000 or fewer SVPs per ml, wherein the SVPs have a diameter greater than 2 μm. In a specific embodiment, the liquid formulation contains 800 or fewer SVPs per ml, wherein the SVPs have a diameter greater than 2 μm. In a specific embodiment, the liquid formulation contains 600 or fewer SVPs per ml, wherein the SVPs have a diameter greater than 2 μm. In a specific embodiment, the liquid formulation contains 400 or fewer SVPs per ml, wherein the SVPs have a diameter greater than 2 μm.In a specific embodiment, the liquid formulation contains 200 or fewer SVPs per ml, wherein the SVPs have a diameter greater than 2 μm.

[0161] Any method known in the art may be used in conjunction with the method provided herein to measure the size and concentration of SVP in liquid formulations. In certain embodiments, the size of SVP is measured by micro-flow imaging [MFI, e.g., as disclosed in Example 3 of this application]. In certain embodiments, the concentration of SVP in liquid formulations is measured by MFI. Additional methods that may be used to measure the size and concentration of SVP in liquid formulations include, but are not limited to, resonant mass measurement (RMM), Raman spectroscopy, and nanoparticle tracking analysis (NTA).

[0162] In a specific embodiment, the liquid formulation underwent a freeze-thaw process (F / T). In a specific embodiment, F / T comprises freezing the formulation to about -70 °C and then thawing the formulation to room temperature. In a specific embodiment, the room temperature is 18 °C to 23 °C. In a specific embodiment, the room temperature is about 18 °C, about 19 °C, about 20 °C, about 21 °C, about 21.5 °C, about 22 °C, or about 23 °C.

[0163] In a specific embodiment, F / T comprises freezing the formulation at room temperature (e.g., about 18 °C or about 20 °C) to about -70 °C for 30 hours or less, 25 hours or less, 20 hours or less, 15 hours or less, 10 hours or less, or 5 hours or less. In a specific embodiment, F / T comprises freezing the formulation at room temperature (e.g., about 18 °C or about 20 °C) to about -70 °C for 10 hours or less. In a specific embodiment, F / T comprises freezing the formulation at room temperature (e.g., about 18 °C or about 20 °C) to about -70 °C within about 5 hours.

[0164] In a specific embodiment, F / T comprises thawing the formulation from about -70°C to room temperature (e.g., about 18°C ​​or about 20°C) for 20 hours or less, 15 hours or less, 10 hours or less, or 5 hours or less. In a specific embodiment, F / T comprises thawing the formulation from about -70°C to room temperature (e.g., about 18°C ​​or about 20°C) for 15 hours or less. In a specific embodiment, F / T comprises thawing the formulation from about -70°C to room temperature (e.g., about 18°C ​​or about 20°C) for 10 hours or less. In a specific embodiment, F / T comprises thawing the formulation from about -70°C to room temperature (e.g., about 18°C ​​or about 20°C) within about 11 hours. In a specific embodiment, F / T includes thawing the formulation from about -70°C to room temperature (e.g., about 18°C ​​or about 20°C) within about 9 hours.

[0165] In a specific embodiment, F / T comprises freezing the formulation from room temperature (e.g., about 18 °C or about 20 °C) to about -70 °C for 10 hours or less and thawing the formulation from about -70 °C to room temperature (e.g., about 18 °C or about 20 °C) for 15 hours or less. In a specific embodiment, F / T comprises freezing the formulation from room temperature (e.g., about 18 °C or about 20 °C) to about -70 °C for 10 hours or less and thawing the formulation from about -70 °C to room temperature (e.g., about 18 °C or about 20 °C) for 10 hours or less. In a specific embodiment, F / T comprises freezing the formulation from room temperature (e.g., about 18 °C or about 20 °C) to about -70 °C within about 5 hours and thawing the formulation from about -70 °C to room temperature (e.g., about 18 °C or about 20 °C) within about 11 hours. In a specific embodiment, F / T comprises freezing the formulation from room temperature (e.g., about 18 °C or about 20 °C) to about -70 °C within about 5 hours and thawing the formulation from about -70 °C to room temperature (e.g., about 18 °C or about 20 °C) within about 9 hours.

[0166] In a specific embodiment, the liquid formulation that has undergone F / T contains 2,000 or fewer SVPs per ml, wherein the SVPs have a diameter greater than 2 μm, and F / T comprises freezing the formulation from room temperature (e.g., about 18 °C or about 20 °C) to about -70 °C within about 5 hours and thawing the formulation from about -70 °C to room temperature (e.g., about 18 °C or about 20 °C) within about 9 hours or about 11 hours. In a specific embodiment, the liquid formulation that has undergone F / T contains 1,000 or fewer SVPs per ml, wherein the SVPs have a diameter greater than 2 μm, and F / T comprises freezing the formulation from room temperature (e.g., about 18 °C or about 20 °C) to about -70 °C within about 5 hours and thawing the formulation from about -70 °C to room temperature (e.g., about 18 °C or about 20 °C) within about 9 or 11 hours.

[0167] In a specific embodiment, F / T comprises three or fewer cycles of freezing and thawing the liquid formulation. In a specific embodiment, F / T comprises two or fewer cycles of freezing and thawing the liquid formulation. In a specific embodiment, F / T comprises three or fewer cycles of freezing and thawing the liquid formulation. In a specific embodiment, F / T comprises one or fewer cycles of freezing and thawing the liquid formulation.

[0168] In a specific embodiment, the liquid formulation underwent a mixing process. In a specific embodiment, the power per unit volume (P / V) of the mixing process is 1000 W / m² 3 The following applies. In a specific embodiment, the P / V of the mixing process is 500 W / m² 3 The following applies. In a specific embodiment, the P / V of the mixing process is 500 W / m² 3 Up to 1000 W / m 3In a specific embodiment, the mixing time of the mixing process is 30 minutes or less. In a specific embodiment, the mixing time of the mixing process is 25 minutes or less. In a specific embodiment, the mixing time of the mixing process is 20 minutes or less. In a specific embodiment, the mixing time of the mixing process is 15 minutes or less. In a specific embodiment, the mixing time of the mixing process is 10 minutes or less. In a specific embodiment, the mixing time of the mixing process is 5 minutes to 30 minutes.

[0169] In a specific embodiment, the P / V of the mixing process is 1000 W / m 3 The mixing time of the mixing process is 30 minutes or less. In a specific embodiment, the P / V of the mixing process is 500 W / m² 3 Up to 1000 W / m 3 And the mixing time of the mixing process is 30 minutes or less. In a specific embodiment, the P / V of the mixing process is 500 W / m² 3 The mixing time of the mixing process is 30 minutes or less. In a specific embodiment, the P / V of the mixing process is 1000 W / m² 3 The mixing time of the mixing process is 5 to 30 minutes. In a specific embodiment, the P / V of the mixing process is 500 W / m² 3 Up to 1000 W / m 3 The mixing time of the mixing process is 5 to 30 minutes. In a specific embodiment, the P / V of the mixing process is 500 W / m² 3 The mixing time of the mixing process is 5 to 30 minutes.

[0170] In a specific embodiment, the liquid formulation subjected to the mixing process comprises 4,000 or fewer SVPs per ml, 3,000 or fewer SVPs per ml, or 2,000 or fewer SVPs per ml, wherein the SVPs have a diameter greater than 2 μm, and the P / V of the mixing process is 500 W / m² 3 The mixing time of the mixing process is 30 minutes or less.

[0171] In certain embodiments, the liquid formulation having a low level of SVP disclosed herein further comprises a surfactant (e.g., the surfactant of Section 5.2.1), wherein the liquid formulation has a pH of 5.0 to 6.5. In certain embodiments, the liquid formulation further comprises an isotonic agent (e.g., the isotonic agent of Section 5.2.2), a solubilizer (e.g., the solubilizer of Section 5.2.3), and / or a buffer (e.g., the buffer of Section 5.2.4). In certain embodiments, the viscosity of the liquid formulation is less than 20.0 mPa·s at 20 °C. In certain embodiments, the liquid formulation has an osmotic concentration of 300 mOsmol / kg to 400 mOsmol / kg. In certain embodiments, the liquid formulation is a liquid aqueous pharmaceutical formulation.

[0172] 5.2.9 Additional Advantages

[0173] In a specific embodiment, the liquid formulation disclosed herein comprises risankizumab at a concentration of about 180 mg / ml, wherein the liquid formulation further has one or more of the following features.

[0174] (1) Liquid formulations contain low levels of phospholipase A2 (PLA2) (e.g., less than 250 pg per mg of risankizumab),

[0175] (2) A low percentage (e.g., less than 5.4%) of the total species of risankizumab having N-glycosylation in liquid formulations have a gomannose N-glycan determined by fluorescence detection (HILIC-FL) following 2-aminobenzoamide (2-AB) labeling and hydrophilic interaction liquid chromatography,

[0176] (3) A low percentage (e.g., less than 10.3%) of the total species of risankizumab with N-glycosylation in liquid formulations have a gomannose N-glycan determined by RapiFluor labeling and HILIC-FL,

[0177] (4) A low percentage (e.g., less than 5.3%) of the total species of risankizumab with N-glycosylation in liquid formulations have M5 as determined by 2-AB labeling and HILIC-FL,

[0178] (5) A low percentage of the total species of risankizumab with N-glycosylation (e.g., less than 5.7%) have M5 as determined by RapiFluor labeling and HILIC-FL,

[0179] (6) A low percentage (e.g., less than 2.6%) of the total species of risankizumab with N-glycosylation in liquid formulations have M6 as determined by 2-AB labeling and HILIC-FL,

[0180] (7) A low percentage (e.g., less than 2.5%) of the total species of risankizumab with N-glycosylation in liquid formulations have M6 as determined by RapiFluor labeling and HILIC-FL,

[0181] (8) A low percentage (e.g., less than 2.0%) of the total species of risankizumab with N-glycosylation in liquid formulations have M7 as determined by 2-AB labeling and HILIC-FL,

[0182] (9) A low percentage (e.g., less than 2.1%) of the total species of risankizumab with N-glycosylation in liquid formulations have M7 as determined by RapiFluor labeling and HILIC-FL,

[0183] (10) A high percentage (e.g., greater than 84.4%) of the total species of risankizumab with N-glycosylation in liquid formulations have fucosylated bianteneric oligosaccharides determined by 2-AB labeling and HILIC-FL,

[0184] (11) A high percentage (e.g., greater than 79.5%) of the total species of risankizumab with N-glycosylation in liquid formulations have fucosylated bianteneric oligosaccharides as determined by RapiFluor labeling and HILIC-FL,

[0185] (12) A high percentage (e.g., greater than 2.5%) of the total species of risankizumab having N-glycosylation in liquid formulations have sialylated glycans, where the level of sialylated glycans is selectively determined by RapiFluor labeling and HILIC-FL,

[0186] (13) A low percentage of risankizumab (e.g., less than 1.2%) is nonglycosylated, optionally where the level of nonglycosylated species of risankizumab is determined by tryptic peptide mapping,

[0187] (14) The incidence of post-treatment anti-drug antibodies (ADA) is low (e.g., less than 4.7%) after administration of a single subcutaneous dose of 150 mg of the liquid formulation to human subjects.

[0188] (15) In liquid formulations, a high percentage (e.g., at least 99.1%) of risankizumab species exist as monomers,

[0189] (16) In liquid formulations, a low percentage of risankizumab species (e.g., 0.4% or less) is present as high molecular weight (HMW) species, where HMW species are species with a higher molecular weight than monomers,

[0190] (17) In liquid formulations, a high percentage of risankizumab species (e.g., greater than 97.5%) is present as a major peak measured by capillary gel electrophoresis (CGE-NR) under non-reducing conditions,

[0191] (18) In liquid formulations, a low percentage of risankizumab species (e.g., less than 2.2%) are present as low molecular weight (LMW) species as measured by CGE-NR, where LMW species are species with a shorter retention time than the main peak and / or

[0192] (19) Contains poloxamer 188 (P188). A detailed description of these features can be found below. In certain embodiments, the liquid formulation further comprises components and / or features disclosed in Sections 5.2.1 through 5.2.8, e.g., components having a low level of SVP disclosed in Sections 5.2.1 through 5.2.7 and / or Section 5.2.8.

[0193] (a) Risankizumab formulation with reduced hitchhiker protein

[0194] In a specific embodiment, the liquid formulation disclosed herein comprises risankizumab at a concentration of about 180 mg / ml and an amount of PLA2 of less than 250 pg per mg of risankizumab.

[0195] In a specific embodiment, the liquid formulation disclosed herein comprises risankizumab at a concentration of about 180 mg / ml, 10,000 or fewer SVPs per ml, and less than 250 pg of PLA2 per mg of risankizumab.

[0196] As used herein, the terms 'phospholipase A2' or 'PLA2' refer to a known 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, which represent another class of lipid mediators (see [Murakami and Kudo (2002)] J. Biochem[131:285-292]). At least 16 groups of phospholipase A2 exist. Dennis and colleagues classified them into six groups based on their characteristics: secretory phospholipase A2 (sPLA2 groups I, II, III, V, IX, X, XI, XII, XIII, and XIV), cytoslurry phospholipase A2 (group IV cPLA2), calcium-independent phospholipase A2 (group VI iPLA2), PAF acetylhydrolase (GVII and GVIII PAF-AH PLA2), lysosomal phospholipase A2 (group XV LPLA2), and adipose tissue-specific phospholipase A2 (GXVI AdPLA) (Reference [Shayman and Tesmer (2019) Molecular and Cell Biology of Lipids 1864:932-940]). In certain embodiments, PLA2 according to the present disclosure can catalyze the hydrolysis of surfactants such as polysorbate 20 (PS20). Exemplary PLA2 according to the present disclosure includes, but is not limited to, PLA2G15, PLA2G7, and PLA2G2.

[0197] The term 'PLA2G15' as used herein, also known as 'PLA2 Group XV', refers to a unique member of the PLA2 family (Reference [Shayman et al. (2011) Prog. Lipid Res . 50:1-13]). PLA2G15 is localized to lysosomes and late endosomes within cells, is pH-optimized, and acts as PLA2 (Reference [Abe and Shayman (2007) J. Lipid Res[Hiraoka et al. 48:2255-2263]). The primary structure of PLA2G15 is highly conserved among mice, bovine, and humans. Six exons are present in the PLA2G15 gene. The primary structure of human and mouse PLA2G15 consists of 412 amino acids (407 for the bovine enzyme). The enzyme contains a common sequence including a signal peptide cleavage site and the lipase motif AXSXG, which is characteristic of serine hydrolase. Serine is part of a catalytic triad that also includes aspartic acid and histidine. The amino-terminal 33-amino acid signal peptide is present on mouse and human peptides along with a cleavage site between proline 33 and alanine 34. Additionally, four N-linked glycosylation sites are present in mouse and human proteins (three in bovine proteins) (Reference [Hiraoka et al. (2005) J. Lipid Res . 46:2441-2447]). The structure and function of PLA2G15 are described in the literature [Shayman and Tesmer, Molecular and Cell Biology of Lipids

[2019] 1864:932-940] is additionally described, the contents of which are incorporated herein by reference in their entirety.

[0198] Representative human PLA2G15 cDNA and human PLA2G15 protein sequences are known in the art and are publicly available at 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 is shorter and has a distinct C-terminus compared to isoform 1. Nucleic acid and polypeptide sequences of PLA2G15 homologs in non-human organisms are known, 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 NP_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 Includes NP_001373635.1).Representative sequences of PLA2G15 homologs are presented below.

[0199] Anti-PLA2G15 antibodies suitable for detecting PLA2G15 protein are known in the art, 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, ABIN2580837, ABIN2580838 and ABIN2580836 (available on the World Wide Web antibodies-online.com), antibody catalog numbers sc-376078, sc-529817, sc-543705, sc-522840, sc-376078 AC, sc-376078 HRP, This includes sc-376078 FITC, sc-376078 PE, and sc-376078 AF488 (Santa Cruz Biotechnology, Dallas, TX). Additionally, reagents for detecting PLA2G15 expression are known. A number of clinical tests for PLA2G15 are available from the NIH Genetic Testing Registry (GTR®) (e.g., GTR test ID: GTR000543805.3, provided by Fulgent Clinical Diagnostics Lab (Temple City, CA)).

[0200] Mouse PLA2G15 isoform 1, amino acid sequence (NP_598553.1)

[0201] MDRHLCTCRETQLRSGLLLPLFLFLMMLADLTLPAQRHPPVVLVPGDLGNQLEAKLDKPKVVHYLCSKKTDSYFTLWLNLELLLPVIIDCWIDNIRLVYNRTSRA TQFPDGVDVRVPGFGETFSMEFLDPSKRNVGSYFYTMVESLVGWGYTRGEDVRGAPYDWRRAPNENGPYFLALREMIIEEMYQMYGGPVVLVAHSMGNVYMLYFL QRQPQVWKDKYIHAFVSLGAPWGGVAKTLRVLASGDNNRIPVIGPLKIREQQRSAVSTSWLLPYNHTWSHEKVFVYTPTTNYTLRDYHRFFRDIGFEDGWFMRQ DTEGLVEAMTPPGVELHCLYGTGVPTPNSFYYESFPDRDPKICFGDGDGTVNLESVLQCQAWQSRQEHRVSLQELPGSEHIEMLANATTLAYLKRVLLEP[SEQ ID NO: 11]

[0202] Mouse PLA2G15 isoform 2-amino acid sequence (NP_001344248.1)

[0203] MDRHLCTCRETQLRSGLLLPLFLLMLADLTLPAQRHPPVVLVPGDLGNQLEAKLDKPKVVHYLCSKKTDSYFTLWLNLELLLPVIIDCWIDNIRLVYNRTSRATQFPDGVDVRVPGFGE TFSMEFLDPSKRNVGSYFYTMVESLVGWGYTRGEDVRGAPYDWRRAPTAATSLEGQIYPCLRLTGGALGGRGQDAACPGLRRQQSHSRHWATEDPGTAAICRLYQLATAIQPHLVT[SEQ ID NO: 12]

[0204] Chinese Hamster PLA2G15 Isoform X1 Amino Acid Sequence (XP_003504359.1)

[0205] MDRHHLTCRATQLRSGLLVPLLLLMMLADLALSVQRHPPVVLVPGDLGNQLEAKLDKPKVVHYLCSKRTDSYFTLWLNLELLLPVIIDCWIDNIRLVYNRTSRATQFPDGVDVRVPGFGETFSLEFLDPSKRTVGSYFHTMVESLVGWGYTRGEDLRGAPYDWRRAPNENGPYFLALREMIEEMYQMYGGPVVLVAHSMGNMYTLYFLQRQPQAWKDKYIHAFISLGAPWGGVAKTLRVLASGDNNRIPVIGPLKIREQQRSAVSTSWLLPYNHTWSHDKVFVHTPTTNYTLRDYHQFFQDIRFEDGWFMRQDTEGLVEAMMPPGVELHCLYGTGVPTPDSFYYESFPDRDPKICFGDGDGTVNLESVLQCQAWQSRQEHKVSLQELPGSEHIEMLANATTLAYLKRVLFEP[서열번호 13]

[0206] 중국 햄스터 PLA2G15 이소형 X2 아미노산 서열(XP_027293711.1)

[0207] MDRHHLTCRATQLRSGLLVPLLLLMMLADLALSVQRHPPVVLVPGDLGNQLEAKLDKPKVVHYLCSKRTDSYFTLWLNLELLLPVIIDCWIDNIRLVYNRTSRATQFPDGVDVRVPGFGETFSLEFLDPSKRTVGSYFHTMVESLVGWGYTRGEDLRGAPYDWRRAPTATTGLEGQVYPRLHFTGCALGGRGQDPARPGLRRQQSHPCYWAT[서열번호 14]

[0208] Anti-PLA2G15 antibodies suitable for detecting PLA2G15 protein are known in the art, 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, ABIN2580837, ABIN2580838 and ABIN2580836 (available on the World Wide Web antibodies-online.com), antibody catalog numbers sc-376078, sc-529817, sc-543705, sc-522840, sc-376078 AC, sc-376078 HRP, This includes sc-376078 FITC, sc-376078 PE, and sc-376078 AF488 (Santa Cruz Biotechnology, Dallas, TX). Additionally, reagents for detecting PLA2G15 expression are known. A number of clinical tests for PLA2G15 are available from the NIH Genetic Testing Registry (GTR®) (e.g., GTR test ID: GTR000543805.3, provided by Fulgent Clinical Diagnostics Lab (Temple City, CA)).

[0209] In a specific embodiment, a PLA2 polypeptide molecule in a liquid formulation disclosed herein (inclusive of the term 'PLA2' as used herein) comprises an amino acid sequence having 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 identity over its entire length with any one of SEQ ID NOs 11 to 14. Such a PLA2 polypeptide molecule may have the function of the full-length PLA2 disclosed herein. The PLA2 polypeptide molecule disclosed herein may have or may not have a signal peptide, and / or may include or be composed only of a proprotein, and / or may include or be composed only of a mature protein.

[0210] In a specific embodiment, the liquid formulation disclosed herein contains less than 250 pg of PLA2 per mg of risankizumab. In specific embodiments, the liquid formulations disclosed herein contain less than 240 pg PLA2, less than 220 pg PLA2, less than 200 pg PLA2, less than 180 pg PLA2, less than 160 pg PLA2, less than 140 pg PLA2, less than 120 pg PLA2, less than 100 pg PLA2, less than 90 pg PLA2, less than 80 pg PLA2, less than 70 pg PLA2, less than 60 pg PLA2, less than 50 pg PLA2, less than 40 pg PLA2, less than 30 pg PLA2, less than 25 pg PLA2, less than 20 pg PLA2, less than 15 pg PLA2, less than 10 pg PLA2, less than 9 pg PLA2, less than 8 pg PLA2, less than 7 pg PLA2, 6 pg of risankizumab per mg. PLA2 less than pg, PLA2 less than 5 pg, PLA2 less than 4.4 pg, PLA2 less than 4 pg, PLA2 less than 3 pg, PLA2 less than 2 pg, PLA2 less than 1 pg, PLA2 less than 0.5 pg, PLA2 less than 0.1 pg, PLA2 less than 0.05 pg, or 0.Less than 0.1 pg of PLA2, or, for example, about 200 pg to about 249 pg PLA2, about 160 pg to about 200 pg PLA2, about 120 pg to about 160 pg PLA2, about 100 pg to about 120 pg PLA2, about 80 pg to about 100 pg PLA2, about 60 pg PLA2 to about 80 pg PLA2, about 40 pg to about 60 pg PLA2, about 25 pg to about 40 pg PLA2, about 10 pg to about 25 pg PLA2, about 5 pg to about 10 pg PLA2, about 4 pg PLA2 to about 10 pg PLA2 comprises about 1 pg PLA2 to about 5 pg PLA2, about 1 pg PLA2 to about 4 pg PLA2, about 1 pg PLA2 to about 3 pg PLA2, about 1 pg PLA2 to about 2 pg PLA2, about 0.5 pg PLA2 to about 1 pg PLA2, about 0.1 pg PLA2 to about 0.5 pg PLA2, about 0.05 pg PLA2 to about 0.1 pg PLA2, or any range in between. In certain embodiments, the liquid formulation disclosed herein contains PLA2 in an amount below the detection lower limit of a PLA2 detection test, for example, about 9 pg PLA2 or less per mg of risankizumab or about 4.4 pg PLA2 or less per mg of risankizumab. In a specific embodiment, the liquid formulation disclosed herein contains about 70 pg PLA2 to about 240 pg PLA2 per mg of risankizumab.

[0211] In specific embodiments, the liquid formulations disclosed herein contain, per mg of risankizumab, about 240 pg PLA2, about 220 pg PLA2, about 200 pg PLA2, about 180 pg PLA2, about 160 pg PLA2, about 140 pg PLA2, about 120 pg PLA2, about 100 pg PLA2, about 90 pg PLA2, about 80 pg PLA2, about 70 pg PLA2, about 60 pg PLA2, about 50 pg PLA2, about 40 pg PLA2, about 30 pg PLA2, about 25 pg PLA2, about 20 pg PLA2, about 15 pg PLA2, about 10 pg PLA2, about 9 pg PLA2, about 8 pg PLA2, about 7 pg PLA2, and about 6 It contains PLA2, about 5 pg PLA2, about 4.4 pg PLA2, about 4 pg PLA2, about 3 pg PLA2, about 2 pg PLA2, about 1 pg PLA2, about 0.5 pg PLA2, about 0.1 pg PLA2, about 0.05 pg PLA2, or about 0.01 pg PLA2.

[0212] In specific embodiments, the liquid formulations disclosed herein contain less than 250 pg of PLA2 per mg of risankizumab, but greater than 240 pg, greater than 220 pg, greater than 200 pg, greater than 180 pg, greater than 160 pg, greater than 140 pg, greater than 120 pg, greater than 100 pg, greater than 90 pg, greater than 80 pg, greater than 70 pg, greater than 60 pg, greater than 50 pg, greater than 40 pg, greater than 30 pg, greater than 25 pg, greater than 20 pg, greater than 15 pg, and greater than 10 pg Includes PLA2, PLA2 exceeding 9 pg, PLA2 exceeding 8 pg, PLA2 exceeding 7 pg, PLA2 exceeding 6 pg, PLA2 exceeding 5 pg, PLA2 exceeding 4 pg, PLA2 exceeding 3 pg, PLA2 exceeding 2 pg, PLA2 exceeding 1 pg, PLA2 exceeding 0.5 pg, PLA2 exceeding 0.1 pg, PLA2 exceeding 0.05 pg, or PLA2 exceeding 0.01 pg.

[0213] The PLA2 in the liquid formulation disclosed herein may be PLA2G2, PLA2G15, or a combination thereof. In certain embodiments, the PLA2 in the liquid formulation disclosed herein is PLA2G15. In certain embodiments, the amount of PLA2 in the liquid formulation may be determined using methods known in the art, for example, by mass spectrometry or ELISA. In certain embodiments, the amount of PLA2 in the liquid formulation is determined by ELISA, for example, the ELISA described in Example 12.

[0214] In certain embodiments, PLA2 in the liquid formulation disclosed herein is derived from the CHO cell line.

[0215] (b) Risankizumab formulations having reduced gomannose N-glycan, high levels of fucosylated oligosaccharides, high levels of sialylated glycan, high levels of glycosylation, increased purity and / or reduced immunogenicity

[0216] It is known that N-glycosylation in antibody structure affects the stability, protein folding, structural integrity, and pharmacokinetics of antibody therapeutics. While we do not wish to be bound by any theory, the novel manufacturing process developed herein can generate antibodies with a remarkably favorable N-glycosylation profile that confers improved antibody stability and solubility, among other benefits. For example, it has been reported that N-glycosylation can stabilize the CH2 domain of IgG, whereas deglycosylation can make antibodies less thermally stable and more sensitive to unfolding. Furthermore, deglycosylated antibodies have a greater tendency to agglutinate (e.g., the literature [K. Zheng, C. Bantog, R. Bayer, MAbs, [See 3, 568-576 (2011)]). Accordingly, a high degree of glycosylation or a small percentage of non-glycosylation antibodies in the formulation can contribute to high stability and low aggregation. Consistently, in certain embodiments, the formulations provided herein contain a high percentage of monomers having reduced high molecular weight or low molecular weight antibody species.

[0217] Monoclonal antibodies possess conserved N-linking glycosylation in the Fc portion of position N297 (or Asn297). The sugar chain attached to N297 is referred to as an 'N-linking oligosaccharide' or 'N-linking glycan'. It is known that three major classes of N-linking oligosaccharides exist: the 'gomannose type', the 'hybrid type', and the 'complex type'. The sugar chains of each type are more heterogeneous in terms of their sugar composition. The three types of N-linking oligosaccharides share the same internal core structure of mannose 3 N-acetylglucosamine 2 (i.e., three mannose sugars and two N-acetylglucosamine sugars). The end of the sugar chain attached to the ASN on the antibody is referred to as the 'reducing end' of the sugar chain, and the opposite end is referred to as the 'non-reducing end'. Gomannose-type sugar chains contain additional mannose residues (usually 2 to 6 in vertebrate cells) at the non-reducing ends of the core structure. Complex-type sugar chains contain additional outer sugars such as N-acetylglucosamine (GlcNAc), galactose (Gal), fucose (Fuc), and sialic acid (SA). Hybrid-type sugar chains have one 'arm' similar to the gomannose type (additional mannose residues) and one 'arm' similar to the complex type (additional GlcNAc and other outer sugar residues). Gomannose-type, hybrid-type, and complex-type N-linked oligosaccharides are synthesized sequentially from a common large gomannose precursor during a biosynthetic process called 'N-linked oligosaccharide processing,' which involves multiple enzymes. The precursor oligosaccharides are typically processed in a specific order, first into gomannose-type oligosaccharides, then into hybrid-type oligosaccharides, and finally into complex-type oligosaccharides. Most N-linked oligosaccharides produced in mammalian cells do not maintain a gomannose or hybrid structure but are converted into complex types of oligosaccharides, with the complex type constituting the dominant form in mammals. Literature [AD.Elbein,. CRC Crit. Rev. Biochem See . 16(1), 21-49 (1984)].

[0218] The present disclosure found that, as a result of the improved risankizumab active ingredient manufacturing process disclosed in Example 6, more gomannose-type sugar chains are converted into structurally more complex hybrid and complex-type sugar chains. While not intending to be bound by any theory, gomannose glycans are often associated with early glycoprotein biosynthesis and are usually more susceptible to enzymatic degradation, whereas certain complex-type glycans with branched and diverse structures may provide some protection against enzymatic cleavage and thus be more stable. Complex-type glycans may provide greater stability due to their branched and diverse structures, which provide a protective shield against enzymatic cleavage and degradation. This enhanced stability is particularly advantageous in the context of therapeutic antibodies.

[0219] (i) Risankizumab formulations having reduced gomannose N-glycan

[0220] In a specific embodiment, the liquid formulation disclosed herein comprises risankizumab at a concentration of about 180 mg / ml and 10,000 or fewer SVPs per ml, wherein less than 5.4% of the total risankizumab species having N-glycosylation in the liquid formulation have high mannose N-glycans determined by 2-AB labeling and HILIC-FL.

[0221] In specific embodiments, in the liquid formulations disclosed herein, the total risankizumab species having N-glycosylation is less than 5.3%, less than 5.2%, less than 5.1%, less than 5.0%, less than 4.9%, less than 4.8%, less than 4.7%, less than 4.6%, less than 4.5%, less than 4.4%, less than 4.3%, less than 4.2%, less than 4.1%, less than 4.0%, less than 3.9%, less than 3.8%, less than 3.7%, or, for example, 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 Any range in between, including 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%, has gomannose N-glycans determined by 2-AB labeling and HILIC-FL.

[0222] In specific embodiments, less than 5.4% of the total risankizumab species having N-glycosylation in the liquid formulations disclosed herein, but greater than 5.3%, greater than 5.2%, greater than 5.1%, greater than 5.0%, greater than 4.9%, greater than 4.8%, greater than 4.7%, greater than 4.6%, greater than 4.5%, greater than 4.4%, greater than 4.3%, greater than 4.2%, greater than 4.1%, greater than 4.0%, greater than 3.9%, greater than 3.8%, greater than 3.7%, greater than 3.6%, or greater than 3.5% have gomannose N-glycans determined by 2-AB labeling and HILIC-FL.

[0223] In specific embodiments, 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 risankizumab species having N-glycosylation in the liquid formulations disclosed herein have a gomannose N-glycan determined by 2-AB labeling and HILIC-FL.

[0224] In a specific embodiment, the liquid formulation disclosed herein comprises risankizumab at a concentration of about 180 mg / ml and 10,000 or fewer SVPs per ml, wherein less than 10.3% of the total risankizumab species having N-glycosylation in the liquid formulation have a gomannose N-glycan determined by RapiFluor labeling and HILIC-FL.

[0225] In specific embodiments, in the liquid formulations disclosed herein, the total risankizumab species having N-glycosylation is less than 10.2%, less than 10.1%, less than 10.0%, less than 9.9%, less than 9.8%, less than 9.7%, less than 9.6%, less than 9.5%, less than 9.4%, less than 9.3%, less than 9.2%, less than 9.1%, less than 9.0%, less than 8.9%, less than 8.8%, less than 8.7%, less than 8.6%, less than 8.5%, less than 8.4%, less than 8.3%, less than 8.2%, less than 8.1%, less than 8.0%, less than 7.9%, less than 7.8%, less than 7.7%, less than 7.6%, less than 7.5%, less than 7.4%, less than 7.3%, less than 7.2%, Less than 7.1%, less than 7.0%, less than 6.9%, less than 6.8%, less than 6.7%, less than 6.6%, less than 6.5%, less than 6.4%, less than 6.3%, less than 6.2%, less than 6.1%, less than 6.0%, less than 5.9%, less than 5.8%, less than 5.7%, less than 5.6%, less than 5.5%, less than 5.4%, less than 5.3%, less than 5.2%, less than 5.1%, less than 5.0%, less than 4.9%, less than 4.8%, less than 4.7%, less than 4.6%, less than 4.5% or less than 4.4% or, for example, about 4.3% to about 10.2%, about 4.3% to about 9.0%, about 4.3% to about 8.0%, about Any range in between, including 4.3% to about 7.0%, about 4.3% to about 6.0%, about 4.3% to about 5.0%, about 4.3% to about 4.9%, about 5.0% to about 10.0%, about 5.0% to about 8.0%, about 5.0% to about 6.0%, about 7.0% to about 10.0%, or about 7.0% to about 8.0%, has gomannose N-glycans determined by RapiFluor labeling and HILIC-FL.

[0226] In specific embodiments, in the liquid formulations disclosed herein, less than 10.3% of the total risankizumab species having N-glycosylation, but greater than 10.2%, greater than 10.1%, greater than 10.0%, greater than 9.9%, greater than 9.8%, greater than 9.7%, greater than 9.6%, greater than 9.5%, greater than 9.4%, greater than 9.3%, greater than 9.2%, greater than 9.1%, greater than 9.0%, greater than 8.9%, greater than 8.8%, greater than 8.7%, greater than 8.6%, greater than 8.5%, greater than 8.4%, greater than 8.3%, greater than 8.2%, greater than 8.1%, greater than 8.0%, greater than 7.9%, greater than 7.8%, greater than 7.7%, greater than 7.6%, greater than 7.5%, greater than 7.4%, and 7.3% Exceeding 7.2%, exceeding 7.1%, exceeding 7.0%, exceeding 6.9%, exceeding 6.8%, exceeding 6.7%, exceeding 6.6%, exceeding 6.5%, exceeding 6.4%, exceeding 6.3%, exceeding 6.2%, exceeding 6.1%, exceeding 6.0%, exceeding 5.9%, exceeding 5.8%, exceeding 5.7%, exceeding 5.6%, exceeding 5.5%, exceeding 5.4%, exceeding 5.3%, exceeding 5.2%, exceeding 5.1%, exceeding 5.0%, exceeding 4.9%, exceeding 4.8%, exceeding 4.7%, exceeding 4.6%, exceeding 4.5%, exceeding 4.4%, exceeding 4.3%, or exceeding 4.2% have gomannose N-glycans determined by RapiFluor labeling and HILIC-FL.

[0227] In specific embodiments, the total risankizumab species having N-glycosylation in the liquid formulations disclosed herein account for about 10.2%, about 10.1%, about 10.0%, about 9.9%, about 9.8%, about 9.7%, about 9.6%, about 9.5%, about 9.4%, about 9.3%, about 9.2%, about 9.1%, about 9.0%, about 8.9%, about 8.8%, about 8.7%, about 8.6%, about 8.5%, about 8.4%, about 8.3%, about 8.2%, about 8.1%, about 8.0%, about 7.9%, about 7.8%, about 7.7%, about 7.6%, about 7.5%, about 7.4%, about 7.3%, about 8.2%, about 8.2%, about 7.2%, about 7.9%, about 7.8%, about 7.7%, about 7.6%, about 7.5%, about 7.4%, about 7.3%, about 7.2%, about 7.1%, approximately 7.0%, approximately 6.9%, approximately 6.8%, approximately 6.7%, approximately 6.6%, approximately 6.5%, approximately 6.4%, approximately 6.3%, approximately 6.2%, approximately 6.1%, approximately 6.0%, approximately 5.9%, approximately 5.8%, approximately 5.7%, approximately 5.6%, approximately 5.5%, approximately 5.4%, approximately 5.3%, approximately 5.2%, approximately 5.1%, approximately 5.0%, approximately 4.9%, approximately 4.8%, approximately 4.7%, approximately 4.6%, approximately 4.5%, approximately 4.4%, or approximately 4.3% have gomannose N-glycans determined by RapiFluor labeling and HILIC-FL.

[0228] In a specific embodiment, a risankizumab species having a gomannose N-glycan comprises one or more gomannose N-glycans selected from the group consisting of mannose 5 N-glycan (M5), mannose 6 N-glycan (M6), and mannose 7 N-glycan (M7). In a specific embodiment, a risankizumab species having a gomannose N-glycan comprises M5, M6, and M7.

[0229] In a specific embodiment, the liquid formulation disclosed herein comprises risankizumab at a concentration of about 180 mg / ml and 10,000 or fewer SVPs per ml, wherein less than 5.3% of the total risankizumab species having N-glycosylation in the liquid formulation have M5 determined by 2-AB labeling and HILIC-FL.

[0230] In specific embodiments, in the liquid formulations disclosed herein, less than 5.2%, less than 5.1%, less than 5.0%, less than 4.9%, less than 4.8%, less than 4.7%, less than 4.6%, less than 4.5%, less than 4.4%, less than 4.3%, less than 4.2%, less than 4.1%, less than 4.0%, less than 3.9%, less than 3.8%, less than 3.7%, less than 3.6%, less than 3.5%, less than 3.4%, less than 3.3%, less than 3.2%, less than 3.1%, less than 3.0%, less than 2.9%, or less than 2.8%, or, for example, about 2.7% to about 5.2%, about 3.1% to about 5.2%, about 3.5% to about 5.2%, Any range in between, including 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%, has an M5 determined by 2-AB labeling and HILIC-FL.

[0231] In specific embodiments, in the liquid formulations disclosed herein, less than 5.3% of the total risankizumab species having N-glycosylation, but greater than 5.2%, greater than 5.1%, greater than 5.0%, greater than 4.9%, greater than 4.8%, greater than 4.7%, greater than 4.6%, greater than 4.5%, greater than 4.4%, greater than 4.3%, greater than 4.2%, greater than 4.1%, greater than 4.0%, greater than 3.9%, greater than 3.8%, greater than 3.7%, greater than 3.6%, greater than 3.5%, greater than 3.4%, greater than 3.3%, greater than 3.2%, greater than 3.1%, greater than 3.0%, greater than 2.9%, greater than 2.8%, greater than 2.7%, or greater than 2.6% have M5 determined by 2-AB labeling and HILIC-FL.

[0232] In specific embodiments, 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 risankizumab species having N-glycosylation in the liquid formulations disclosed herein have M5 determined by 2-AB labeling and HILIC-FL.

[0233] In a specific embodiment, the liquid formulation disclosed herein comprises risankizumab at a concentration of about 180 mg / ml and 10,000 or fewer SVPs per ml, wherein less than 5.7% of the total risankizumab species having N-glycosylation in the liquid formulation have M5 as determined by RapiFluor labeling and HILIC-FL.

[0234] In specific embodiments, in the liquid formulations disclosed herein, the total risankizumab species having N-glycosylation is less than 5.6%, less than 5.5%, less than 5.4%, less than 5.3%, less than 5.2%, less than 5.1%, less than 5.0%, less than 4.9%, less than 4.8%, less than 4.7%, less than 4.6%, less than 4.5%, less than 4.4%, less than 4.3%, less than 4.2%, less than 4.1%, less than 4.0%, less than 3.9%, less than 3.8%, less than 3.7%, less than 3.6%, less than 3.5%, less than 3.4%, less than 3.3%, or, for example, about 3.2% to about 5.6%, about 3.2% to about 5.0%, about 3.2% to about 4.5%, about 3.2% to about Any range in between, including 4.0%, about 3.2% to about 3.7%, about 3.5% to about 5.0%, about 3.5% to about 4.5%, about 3.5% to about 4.5%, or about 3.5% to about 4.0%, has an M5 determined by RapiFluor labeling and HILIC-FL.

[0235] In specific embodiments, in the liquid formulations disclosed herein, less than 5.7% of the total risankizumab species having N-glycosylation, but greater than 5.6%, greater than 5.5%, greater than 5.4%, greater than 5.3%, greater than 5.2%, greater than 5.1%, greater than 5.0%, greater than 4.9%, greater than 4.8%, greater than 4.7%, greater than 4.6%, greater than 4.5%, greater than 4.4%, greater than 4.3%, greater than 4.2%, greater than 4.1%, greater than 4.0%, greater than 3.9%, greater than 3.8%, greater than 3.7%, greater than 3.6%, greater than 3.5%, greater than 3.4%, greater than 3.3%, greater than 3.2%, or greater than 3.1% have an M5 determined by RapiFluor labeling and HILIC-FL.

[0236] In specific embodiments, 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%, about 5.2%, about 5.3%, about 5.4%, about 5.5%, or about 5.6% of the total risankizumab species having N-glycosylation in the liquid formulations disclosed herein have an M5 determined by RapiFluor labeling and HILIC-FL.

[0237] In a specific embodiment, the liquid formulation disclosed herein comprises risankizumab at a concentration of about 180 mg / ml and 10,000 or fewer SVPs per ml, wherein less than 2.6% of the total risankizumab species having N-glycosylation in the liquid formulation have M6 as determined by 2-AB labeling and HILIC-FL.

[0238] In specific embodiments, in the liquid formulations disclosed herein, the total risankizumab species having N-glycosylation is less than 2.5%, less than 2.4%, less than 2.3%, less than 2.2%, less than 2.1%, less than 2.0%, less than 1.9%, less than 1.8%, less than 1.7%, less than 1.6%, less than 1.5%, less than 1.4%, less than 1.3%, less than 1.2%, less than 1.1%, less than 1.0%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, or less than 0.5%, or, for example, 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 Any range in between, including 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%, has M6 determined by 2-AB labeling and HILIC-FL.

[0239] In specific embodiments, in the liquid formulations disclosed herein, less than 2.6% of the total risankizumab species having N-glycosylation, but greater than 2.5%, greater than 2.4%, greater than 2.3%, greater than 2.2%, greater than 2.1%, greater than 2.0%, greater than 1.9%, greater than 1.8%, greater than 1.7%, greater than 1.6%, greater than 1.5%, greater than 1.4%, greater than 1.3%, greater than 1.2%, greater than 1.1%, greater than 1.0%, greater than 0.9%, greater than 0.8%, greater than 0.7%, greater than 0.6%, greater than 0.5%, greater than 0.4%, or greater than 0.3% have M6 determined by 2-AB labeling and HILIC-FL.

[0240] In specific embodiments, 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 risankizumab species having N-glycosylation in the liquid formulations disclosed herein have M6 determined by 2-AB labeling and HILIC-FL.

[0241] In a specific embodiment, the liquid formulation disclosed herein comprises risankizumab at a concentration of about 180 mg / ml and 10,000 or fewer SVPs per ml, wherein less than 2.5% of the total risankizumab species having N-glycosylation in the liquid formulation disclosed herein have M6 as determined by RapiFluor labeling and HILIC-FL.

[0242] In specific embodiments, in the liquid formulations disclosed herein, the total risankizumab species having N-glycosylation is less than 2.4%, less than 2.3%, less than 2.2%, less than 2.1%, less than 2.0%, less than 1.9%, less than 1.8%, less than 1.7%, less than 1.6%, less than 1.5%, less than 1.4%, less than 1.3%, less than 1.2%, less than 1.1%, less than 1.0%, less than 0.9%, less than 0.8%, or less than 0.7%, or, for example, about 0.6% to about 2.4%, about 0.6% to about 2.0%, about 0.6% to about 1.5%, about 0.6% to about 1.0%, about 0.6% to about 0.9%, about 0.6% to about 0.8%, or about 0.6% to about Any range in between, including 0.7%, about 1.0% to about 2.0%, or about 1.0% to about 1.5%, has M6 determined by RapiFluic labeling and HILIC-FL.

[0243] In specific embodiments, less than 2.5% of the total risankizumab species having N-glycosylation in the liquid formulations disclosed herein, but greater than 2.4%, greater than 2.3%, greater than 2.2%, greater than 2.1%, greater than 2.0%, greater than 1.9%, greater than 1.8%, greater than 1.7%, greater than 1.6%, greater than 1.5%, greater than 1.4%, greater than 1.3%, greater than 1.2%, greater than 1.1%, greater than 1.0%, greater than 0.9%, greater than 0.8%, greater than 0.7%, greater than 0.6%, or greater than 0.5% have M6 determined by RapiFluor labeling and HILIC-FL.

[0244] In a specific embodiment, 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%, or about 0.6% of the total risankizumab species having N-glycosylation in the liquid formulation have M6 determined by RapiFluor labeling and HILIC-FL.

[0245] In a specific embodiment, the liquid formulation disclosed herein comprises risankizumab at a concentration of about 180 mg / ml and 10,000 or fewer SVPs per ml, wherein less than 2.0% of the total risankizumab species having N-glycosylation in the liquid formulation have M7 as determined by 2-AB labeling and HILIC-FL.

[0246] In specific embodiments, in the liquid formulations disclosed herein, the total risankizumab species having N-glycosylation is less than 1.9%, less than 1.8%, less than 1.7%, less than 1.6%, less than 1.5%, less than 1.4%, less than 1.3%, less than 1.2%, less than 1.1%, less than 1.0%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, or less than 0.5%, or, for example, 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 Any range in between, including 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%, has M7 determined by 2-AB labeling and HILIC-FL.

[0247] In specific embodiments, less than 2.0% of the total risankizumab species having N-glycosylation in the liquid formulations disclosed herein, but greater than 1.9%, greater than 1.8%, greater than 1.7%, greater than 1.6%, greater than 1.5%, greater than 1.4%, greater than 1.3%, greater than 1.2%, greater than 1.1%, greater than 1.0%, greater than 0.9%, greater than 0.8%, greater than 0.7%, greater than 0.6%, greater than 0.5%, greater than 0.4%, or greater than 0.3% have M7 determined by 2-AB labeling and HILIC-FL.

[0248] In specific embodiments, 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 risankizumab species having N-glycosylation in the liquid formulations disclosed herein have M7 determined by 2-AB labeling and HILIC-FL.

[0249] In a specific embodiment, the liquid formulation disclosed herein comprises risankizumab at a concentration of about 180 mg / ml and 10,000 or fewer SVPs per ml, wherein less than 2.1% of the total risankizumab species having N-glycosylation in the liquid formulation have M7 as determined by RapiFluor labeling and HILIC-FL.

[0250] In specific embodiments, in the liquid formulations disclosed herein, the total risankizumab species having N-glycosylation is less than 2.0%, less than 1.9%, less than 1.8%, less than 1.7%, less than 1.6%, less than 1.5%, less than 1.4%, less than 1.3%, less than 1.2%, less than 1.1%, less than 1.0%, less than 0.9%, less than 0.8%, less than 0.7%, or less than 0.6%, or, for example, about 0.5% to about 2.0%, about 0.5% to about 1.5%, about 0.5% to about 1.0%, about 0.5% to about 0.9%, about 0.5% to about 0.8%, about 0.5% to about 0.7%, about 0.5% to about 0.6%, about 1.0% to about 2.0%, or about Any range in between, including 1.0% to about 1.5%, has M7 determined by RapiFluic labeling and HILIC-FL.

[0251] In specific embodiments, in the liquid formulations disclosed herein, less than 2.1% of the total risankizumab species having N-glycosylation, but greater than 2.0%, greater than 1.9%, greater than 1.8%, greater than 1.7%, greater than 1.6%, greater than 1.5%, greater than 1.4%, greater than 1.3%, greater than 1.2%, greater than 1.1%, greater than 1.0%, greater than 0.9%, greater than 0.8%, greater than 0.7%, greater than 0.6%, greater than 0.5%, or greater than 0.4% have M7 determined by RapiFluor labeling and HILIC-FL.

[0252] In specific embodiments, 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%, or about 0.5% of the total risankizumab species having N-glycosylation in the liquid formulations disclosed herein have M7 determined by RapiFluor labeling and HILIC-FL.

[0253] In a specific embodiment, 2-AB and HILIC-FL are performed as described in Example 14. In a specific embodiment, RapiFluor labeling and HILIC-FL are performed as described in Example 15.

[0254] (ii) Risankizumab formulations having higher levels of fucosylated oligosaccharides

[0255] In a specific embodiment, the liquid formulation disclosed herein comprises risankizumab at a concentration of about 180 mg / ml and no more than 10,000 SVPs per ml, wherein more than 84.4% of the total risankizumab species having N-glycosylation in the liquid formulation have fucosylated bianteneric oligosaccharides determined by 2-AB labeling and HILIC-FL.

[0256] In specific embodiments, in the liquid formulations disclosed herein, greater than 85%, greater than 85.5%, greater than 86%, greater than 86.5%, greater than 87%, greater than 87.5%, greater than 88%, greater than 88.5%, greater than 89%, greater than 89.5%, greater than 90%, or greater than 90.5% of the total risankizumab species having N-glycosylation, or any range between these, have fucosylated bianteneric oligosaccharides determined by 2-AB labeling and HILIC-FL or any range between these.

[0257] In a specific embodiment, about 85% to about 91%, about 86% to about 91%, about 87% to about 91%, about 88% to about 91%, about 89% to about 91%, about 89.5% to about 91%, or about 89.8% to about 90.9% of the total risankizumab species having N-glycosylation in the liquid formulation disclosed herein have fucosylated bianteneric oligosaccharides determined by 2-AB labeling and HILIC-FL. In a specific embodiment, about 85%, about 85.5%, about 86%, about 86.5%, about 87%, about 87.5%, about 88%, about 88.5%, about 89%, about 89.5%, about 89.8%, about 90.0%, about 90.5%, about 90.9%, or about 91% of the total risankizumab species having N-glycosylation have fucosylated bianteneric oligosaccharides determined by 2-AB labeling and HILIC-FL.

[0258] In a specific embodiment, the liquid formulation disclosed herein comprises risankizumab at a concentration of about 180 mg / ml and no more than 10,000 SVPs per ml, wherein more than 79.5% of the total risankizumab species having N-glycosylation in the liquid formulation have fucosylated bianteneric oligosaccharides as determined by RapiFluor labeling and HILIC-FL.

[0259] In specific embodiments, in the liquid formulations disclosed herein, greater than 80%, greater than 81%, greater than 82%, greater than 83%, greater than 84%, greater than 85%, greater than 86%, greater than 87%, greater than 88%, or greater than 88.5% of the total risankizumab species having N-glycosylation have fucosylated bianteneric oligosaccharides determined by RapiFluor labeling and HILIC-FL.

[0260] In a specific embodiment, about 80% to about 89%, about 82% to about 89%, about 84% to about 89%, about 86% to about 89%, about 88% to about 89%, or about 88.0% to about 88.9% of the total risankizumab species having N-glycosylation in the liquid formulation have fucosylated biantenary oligosaccharides determined by RapiFluor labeling and HILIC-FL. In a specific embodiment, 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 risankizumab species having N-glycosylation in the liquid formulation disclosed herein have fucosylated biantenary oligosaccharides determined by RapiFluor labeling and HILIC-FL.

[0261] In a specific embodiment, 2-AB and HILIC-FL are performed as described in Example 14. In a specific embodiment, RapiFluor labeling and HILIC-FL are performed as described in Example 15.

[0262] (iii) Risankizumab formulations having higher levels of sialylated glycans

[0263] In a specific embodiment, the liquid formulation disclosed herein comprises risankizumab at a concentration of about 180 mg / ml and 10,000 or fewer SVPs per ml, wherein more than 2.5% of the total risankizumab species having N-glycosylation in the liquid formulation have sialylated glycans, optionally the level of sialylated glycans is determined by RapiFluor labeling and HILIC-FL.

[0264] In specific embodiments, in the liquid formulations disclosed herein, greater than 2.6%, greater than 2.7%, greater than 2.8%, greater than 2.9%, greater than 3.0%, greater than 3.1%, greater than 3.2%, greater than 3.2%, greater than 3.3%, greater than 3.4%, greater than 3.5%, greater than 3.6%, greater than 3.7%, or greater than 3.8% or any range in between of the total risankizumab species having N-glycosylation has sialylated glycans, and optionally the level of sialylated glycans is determined by RapiFluor labeling and HILIC-FL.

[0265] In a specific embodiment, in the liquid formulation disclosed herein, about 2.6% to about 3.9%, about 3% to about 4%, about 3.5% to about 4.5%, about 3.8% to about 4.2%, or about 3.9% to about 4.1% of the total risankizumab species having N-glycosylation have sialylated glycans, and optionally the level of sialylated glycans is determined by RapiFluor labeling and HILIC-FL.

[0266] In a specific embodiment, in the liquid formulation disclosed herein, about 2.6%, 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%, or about 4.0% of the total risankizumab species having N-glycosylation have sialylated glycans, and optionally the level of sialylated glycans is determined by RapiFluor labeling and HILIC-FL.

[0267] In a specific embodiment, RapiFluor labeling and HILIC-FL are performed as described in Example 15.

[0268] (iv) Risankizumab formulations with higher levels of glycosylation

[0269] In a specific embodiment, the liquid formulation disclosed herein comprises risankizumab at a concentration of about 180 mg / ml, 10,000 or fewer SVPs per ml, and less than 1.2% of nonglycosylated risankizumab species, optionally the level of nonglycosylated risankizumab species is determined by triptic peptide mapping.

[0270] In certain embodiments, the liquid formulations disclosed herein contain less than 1.1%, less than 1.0%, or less than 0.9% of non-glycosylated risankizumab species, optionally the level of non-glycosylated risankizumab species is determined by triptic peptide mapping or any range in between.

[0271] In a specific embodiment, the liquid formulation disclosed herein comprises about 0.8% to about 1.1% or about 0.8% to about 1.0% (e.g., about 0.8%, about 0.9%, about 1.0%, or about 1.1%) of a non-glycosylated risankizumab species.

[0272] In a specific embodiment, non-glycosylated risankizumab is determined by triptic peptide mapping (e.g., triptic peptide mapping analysis described in Example 16).

[0273] (v) Increased purity risankizumab formulation

[0274] In a specific embodiment, the liquid formulation disclosed herein comprises risankizumab at a concentration of about 180 mg / ml and 10,000 or fewer SVPs per ml, wherein at least 99.1% of the risankizumab species are present as monomers, optionally the monomer levels are determined by ultra-performance size exclusion chromatography (UP-SEC).

[0275] In a specific embodiment, in the liquid formulation disclosed herein, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, or at least 99.7% of the risankizumab species, or any range in between, such as, for example, 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%, is present as a monomer, and optionally, the level of the monomer is determined by UP-SEC.

[0276] In a specific embodiment, in the liquid formulation disclosed herein, about 99.2% to about 99.7%, about 99.3% to about 99.7%, or about 99.4% to about 99.6% of the risankizumab species (e.g., about 99.2%, about 99.3%, about 99.4%, about 99.5%, about 99.6%, or about 99.7%) is present as a monomer, and optionally, the level of the monomer is determined by UP-SEC.

[0277] In a specific embodiment, the liquid formulation disclosed herein comprises risankizumab at a concentration of about 180 mg / ml and 10,000 or fewer SVPs per ml, wherein 0.4% or fewer of the risankizumab species in the liquid formulation are present as high molecular weight (HMW) species, HMW species are species having a molecular weight higher than that of monomers, and optionally, the level of HMW species is determined by UP-SEC.

[0278] In specific embodiments, in the liquid formulations disclosed herein, 0.35% or less, 0.3% or less, 0.25% or less, 0.2% or less, 0.15% or less, or any range in between is present as HMW species, and optionally, the level of HMW species is determined by UP-SEC.

[0279] In a specific embodiment, in the liquid formulation disclosed herein, 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% (e.g., about 0.1%, about 0.2%, about 0.3%, or about 0.4%) of risankizumab species are present as HMW species, and optionally, the level of HMW species is determined by UP-SEC.

[0280] In a specific embodiment, the liquid formulation disclosed herein comprises risankizumab at a concentration of about 180 mg / ml and 10,000 or fewer SVPs per ml, wherein more than 97.5% of the risankizumab species are present as a major peak as measured by CGE-NR.

[0281] In specific embodiments of the liquid formulations disclosed herein, greater than 97.6%, greater than 97.7%, greater than 97.8%, greater than 97.9%, greater than 98.0%, greater than 98.1%, greater than 98.2%, greater than 98.3%, or greater than 98.4%, or any range in between, of the risankizumab species is present as a major peak measured by CGE-NR.

[0282] In specific embodiments, 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 risankizumab species in the liquid formulations disclosed herein (e.g., about 97.6%, about 97.7%, about 97.8%, about 97.9%, about 98.0%, about 98.1%, about 98.2%, about 98.3%, or about 98.4%) is the major as measured by CGE-NR. It exists as a peak.

[0283] In a specific embodiment, the liquid formulation disclosed herein comprises risankizumab at a concentration of about 180 mg / ml and 10,000 or fewer SVPs per ml, wherein less than 2.2% of the risankizumab species in the formulation are present as low molecular weight (LMW) species as measured by CGE-NR, and LMW species are species having a retention time shorter than the major peak.

[0284] In specific embodiments, in the liquid formulations disclosed herein, less than 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 species, or any range between these, is present as the LMW species as measured by CGE-NR.

[0285] In specific embodiments of the liquid formulations disclosed herein, 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% (e.g., about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, or about 2.1%) of risankizumab species are present as LMW species as measured by CGE-NR.

[0286] (vi) Lisankizumab formulations with reduced immunogenicity

[0287] In a specific embodiment, the liquid formulation disclosed herein comprises risankizumab at a concentration of about 180 mg / ml and 10,000 or fewer SVPs per ml, wherein the incidence of anti-drug antibodies (ADA) occurring after treatment following administration of a single subcutaneous 150 mg dose of the risankizumab liquid formulation to a subject (e.g., human subject) is less than 4.7%.

[0288] In a specific embodiment, the incidence of ADA occurring after treatment in a subject (e.g., human subject) after administration of a single subcutaneous 150 mg dose of the risankizumab liquid formulation disclosed herein is less than 4.5%, less than 4.0%, less than 3.5%, less than 3.0%, less than 2.5%, less than 2.0%, less than 1.5%, less than 1.0%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.01%, less than 0.001%, or less than 0.0001%.

[0289] In a specific embodiment, the incidence of ADA occurring after treatment in a subject (e.g., human subject) after administration of a single subcutaneous 150 mg dose of the risankizumab liquid formulation disclosed herein is about 0% to about 4%, about 0% to about 3%, about 0% to about 2%, or about 0% to about 1% (e.g., about 0%, about 1%, about 2%, about 3%, or about 4%).

[0290] In a specific embodiment, the incidence of ADA occurring after treatment is approximately 0.0%. In a specific embodiment, there is no occurrence of ADA occurring after treatment.

[0291] In certain embodiments, the presence of ADA is determined using a validated potency-based cross-linked electrochemiluminescence immunoassay. The validated potency-based cross-linked electrochemiluminescence immunoassay can be performed as described in Example 18.

[0292] (c) Risankizumab formulation containing poloxamer 188

[0293] In one aspect, the present disclosure relates to a liquid formulation comprising (1) about 180 mg / ml risankizumab and (2) poloxamer 188 (P188), optionally the liquid formulation does not comprise polysorbate 20 (PS20) and / or polysorbate 80 (PS80).

[0294] In certain embodiments, the liquid formulations described herein further comprise, for example, an amount of phospholipase A2 (PLA2) exceeding 250 pg per mg of risankizumab.

[0295] In specific embodiments, the liquid formulations described herein contain risankizumab in amounts greater than 260 pg, greater than 270 pg, greater than 280 pg, greater than 290 pg, greater than 300 pg, greater than 310 pg, greater than 320 pg, greater than 330 pg, greater than 340 pg, greater than 350 pg, greater than 360 pg, greater than 380 pg, greater than 400 pg, greater than 450 pg, greater than 500 pg, greater than 550 pg, greater than 600 pg, greater than 650 pg, greater than 700 pg, greater than 750 pg, greater than 800 pg, greater than 900 pg, or greater than 1000 pg per mg of risankizumab, e.g., 250 pg to 1100 pg, 260 pg to 1100 pg, 270 pg to 1100 pg, 280 pg to 1100 pg, 290 pg to 1100 pg, 300 pg to 1100 pg, 310 pg to 1100 pg, 320 pg to 1100 pg, 340 pg to 1100 pg, 360 pg to 1100 pg, 250 pg to 1000 pg, 250 pg to 900 pg, 250 pg to 800 pg, 250 pg to 700 pg, 250 pg to 600 pg, 250 pg to 500 pg, 250 pg to 400 pg, 250 pg to 1030 pg, 290 pg to 1090 It contains an amount of PLA2 in any range between pg, 360 pg to 450 pg, or 310 pg to 920 pg.

[0296] In specific embodiments, the liquid formulations described herein contain an amount of PLA2 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 per mg of risankizumab.

[0297] PLA2 in the liquid formulations described herein may be PLA2G2, PLA2G15, or a combination thereof. In certain embodiments, PLA2 is PLA2G15.

[0298] The amount of PLA2 in the liquid formulations described herein may be determined using methods known in the art, for example, by mass spectrometry or by ELISA. In a specific embodiment, the amount of PLA2 in the liquid formulations described herein is determined by ELISA, for example, using the ELISA method described in Example 9.

[0299] In a specific embodiment, the liquid formulation does not contain PS20. In a specific embodiment, the liquid formulation does not contain PS80. In a specific embodiment, the liquid formulation does not contain PS20 and does not contain PS80.

[0300] (d) Additional intended features

[0301] Reducing hitchhiker protein in risankizumab formulations can favorably increase the stability of the formulation (e.g., reduced particle formation, increased shelf life of risankizumab finished products, etc.). In certain embodiments, particles visible to the naked eye or glittering particles are not observed in the liquid risankizumab formulations described herein over a period of 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) at 2° to 40°C (e.g., 4°C to 35°C, 4°C to 25°C, 4°C to 10°C, 2°C to 8°C, or any temperature within the aforementioned range, e.g., about 2°C, about 4°C, about 5°C, about 8°C, about 25°C, about 40°C, etc.). In certain embodiments, visible particles or glittering particles are not observed in the liquid risankizumab formulations described herein over a period of about 24 months at 4°C.

[0302] In certain embodiments, the liquid risankizumab formulations described herein comprise a surfactant (e.g., PS20) having increased stability. The stability of the surfactant in the liquid risankizumab formulations described herein can be evaluated by directly measuring the amount of surfactant in the liquid risankizumab formulation after storage at a specific 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 certain period (e.g., 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, 30 months, 36 months, etc.). Alternatively, the stability of the surfactant in the liquid risankizumab formulations described herein can be evaluated by measuring the amount of surfactant degradation products (e.g., amount of free fatty acids) in the risankizumab formulations after storage at a specific 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 certain period (e.g., 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, 30 months, 36 months, etc.). In a specific embodiment, the storage time 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 months or more, or any range in between, including, for example, 3 months to 36 months, 12 months to 24 months, etc.

[0303] In a specific embodiment, the liquid risankizumab formulation described herein comprises, for example, PS20 at a concentration of 0.20 mg / mL, and the stability of PS20 in this liquid risankizumab formulation is increased.

[0304] In a specific embodiment, the stability of PS20 is evaluated by directly measuring the amount of PS20 in the risankizumab formulation after storage at a specific 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 certain period (e.g., 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, 30 months, 36 months, etc.). The amount of PS20 may be measured using any method known in the art, for example, using a High Performance Liquid Chromatography Charged Aerosol Detector (HPLC-CAD), e.g., the HPLC-CAD described in Example 13. In a specific embodiment, the storage 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 months or more, or any range in between, including, for example, 3 months to 36 months, 12 months to 24 months, etc.

[0305] In a specific embodiment, at least 80% of the initial amount of PS20 (e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or about 100%) is retained after storage at 5°C for 6 months. In a specific embodiment, at least 70% of the initial amount of PS20 (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%) is retained after storage at 5°C for 24 months. In a specific embodiment, at least 60% of the initial amount of PS20 (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%) is retained after storage at 25°C for 6 months. In a specific embodiment, at least 40% of the initial amount of PS20 (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%) is retained after storage at 40°C for 6 months.

[0306] In certain embodiments, the liquid risankizumab formulations described herein contain PS80, and the stability of PS80 in these liquid risankizumab formulations is increased.

[0307] In a specific embodiment, the stability of PS80 is evaluated by directly measuring the amount of PS80 in the risankizumab composition after storage at a specific 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 certain period (e.g., 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, 30 months, 36 months, etc.). The amount of PS80 may be measured using any method known in the art, for example, using HPLC-CAD, e.g., the HPLC-CAD described in Example 13. In a specific embodiment, the 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 months or more, or any range in between, for example, 3 months to 36 months, 12 months to 24 months, etc.

[0308] For example, in a specific embodiment, at least 80% of the initial amount of PS80 (e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or about 100%) is retained after storage at 5°C for 6 months. In a specific embodiment, at least 60% of the initial amount of PS80 (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%) is retained after storage at 25°C for 6 months. In a specific embodiment, at least 60% of the initial amount of PS80 (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%) is retained after storage at 40°C for 6 months.

[0309] In some embodiments, the liquid risankizumab formulations described herein contain poloxamer 188 (P188). In certain embodiments, the P188-containing liquid risankizumab formulations do not contain PS20 and / or PS80. In certain embodiments, the stability of P188 in the liquid risankizumab formulations is higher or more constant compared to the stability of PS20 or PS80 in PS20 or PS80-containing liquid risankizumab formulations over time (in some cases).

[0310] In a specific embodiment, the stability of P188 is evaluated by directly measuring the amount of P188 in the liquid risankizumab formulation after storage at a specific 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 certain period (e.g., 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, 30 months, 36 months, etc.). The amount of P188 may be measured using any method known in the art, for example, using the Pluronic F-68 colorimetric assay, e.g., the Pluronic F-68 colorimetric assay described in Example 19. In a specific embodiment, the 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 months or more, or any range in between, for example, 3 months to 36 months, 12 months to 24 months, 3 months to 6 months, etc.

[0311] For example, in a specific embodiment, at least 85% of the initial amount of P188 (e.g., at least 90%, at least 95%, at least 98%, at least 99%, or about 100%) is retained after storage at 5°C for 3 months. In a specific embodiment, at least 65% of the initial amount of P188 (e.g., 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%) is retained after storage at 25°C for 3 months. In a specific embodiment, at least 60% of the initial amount of P188 (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%) is retained after storage at 40°C for 3 months.

[0312] For example, in a specific embodiment, at least 80% of the initial amount of P188 (e.g., at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or about 100%) is retained after storage at 5°C for 6 months. In a specific embodiment, at least 60% of the initial amount of P188 (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%) is retained after storage at 25°C for 6 months. In a specific embodiment, at least 60% of the initial amount of P188 (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%) is retained after storage at 40°C for 6 months.

[0313] The amino acid sequence of risankizumab is provided in the table below.

[0314]

[0315] According to USAN, risankizumab has the following chemical names.

[0316] 1. Immunoglobulin G1, anti-(human interleukin 23 subunit p19)(human-musculus( Mus musculus ) (Reprint), Human-Mus Musculus κ- Disulfide of the chain, dimer

[0317] 2. Immunoglobulin G1-kappa, anti-(human interleukin-23 subunit alpha (IL-23-A, interleukin-23 subunit p19, IL-23p19)); humanized monoclonal antibody; γ1 heavy chain (1-449)[humanized VH(Homo sapiens( 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')-kappa light chain (1'-214') with disulfide [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'')-bisdisulfide.

[0318] According to INN (see literature [WHO Drug Information, 29 (2), 254-255]), risankizumab has the following chemical name.

[0319] Immunoglobulin G1-kappa, anti-[Homo sapiens IL23A (interleukin 23 subunit alpha, IL-23A, IL23 subunit p19 IL23p19)], humanized monoclonal antibody; Gamma 1 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), CH2 L1.3>A(237), L1.2>A(238)(234-343), CH3(344-448), CHS K2>del(449))(121-449)],(223-214')-Disulfide with 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')]; dimer(229-229":232-232")-bisdisulfide.

[0320] 5.3 Treatment Methods

[0321] The present disclosure further provides a method for treating a disease, comprising administering the liquid formulation disclosed herein to a subject in need. In certain embodiments, the disease is an autoimmune disease. In certain embodiments, the disease is an inflammatory disease.

[0322] In a specific embodiment, the method is intended to treat Crohn's disease. In a specific embodiment, the method is intended to treat moderate to severe active Crohn's disease. In a specific embodiment, the method is intended to treat moderate to severe active Crohn's disease in adults. In a specific embodiment, the method is intended to induce remission of Crohn's disease.

[0323] In a specific embodiment, the method is intended to treat psoriasis. In a specific embodiment, the method is intended to treat psoriatic arthritis. In a specific embodiment, the method is intended to treat active psoriatic arthritis in adults. In a specific embodiment, the method is intended to treat plaque psoriasis. In a specific embodiment, the method is intended to treat moderate to severe plaque psoriasis. In a specific embodiment, the method is intended to treat moderate to severe plaque psoriasis in adults that is a candidate for systemic therapy or phototherapy.

[0324] 5.4 Method to reduce the formation of invisible particles (SVP)

[0325] The present disclosure further provides a method for reducing the formation of SVP in a liquid formulation (e.g., the liquid formulation of Section 5.2) comprising risankizumab at a concentration of about 180 mg / ml. In certain embodiments, the method comprises the freeze-thaw process (F / T) disclosed herein. In certain embodiments, the method comprises the mixing process disclosed herein. In certain embodiments, the method comprises the F / T and mixing process disclosed herein.

[0326] In a specific embodiment, F / T comprises freezing the formulation to about -70 °C and then thawing the formulation to room temperature. In a specific embodiment, the room temperature is 18 °C to 23 °C. In a specific embodiment, the room temperature is about 18 °C, about 19 °C, about 20 °C, about 21 °C, about 21.5 °C, about 22 °C, or about 23 °C.

[0327] In a specific embodiment, F / T comprises freezing the formulation at room temperature (e.g., about 18 °C or about 20 °C) to about -70 °C for 30 hours or less, 25 hours or less, 20 hours or less, 15 hours or less, 10 hours or less, or 5 hours or less. In a specific embodiment, F / T comprises freezing the formulation at room temperature (e.g., about 18 °C or about 20 °C) to about -70 °C for 10 hours or less. In a specific embodiment, F / T comprises freezing the formulation at room temperature (e.g., about 18 °C or about 20 °C) to about -70 °C within about 5 hours.

[0328] In a specific embodiment, F / T comprises thawing the formulation from about -70°C to room temperature (e.g., about 18°C ​​or about 20°C) for 20 hours or less, 15 hours or less, 10 hours or less, or 5 hours or less. In a specific embodiment, F / T comprises thawing the formulation from about -70°C to room temperature (e.g., about 18°C ​​or about 20°C) for 15 hours or less. In a specific embodiment, F / T comprises thawing the formulation from about -70°C to room temperature (e.g., about 18°C ​​or about 20°C) for 10 hours or less. In a specific embodiment, F / T comprises thawing the formulation from about -70°C to room temperature (e.g., about 18°C ​​or about 20°C) within about 11 hours. In a specific embodiment, F / T includes thawing the formulation from about -70°C to room temperature (e.g., about 18°C ​​or about 20°C) within about 9 hours.

[0329] In a specific embodiment, F / T comprises freezing the formulation from room temperature (e.g., about 18 °C or about 20 °C) to about -70 °C for 10 hours or less and thawing the formulation from about -70 °C to room temperature (e.g., about 18 °C or about 20 °C) for 15 hours or less. In a specific embodiment, F / T comprises freezing the formulation from room temperature (e.g., about 18 °C or about 20 °C) to about -70 °C for 10 hours or less and thawing the formulation from about -70 °C to room temperature (e.g., about 18 °C or about 20 °C) for 10 hours or less. In a specific embodiment, F / T comprises freezing the formulation from room temperature (e.g., about 18 °C or about 20 °C) to about -70 °C within about 5 hours and thawing the formulation from about -70 °C to room temperature (e.g., about 18 °C or about 20 °C) within about 11 hours. In a specific embodiment, F / T comprises freezing the formulation from room temperature (e.g., about 18 °C or about 20 °C) to about -70 °C within about 5 hours and thawing the formulation from about -70 °C to room temperature (e.g., about 18 °C or about 20 °C) within about 9 hours.

[0330] In a specific embodiment, F / T comprises three or fewer cycles of freezing and thawing the liquid formulation. In a specific embodiment, F / T comprises two or fewer cycles of freezing and thawing the liquid formulation. In a specific embodiment, F / T comprises three or fewer cycles of freezing and thawing the liquid formulation. In a specific embodiment, F / T comprises one or fewer cycles of freezing and thawing the liquid formulation.

[0331] In a specific embodiment, the power per unit volume (P / V) of the mixing process is 1000 W / m² 3 The following applies. In a specific embodiment, the P / V of the mixing process is 500 W / m² 3 The following applies. In a specific embodiment, the P / V of the mixing process is 500 W / m² 3 Up to 1000 W / m 3In a specific embodiment, the mixing time of the mixing process is 30 minutes or less. In a specific embodiment, the mixing time of the mixing process is 25 minutes or less. In a specific embodiment, the mixing time of the mixing process is 20 minutes or less. In a specific embodiment, the mixing time of the mixing process is 15 minutes or less. In a specific embodiment, the mixing time of the mixing process is 10 minutes or less. In a specific embodiment, the mixing time of the mixing process is 5 minutes to 30 minutes.

[0332] In a specific embodiment, the P / V of the mixing process is 1000 W / m 3 The mixing time of the mixing process is 30 minutes or less. In a specific embodiment, the P / V of the mixing process is 500 W / m² 3 Up to 1000 W / m 3 And the mixing time of the mixing process is 30 minutes or less. In a specific embodiment, the P / V of the mixing process is 500 W / m² 3 The mixing time of the mixing process is 30 minutes or less. In a specific embodiment, the P / V of the mixing process is 1000 W / m² 3 The mixing time of the mixing process is 5 to 30 minutes. In a specific embodiment, the P / V of the mixing process is 500 W / m² 3 Up to 1000 W / m 3 The mixing time of the mixing process is 5 to 30 minutes. In a specific embodiment, the P / V of the mixing process is 500 W / m² 3 The mixing time of the mixing process is 5 to 30 minutes.

[0333] 5.5 Method for purifying risankizumab from hitchhiker protein

[0334] Risankizumab may be recombinantly produced in various host cells (e.g., CHO cells or NS0 cells) by using the method described in the examples disclosed herein (e.g., Example 6), or by using methods known in the art, e.g., cell culture methods using hydrolysate-based or chemically defined media containing a specific range of manganese and / or galactose (e.g., see U.S. Patent No. 9,062,106), or by using recombinant host cells overexpressing β1,4 galactosyl-transferase, or by using host cells having beta-galactosidase knockdown (U.S. Patent No. 9,550,826). U.S. Patents No. 9,062,106 and No. 9,550,826 are incorporated herein by reference in their entirety.

[0335] The risankizumab formulations described herein are Example 6 of the present invention, Fig. 22 , can be produced using the exemplary optimized purification process described in Section 5.5.

[0336] Once a purified solution or mixture containing antibodies is obtained, the separation of antibodies from other proteins produced by cells, e.g., HPs, may be performed using a combination of different purification techniques, comprising, but not limited to, an affinity separation step, an ion exchange separation step, a mixed-mode separation step, and a hydrophobic interaction separation step, either alone or in combination. The separation step separates the mixture of proteins based on biophysical characteristics such as charge, degree of hydrophobicity, and / or size, without limitation, depending on the specific separation mode including chromatographic separation. In specific embodiments, separation may be performed using chromatography including cationic, anionic, hydrophobic interaction, and / or mixed-mode chromatography without limitation. Chromatographic resins are available for each of these techniques, thereby enabling precise adjustment of the purification scheme for the specific proteins involved. The essence of each described separation method is that the protein moves at different speeds through a chromatographic medium, e.g., a resin in a column, to achieve physical separation that increases as it passes further through the chromatographic medium, or the protein can be selectively attached to a chromatographic medium, e.g., a separation resin in a column, and then differentially eluted using different eluents. In a specific embodiment, when the hitchhike protein specifically attaches to the chromatographic medium, e.g., the resin in a column, and the antibody does not, i.e., when the antibody is contained in the eluent, the antibody is separated from the hitchhike protein, whereas in other cases, the antibody of interest may be attached to the chromatographic medium, e.g., the resin in a column, while the hitchhike protein is extruded from the column during a washing cycle.

[0337] (a). 1st recovery

[0338] In a specific embodiment, it is advantageous to apply the sample generated according to the present disclosure to at least the first step of purification and the first recovery.

[0339] The primary recovery may include one or more centrifugation steps to further purify the sample mixture to support the purification of the protein of interest. Centrifugation of the sample may be performed, for example, at 7,000 xg to approximately 12,750 xg, but is not limited thereto. In the context of large-scale purification, such centrifugation may be performed online, for example, but not limited thereto, at a flow rate set to achieve a turbidity level of 150 NTU in the generated supernatant. Subsequently, this supernatant may be collected for further purification.

[0340] In certain embodiments, the primary recovery may also include the use of one or more deep filtration steps to further purify the sample matrix and thereby support the purification of antibodies produced using the cell culture technology of the present disclosure. The deep filter comprises a filtration medium having a density gradient. This density gradient allows larger particles to be trapped near the surface of the filter, while allowing smaller particles to pass through a larger opening at the surface of the filter and be trapped only at a smaller opening closer to the center of the filter. In certain embodiments, the deep filtration step may be a delipid deep filtration step. In certain embodiments, the deep filtration step is used only during the primary recovery step. In certain embodiments, a deep filter comprising a delipid deep filter is used during one or more additional purification steps. Non-limiting examples of the deep filter that may be used with the method disclosed herein include the X0HC deep filter, D0HC deep filter, and Cuno TM Model 30 / 60ZA Deep Filter (3M Corp.) and 0.45 / 0.2 μm Sartopore TM Includes a double-layer filter cartridge.

[0341] (b). Affinity chromatography

[0342] In certain embodiments, it is advantageous to apply risankizumab produced according to the present disclosure to affinity chromatography to further purify antibodies from hitchhiker proteins (e.g., lipases). In certain embodiments, the chromatographic material may bind selectively or specifically to risankizumab. Non-limiting examples of such chromatographic materials include chromatographic materials containing protein A, protein G, e.g., antigens bound by the antibody of interest, and chromatographic materials containing Fc-binding proteins. In certain embodiments, the affinity chromatography step may involve applying a primary recovery sample to a column containing a suitable protein A resin. In certain embodiments, the protein A resin is useful for affinity purification and isolation of various antibody isotypes, in particular IgG1, IgG2, and IgG4. Protein A is a bacterial cell wall protein that binds primarily to mammalian IgG through its Fc region. In its natural state, protein A has five IgG-binding domains as well as other domains of unknown function.

[0343] Suitable protein A resin, e.g., GE Healthcare's MabSelect TM and MabSelect SuRe TM It is commercially available. In certain embodiments, MabSelect TM A suitable column packed with [material] is a column with a diameter of approximately 1.0 cm x a length of approximately 21.6 cm (a bed volume of approximately 17 ml). A column of this size can be used for small-scale purification and is comparable to other columns used for scale-up. For example, a 20 cm x 21 cm column (bed volume of approximately 6.6 L) can be used for larger-scale purification. Regardless of the column size, the column is MabSelect TM or MabSelect SuRe TM It can be filled using a suitable resin such as.

[0344] (c). Ion exchange chromatography

[0345] In certain embodiments, it is advantageous to apply the risankizumab produced according to the present disclosure to ion exchange chromatography to purify risankizumab from hitchhiker proteins (e.g., lipases). Ion exchange separation comprises any method in which two substances are separated based on the difference in their respective ionic charges, and may utilize a cationic exchange material or an anionic exchange material. For example, the use of a cationic exchange material versus an anionic exchange material is based on the localized charge of the proteins. Thus, using an anionic exchange step before using a cationic exchange step, or using a cationic exchange step before using an anionic exchange step, is covered by the present disclosure. Additionally, using only a cationic exchange step, only an anionic exchange step, or any serial combination of the two is covered by the present disclosure.

[0346] In performing the separation, the initial protein mixture may be contacted with an ion exchange material using any various techniques, for example, batch purification techniques or chromatography techniques.

[0347] Anionic 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, e.g., DE23 TM , DE32TM , DE52 TM , CM-23 TM , CM-32 TM and CM-52 TM It is available from Whatman Ltd. in Maidstone, Kent, UK. SEPHADEX®-based and cross-linked ion exchangers are also known. For example, DEAE-, QAE-, CM-, and SP-SEPHADEX® and DEAE-, Q-, CM-, and S-SEPHAROSE® and SEPHAROSE® Fast Fe are all available from Pharmacia AB. In addition, TOYOPEARL TM DEAE-6505 or M and TOYOPEARL TM Both DEAE and CM derivatized ethylene glycol-methacrylate copolymers, such as CM-650S or M, are available from Toso Haas Co., Philadelphia, Pennsylvania. In certain embodiments, Poros TM Cation exchange chromatography using XS resin is used.

[0348] (d). Ultrafiltration / Resolution filtration

[0349] In certain embodiments, it is advantageous to apply the risankizumab produced according to the present disclosure to ultrafiltration and / or volume filtration to purify risankizumab from hitchhiker proteins (e.g., lipases). Ultrafiltration is described in detail in the literature [Microfiltration and Ultrafiltration: Principles and Applications, L. Zeman and A. Zydney (Marcel Dekker, Inc., New York, NY, 1996)] and [Ultrafiltration Handbook, Munir Cheryan (Technomic Publishing, 1986; ISBN No. 87762-456-9)]. One filtration process is tangential flow filtration as described in the Millipore catalog title ['Pharmaceutical Process Filtration Catalogue' pp. 177-202 (Bedford, Mass., 1995 / 96)]. Ultrafiltration is generally considered to mean filtration using a filter having a pore size of less than 0.1 μm. By using a filter with such a small pore size, the volume of the sample can be reduced through the permeation of the sample buffer through the filter while the antibody remains in the filter.

[0350] Volume filtration is a method of using an ultrafilter to remove and exchange salts, sugars, and non-aqueous solvents, to separate and / or free from combined species, to remove low molecular weight substances, and / or to induce rapid changes in the ionic and / or pH environment. Microsolutes are most efficiently removed by adding a solvent to the solution being ultrafiltered at a rate approximately equal to the ultrafiltration rate. This effectively purifies the retained protein by washing microspecies from the solution in a constant volume. In certain embodiments, the volume filtration step is optionally used to remove impurities from the protein preparation as well as to exchange various buffers used in connection with this disclosure, prior to additional chromatography or other purification steps.

[0351] (e). Hydrophobic interaction chromatography

[0352] In certain embodiments, it is advantageous to apply the risankizumab produced according to the present disclosure to hydrophobic interaction chromatography to purify risankizumab from hitchhiker proteins (e.g., lipases). For example, a first eluent obtained from an ion exchange column may be applied to a hydrophobic interaction material to obtain a second eluent having a reduced level of HP. Hydrophobic interaction chromatography (HIC) steps, such as those disclosed herein, are generally performed to purify proteins, including the removal of HP.

[0353] In performing HIC-based separation, the sample mixture is brought into contact with the HIC substance, for example, using batch purification techniques or a column. Prior to HIC purification, it may be desirable to remove any chaotropic agents or highly hydrophobic substances, for example, by passing the mixture through a pre-column.

[0354] While ion exchange chromatography relies on the charge of proteins to isolate them, hydrophobic interaction chromatography utilizes the hydrophobic properties of proteins. Hydrophobic groups on proteins interact with hydrophobic groups on the column. The more hydrophobic a protein is, the stronger the interaction force with the column. Therefore, the HIC step removes host cell-derived impurities (e.g., DNA and other high and low molecular weight products-related species).

[0355] HIC columns generally comprise a base matrix (e.g., cross-linked agarose or synthetic copolymer material) coupled with hydrophobic ligands (e.g., alkyl or aryl groups). Suitable HIC columns are agarose resins substituted with phenyl groups (e.g., phenyl sepharose). TM It includes columns. An example of an HIC column is phenyl sepharose with low or high substitution. TM 6 Fast Flow Columns (Pharmacia LKB Biotechnology, AB, Sweden), Phenyl Sepharose TM High-performance column (Pharmacia LKB Biotechnology, AB, Sweden), Octyl Sepharose TM High-performance column (Pharmacia LKB Biotechnology, AB, Sweden), Fractogel TM EMD Propyl or Fractogel TM EMD Phenyl Column (E. Merck, Germany), Macro-Prep TM Mehyl or Macro-Prep TM t-butyl support (Bio-Rad, California), WP HI-Propyl(C3) TM Column (JT Baker, New Jersey) and Toyopearl TM Includes, but is not limited to, ether, phenyl or butyl columns (TosoHaas, Pennsylvania).

[0356] (f). Multimode (mixed-mode) chromatography

[0357] In certain embodiments, it is advantageous to apply the risankizumab produced according to the present disclosure to multimode chromatography to purify risankizumab from hitchhiker proteins (e.g., lipases). Multimode chromatography is chromatography that utilizes a multimode medium resin. Such resins include multimode chromatography ligands. In certain embodiments, such ligands refer to ligands that can provide at least two different but mutually cooperative sites that interact with the substance to be bound. One of these sites provides an attractive type of charge-charge interaction between the ligand and the substance of interest. The other site typically provides electron acceptor-donor interactions and / or hydrophobic and / or hydrophilic interactions. Electron donor-acceptor interactions include interactions such as hydrogen-bonding, π-π, cation-π, charge transfer, dipole-dipole, induced dipole, etc. Multimode chromatography ligands are also known as 'mixed-mode' chromatography ligands.

[0358] In certain embodiments, the multimode chromatography resin comprises multimode ligands coupled to an organic or inorganic support, sometimes represented as a base matrix, either directly or through a spacer. The support may be in the form of particles, such as spherical particles, monoliths, filters, membranes, surfaces, capillaries, etc. In certain embodiments, the support may be prepared from natural polymers, e.g., cross-linked carbohydrate materials, e.g., agarose, agar, cellulose, dextran, chitosan, konjac, carrageenan, gellan, alginate, etc. To obtain a high adsorption capacity, the support may be porous, and the ligands are subsequently coupled to the pore surface as well as the outer surface. Such natural polymer supports may be prepared according to standard methods, such as reverse suspension gelation (see [S. Hjerten: Biochim Biophys Acta 79(2), 393-398 (1964)]). Alternatively, the support may be prepared from synthetic polymers, e.g., cross-linked synthetic polymers, e.g., styrene or styrene derivatives, divinylbenzene, acrylamide, acrylate esters, methacrylate esters, vinyl esters, vinylamide, etc. These synthetic polymers may be produced according to standard methods, for example, see the literature ['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 are also available from commercial sources such as Amersham Biosciences in Uppsala, Sweden. In certain embodiments, mixed-mode chromatography combines anion exchange (AEX) and hydrophobic interaction (HIC) functionality. One non-limiting example of such mixed-mode chromatography suitable for the present disclosure is CaptoTM Adhere is mixed-mode chromatography.

[0359] 5.6 Exemplary embodiments

[0360] The present disclosure provides the following non-limiting exemplary embodiments.

[0361] 1. (i) Risankizumab at a concentration of approximately 180 mg / ml,

[0362] (ii) water and

[0363] (iii) containing a surfactant,

[0364] A liquid aqueous pharmaceutical formulation suitable for subcutaneous injection having a pH of 5.0 to 6.5.

[0365] 2. A liquid aqueous pharmaceutical formulation comprising risankizumab at a concentration of about 180 mg / ml, comprising 10,000 or fewer invisible particles (SVP) per ml, wherein the SVP have a diameter greater than 2 μm.

[0366] 3. A liquid aqueous pharmaceutical formulation comprising risankizumab at a concentration of about 180 mg / ml, wherein the liquid aqueous pharmaceutical formulation comprises less than 250 pg of phospholipase A2 (PLA2) per mg of said risankizumab.

[0367] 4. A liquid aqueous pharmaceutical formulation comprising risankizumab at a concentration of about 180 mg / ml, comprising 10,000 or fewer invisible particles (SVP) per ml and less than 250 pg of phospholipase A2 (PLA2) per mg of said risankizumab, wherein said SVP has a diameter greater than 2 μm.

[0368] 5. A formulation in which, in embodiment 3 or 4, the PLA2 is PLA2G15.

[0369] 6. A formulation comprising less than 9 pg of PLA2 per mg of risankizumab in any one of embodiments 3 to 5.

[0370] 7. A formulation in any one of embodiments 3 to 6, wherein the level of PLA2 is determined by an enzyme-linked immunoassay (ELISA).

[0371] 8. A formulation in any one of embodiments 3 to 7, wherein no particles visible to the naked eye or glittering particles are observed for 24 months at 4°C.

[0372] 9. A formulation comprising polysorbate 20 (PS20) in any one of embodiments 3 to 8, wherein the concentration of PS20 in the formulation after storage at 5°C for 6 months is at least 80% of the concentration of PS20 in the formulation before storage at 5°C for 6 months.

[0373] 10. A formulation according to embodiment 9, wherein the concentration of PS20 in the formulation after storage at 25°C for 6 months is at least 60% of the concentration of PS20 in the formulation before storage at 25°C for 6 months.

[0374] 11. A formulation according to embodiment 9 or 10, wherein the concentration of PS20 in the formulation after storage at 40°C for 6 months is at least 40% of the concentration of PS20 in the formulation before storage at 4°C for 6 months.

[0375] 12. A formulation in any one of embodiments 9 to 11, wherein the concentration of PS20 is measured using a high-performance liquid chromatography-charged aerosol detector (HPLC-CAD).

[0376] 13. A formulation in any one of embodiments 3 to 12, wherein the incidence of anti-drug antibodies (ADA) occurring after treatment is less than 4.7% after administering a single subcutaneous dose of 150 mg of the formulation to a human subject.

[0377] 14. The formulation of Embodiment 13, wherein there is no occurrence of ADA after treatment.

[0378] 15. A formulation of embodiment 13 or 14, wherein the presence of ADA is determined using a verified potency-based crosslinked electrochemiluminescence immunoassay.

[0379] 16. A formulation in any one of embodiments 3 to 12, wherein at least 99.1% of the risankizumab species in the formulation exists as a monomer.

[0380] 17. A formulation of embodiment 16, wherein the percentage of the risankizumab species present as monomers is determined by ultra-high performance size exclusion chromatography (UP-SEC).

[0381] 18. A formulation in any one of embodiments 3 to 12, wherein 0.4% or less of the risankizumab species in the formulation is present as a high molecular weight (HMW) species, and the HMW species is a species having a higher molecular weight than the monomer.

[0382] 19. A formulation in which the percentage of the risankizumab species present as an HMW species in embodiment 18 is determined by UP-SEC.

[0383] 20. A formulation in any one of embodiments 3 to 12, wherein more than 97.5% of the risankizumab species in the formulation is present as a major peak measured by capillary gel electrophoresis (CGE-NR) under non-reducing conditions.

[0384] 21. A formulation in any one of embodiments 3 to 12, wherein less than 2.2% of the risankizumab species in the formulation is present as a low molecular weight (LMW) species as measured by CGE-NR, and the LMW species is a species having a retention time shorter than the main peak.

[0385] 22. A formulation in any one of embodiments 2 and 4 to 21, wherein the size of the SVP is measured by micro-flow imaging.

[0386] 23. A formulation in any one of embodiments 2 and 4 to 22, wherein the number of SVPs in the formulation is measured by micro-flow imaging.

[0387] 24. A formulation in any one of embodiments 2 and 4 to 23, comprising 9,000 or fewer SVPs per ml, 8,000 or fewer SVPs per ml, 7,000 or fewer SVPs per ml, 6,000 or fewer SVPs per ml, 5,000 or fewer SVPs per ml, 4,000 or fewer SVPs per ml, 3,000 or fewer SVPs per ml, 2,000 or fewer SVPs per ml, 1,000 or fewer SVPs per ml, 800 or fewer SVPs per ml, 600 or fewer SVPs per ml, 400 or fewer SVPs per ml, or 200 or fewer SVPs per ml.

[0388] 25. A method for reducing the formation of invisible particles (SVP) in a liquid aqueous pharmaceutical formulation containing risankizumab at a concentration of about 180 mg / ml, comprising a freeze-thaw process (F / T), wherein the F / T comprises freezing the formulation from about 18°C ​​to about -70°C for 10 hours or less, and then thawing the formulation from about -70°C to about 18°C ​​for 15 hours or less.

[0389] 26. A method according to embodiment 25, wherein the F / T comprises freezing the formulation from about 18°C ​​to about -70°C within about 5 hours, and then thawing the formulation from about -70°C to about 18°C ​​within about 9 hours.

[0390] 27. A method according to embodiment 25 or 26, wherein the F / T comprises three or fewer, two or fewer, or one or fewer cycles of freezing and thawing of the formulation.

[0391] 28. A method for reducing the formation of invisible particles (SVP) in a liquid aqueous pharmaceutical formulation containing risankizumab at a concentration of about 180 mg / ml, comprising a mixing process, wherein the power per volume (P / V) of the mixing process is 1000 W / m² 3 A method wherein the mixing time of the above mixing process is 30 minutes or less.

[0392] 29. A method for reducing the formation of invisible particles (SVP) in a liquid aqueous pharmaceutical formulation containing risankizumab at a concentration of about 180 mg / ml, comprising (i) a freeze-thaw process (F / T) and (ii) a mixing process, wherein (i) the F / T comprises freezing said formulation from about 18 °C to about -70 °C for 10 hours or less, and then thawing said formulation from about -70 °C to about 18 °C for 15 hours or less, and (ii) the power per volume (P / V) of said mixing process is 1000 W / m² 3 A method wherein the mixing time of the above mixing process is 30 minutes or less.

[0393] 30. In embodiment 29, the above A method comprising freezing the above formulation from about 18°C ​​to about -70°C within about 5 hours, and then thawing the above formulation from about -70°C to about 18°C ​​within about 9 hours.

[0394] 31. A method according to embodiment 29 or 30, wherein the F / T comprises three or fewer, two or fewer, or one or fewer cycles of freezing and thawing of the formulation.

[0395] 32. In any one of embodiments 28 to 31, the P / V of the mixing process is 500 W / m² 3 Lee Ha-in, method.

[0396] 33. A method in any one of embodiments 28 to 32, wherein the mixing time of the mixing process is 5 minutes to 30 minutes.

[0397] 34. A liquid aqueous pharmaceutical formulation produced by any one of the methods of embodiments 25 to 33.

[0398] 35. A formulation of any one of embodiments 2 to 24 and 34 or a method of any one of embodiments 25 to 33, wherein the formulation further comprises water and a surfactant, and the formulation has a pH of 5.0 to 6.5 and is suitable for subcutaneous injection.

[0399] 36. A formulation or method of embodiment 1 or 35, wherein the surfactant is PS20.

[0400] 37. A formulation or method of embodiment 36, wherein the PS20 is at a concentration of about 0.2 mg / ml.

[0401] 38. A formulation or method in any one of embodiments 1 to 24 and 34 to 37, wherein the viscosity of the formulation is less than 20.0 mPa·s at 20 ℃.

[0402] 39. A formulation or method of embodiment 38, wherein the viscosity of the formulation is 14.0 mPa.s to 20.0 mPa.s at 20°C.

[0403] 40. A formulation or method of embodiment 39, wherein the viscosity of the formulation is about 15 mPa.s, about 17 mPa.s, about 17 mPa.s, or about 19 mPa.s at 20°C.

[0404] 41. A formulation or method of any one of embodiments 38 to 40, wherein the viscosity of the formulation is measured by an ALP method or a falling ball viscometer.

[0405] 42. In any one of the formulations of embodiments 1 to 24 and 34 to 41 or any one of the methods of embodiments 25 to 33 and 35 to 41,

[0406] (iv) Topical topics,

[0407] (v) buffer and / or

[0408] (vi) A formulation or method further comprising a solubilizing agent.

[0409] 43. A formulation or method of embodiment 42, wherein the formulation comprises an isotonic agent.

[0410] 44. In the formulation or method of Embodiment 43, the formulation is

[0411] (a) trehalose or

[0412] (b) A formulation or method comprising mannitol.

[0413] 45. A formulation or method of any one of embodiments 42 to 44, wherein the formulation comprises a buffer solution.

[0414] 46. ​​A formulation or method of embodiment 45, wherein the formulation comprises an acetate buffer.

[0415] 47. A formulation or method of embodiment 46, wherein the acetate buffer is at a concentration of about 10 mM or about 9.5 mM.

[0416] 48. In the formulation or method of embodiment 46 or 47, said formulation

[0417] (a) sodium acetate at a concentration of about 9.1 mM and acetic acid at a concentration of about 0.4 mM, or

[0418] (b) sodium acetate at a concentration of about 9.1 mM and acetic acid at a concentration of about 0.9 mM, or

[0419] (c) A formulation or method comprising sodium acetate at a concentration of about 8.7 mM and acetic acid at a concentration of about 1.3 mM.

[0420] 49. A formulation of any one of embodiments 1 to 24 and 34 to 44 or a method of any one of embodiments 25 to 33 and 35 to 44, wherein the formulation does not contain a buffer solution.

[0421] 50. A formulation or method of any one of embodiments 42 to 49, wherein the formulation comprises a solubilizing agent.

[0422] 51. In the formulation or method of embodiment 50, the formulation is

[0423] (a) Proline at a concentration of about 225 mM or

[0424] (b) A formulation or method comprising glycine at a concentration of about 225 mM.

[0425] 52. A formulation of any one of embodiments 1 to 24 and 34 to 51 or a method of any one of embodiments 25 to 33 and 35 to 51, wherein the formulation has a pH of about 5.2, about 5.5, about 5.7, or about 6.2.

[0426] 53. A formulation or method of embodiment 52, wherein the formulation has a pH of about 5.5.

[0427] 54. A formulation of any one of embodiments 1 to 24 and 34 to 53 or a method of any one of embodiments 25 to 33 and 35 to 53, wherein the formulation has an osmotic concentration of 320 mOsmol / kg to 350 mOsmol / kg.

[0428] 55. In any one of the formulations of embodiments 1 to 24 and 34 to 41 or any one of the methods of embodiments 25 to 33 and 35 to 41,

[0429] (i) PS20 at a concentration of about 0.2 mg / ml,

[0430] (ii) trehalose at a concentration of approximately 185 mM and

[0431] (iii) containing acetate buffer at a concentration of 9.5 mM, and

[0432] The above formulation is a formulation or method having a pH of about 5.7.

[0433] 56. In any one of the formulations of embodiments 1 to 24 and 34 to 41 or any one of the methods of embodiments 25 to 33 and 35 to 41,

[0434] (i) PS20 at a concentration of about 0.2 mg / ml,

[0435] (ii) trehalose at a concentration of approximately 185 mM and

[0436] (iii) containing acetate buffer at a concentration of 9.5 mM, and

[0437] The above formulation is a formulation or method having a pH of about 5.5.

[0438] 57. In any one of the formulations of embodiments 1 to 24 and 34 to 41 or any one of the methods of embodiments 25 to 33 and 35 to 41,

[0439] (i) PS20 at a concentration of about 0.2 mg / ml,

[0440] (ii) trehalose at a concentration of approximately 185 mM and

[0441] (iii) containing sodium acetate at a concentration of about 9.1 mM and acetic acid at a concentration of about 0.4 mM,

[0442] The above formulation is a formulation or method having a pH of about 5.7.

[0443] 58. In any one of the formulations of embodiments 1 to 24 and 34 to 41 or any one of the methods of embodiments 25 to 33 and 35 to 41,

[0444] (i) PS20 at a concentration of about 0.2 mg / ml,

[0445] (ii) trehalose at a concentration of approximately 185 mM and

[0446] (iii) containing sodium acetate at a concentration of about 9.1 mM and acetic acid at a concentration of about 0.4 mM,

[0447] The above formulation is a formulation or method having a pH of about 5.5.

[0448] 59. In any one of the formulations of embodiments 1 to 24 and 34 to 41 or any one of the methods of embodiments 25 to 33 and 35 to 41,

[0449] (i) PS20 at a concentration of about 0.2 mg / ml,

[0450] (ii) trehalose at a concentration of approximately 185 mM and

[0451] (iii) containing acetate buffer at a concentration of 10 mM, and

[0452] The above formulation is a formulation or method having a pH of about 5.7.

[0453] 60. In any one of the formulations of embodiments 1 to 24 and 34 to 41 or any one of the methods of embodiments 25 to 33 and 35 to 41,

[0454] (i) PS20 at a concentration of about 0.2 mg / ml,

[0455] (ii) trehalose at a concentration of approximately 185 mM and

[0456] (iii) containing acetate buffer at a concentration of 10 mM, and

[0457] The above formulation is a formulation or method having a pH of about 5.5.

[0458] 61. In any one of the formulations of embodiments 1 to 24 and 34 to 41 or any one of the methods of embodiments 25 to 33 and 35 to 41,

[0459] (i) PS20 at a concentration of about 0.2 mg / ml,

[0460] (ii) trehalose at a concentration of approximately 185 mM and

[0461] (iii) containing sodium acetate at a concentration of about 9.1 mM and acetic acid at a concentration of about 0.9 mM,

[0462] The above formulation is a formulation or method having a pH of about 5.7.

[0463] 62. In any one of the formulations of embodiments 1 to 24 and 34 to 41 or any one of the methods of embodiments 25 to 33 and 35 to 41,

[0464] (i) PS20 at a concentration of about 0.2 mg / ml,

[0465] (ii) trehalose at a concentration of approximately 185 mM and

[0466] (iii) containing sodium acetate at a concentration of about 8.7 mM and acetic acid at a concentration of about 1.3 mM,

[0467] The above formulation is a formulation or method having a pH of about 5.5.

[0468] 63. In any one of the formulations of embodiments 1 to 24 and 34 to 41 or any one of the methods of embodiments 25 to 33 and 35 to 41,

[0469] (i) PS20 at a concentration of about 0.2 mg / ml and

[0470] (ii) containing mannitol at a concentration of about 190 mM, and

[0471] The above formulation is a formulation or method having a pH of about 5.7.

[0472] 64. In any one of the formulations of embodiments 1 to 24 and 34 to 41 or any one of the methods of embodiments 25 to 33 and 35 to 41,

[0473] (i) PS20 at a concentration of about 0.2 mg / ml and

[0474] (ii) containing glycine at a concentration of approximately 225 mM, and

[0475] The above formulation is a formulation or method having a pH of about 5.7.

[0476] 65. In any one of the formulations of embodiments 1 to 24 and 34 to 41 or any one of the methods of embodiments 25 to 33 and 35 to 41,

[0477] (i) PS20 at a concentration of about 0.2 mg / ml and

[0478] (ii) containing proline at a concentration of approximately 225 mM, and

[0479] The above formulation is a formulation or method having a pH of about 5.7.

[0480] 6. Examples

[0481] The following is a description of the various methods and materials used in the study. These are presented to provide a complete disclosure and description of the methods for manufacturing and using the present invention to those skilled in the art, and are not intended to limit the scope of what the inventors consider to be their invention, nor are they intended to indicate that the following experiments have been performed and may be performed. It should be understood that the exemplary descriptions described in the present tense are not necessarily performed, but rather may be performed to generate data, etc., related to the teachings of the present invention. While efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, percentages, etc.), some experimental error and deviation must be taken into account.

[0482] 6.1 Example 1: Screening of Risankizumab Formulations

[0483] In a two-level full factorial design, the stability around the target formulation at 180 mg / ml (Formulation No. 5) was evaluated for nine formulations with three different concentrations of risankizumab and three different pH values, and compared with the commercial 150 mg / ml risankizumab formulation (Formulation No. 10). Table 1 It represents the design of 10 formulations.

[0484]

[0485] Samples were prepared using a semi-automatic sample handling procedure. The pH of the prepared formulation was determined manually, with the pH value being within a range of ± 0.05. The osmolality of the formulation was measured and Table 2 It was presented in.

[0486]

[0487] The viscosity of the formulation was measured by ALP viscosity measurements at 20°C using a falling ball viscometer (20°C) and a liquid handling system. The falling ball viscometer measured the time required for a spherical ball to fall a defined distance under gravity through a tube filled with the formulation being tested. Measurements were collected under temperature-controlled conditions, and the results were given as dynamic viscosity using milli-Pascal seconds [mPa·s], an internationally standardized absolute unit. The ALP (Liquid Characterization) method was performed on a liquid handling system acting as a high-throughput viscometer. Viscosity measurements can be evaluated using the liquid handling system by analyzing mass flow behavior and pressure profiles through a pipette tip during transient flow, and by comparing either the dispensed weight or pressure reading with a calibration curve.

[0488] Figures 1a and 1b As shown in [figure], the viscosity of the formulation measured by ALP at 20 °C was risankizumab concentration-dependent. In particular, when the risankizumab concentration increased from 150 mg / ml to 162 mg / ml, the viscosity of the formulation increased only from about 9 mPas to about 12 mPas, and when the risankizumab concentration increased from 162 mg / ml to 180 mg / ml, the viscosity of the formulation increased to about 18 to 19 mPas. However, when the risankizumab concentration increased from 180 mg / ml to 198 mg / ml, the viscosity of the formulation increased exponentially. Viscosity was higher in the formulation with a pH of 6.2 than in the formulation with a pH of 5.2 to 5.7.

[0489] Viscosity values ​​measured by the falling ball method and the ALP method generally showed similar results. However, at higher risankizumab concentrations, viscosity values ​​were higher when measured by the ALP method than by the falling ball method. Fig. 2 ).

[0490] Size heterogeneity was determined by size-exclusion ultra-high performance liquid chromatography (SE-UHPLC) in risankizumab formulations under freeze-thaw stress (ft), mechanical stress (mech), or no stress conditions. In particular, the percentage of monomers (%) within the formulation (monomer %, Fig. 3a ), percentage (%) of high molecular weight (HMW) species (HMW %, Fig. 3b ) and percentage (%) of low molecular weight (LMW) species (LMW %, Fig. 3c ) was evaluated. Specifically, SE-UHPLC was performed on a UHPLC system equipped with a solvent transfer pump, an autosampler, a column oven, and a diode array detector for collecting UV absorbance. SEC fractionation was performed on a UHPLC system using a conventional SEC column (Waters, ACQUITY UPLC BEH200 SEC 4.6 x 150 mm; 1.7 μm particles, pore size 200 Å) containing a standard mobile phase and small particles. Risankizumab formulations were subjected to four (4) freeze-thaw cycles (ft) induced by shaking or mechanical stress (mech) prior to the SE-UHPLC test. A lower HMW % was observed in the formulation with a pH of 5.70 under all stress conditions ( Fig. 3b The effect of pH on monomer % and HMW % was within a method variability of approximately + / -0.4% ( Figures 3d and 3e ).

[0491] 6.2 Example 2: Feasibility Screening of Risankizumab Formulations

[0492] Four risankizumab formulations (Formulations 1 to 4) containing 180 mg / ml of risankizumab were investigated in this example. The components of Formulations 1 to 4 and a benchmark formulation containing 150 mg / ml of risankizumab Table 3 It was provided in. The viscosity (measured by a falling ball at 20 ℃) ​​and osmolality of the formulation were also measured. Table 3As presented in [here]. The risankizumab formulation was prepared using 200 mg / ml of risankizumab active pharmaceutical ingredient in 0.02 mg / ml NaCl as the starting material. This solution was ultrafiltered to approximately 226 mg / ml (a formulation based on 34.6 mg / ml mannitol) or approximately 240 mg / ml (a formulation based on 42 mg / ml mannitol, trehalose, proline, glycine, and arginine) and mixed with an excipient stock solution concentrated to a target protein concentration of 180 mg / ml. Ultrafiltration (UF) was performed using Vivaspin centrifuge tubes. Protein concentration was measured using OD 280 (a CTech Solo VPE paired with a Cary 60 as the light source). The purpose of the study was to evaluate the viscosity of potential formulations at an intended concentration of 180 mg / ml and physiological osmolality concentrations of 240 to 350 mOsmol / kg. Ideally, the viscosity would be less than 20 mPas to allow for efficient processing and convenient administration.

[0493]

[0494] The stability of formulations 1 to 4 was evaluated. The formulations were subjected to stress at 40°C for 7 days, stress at 40°C for 21 days, or no temperature stress. Samples were collected at t0 after 7 and 21 days of incubation at 40°C / 75% rH. Samples for SVP analysis were analyzed directly, while samples for SE- and CEUHPLC analysis were frozen at -80°C until analysis following the conclusion of the stability study.

[0495] Size heterogeneity in the formulation was evaluated by performing SE-UHPLC. Specifically, SE-UHPLC was performed on a UHPLC system equipped with a solvent transfer pump, an autosampler, a column oven, and a diode array detector for collecting UV absorbance. SEC fractionation was performed on the UHPLC system using a conventional SEC column (Waters, ACQUITY UPLC BEH200 SEC 4.6 x 150 mm; 1.7 μm particles, pore size 200 Å) containing a standard mobile phase and small particles. Monomer percentage (monomer %, Fig. 4a ), percentage of high molecular weight (HMW) species (HMW %, Fig. 4b ) and percentage of low molecular weight (LMW) species (LMW %, Fig. 4c ) was measured in each formulation. The change in monomer % over time ('delta') at 40 ℃ was determined as the value obtained by subtracting T0 from Tx (Fig. 4d ), where Tx is the monomer % at full point day 7 or 21 and T0 is the monomer % at day 0.

[0496] The initial monomer % of the proline-based, glycine-based, and trehalose / acetate-based formulations was similar to the benchmark formulation ( Fig. 4a )(difference within ±0.4% method variability). Proline-based, glycine-based, and trehalose / acetate-based formulations also demonstrated stability similar to the benchmark formulation upon incubation at 40 °C (difference within ±0.4% method variability). The initial monomer % of the mannitol-based formulation at T0 was lower than that of other formulations (95.9% vs. 96.7–97.2%) and remained lower over time ( Fig. 4a A trend of accelerated loss of monomer % was observed for mannitol-based and glycine-based formulations compared to proline-based formulations ( Figs. 4a and 4dMannitol-based and glycine-based formulations showed increased degradation compared to trehalose / acetate-based and proline-based formulations ( Fig. 4d The trehalose / acetate-based and proline-based formulations demonstrated stability similar to the benchmark formulation ( Fig. 4d ).

[0497] Regarding HMW content, Figures 4b and 4e This indicates that the trehalose / acetate-based, glycine-based, and proline-based formulations demonstrated stability similar to the benchmark formulation (difference within ±0.4% method variability). The initial HMW % at T0 for the mannitol-based formulation was higher than that of the other formulations and remained higher over time. A higher increase in HMW content was observed for the mannitol-based and glycine-based formulations.

[0498] Regarding LMW content, the difference in LMW % between formulations was within method variability ( Figures 4c and 4f ). Glycine-based formulations appeared to show a tendency for LMW to increase more significantly over time.

[0499] The percentage of charged protein variants within the tested risankizumab formulations was determined by cation exchange chromatography ultra-high performance liquid chromatography (CEX-UHPLC). CEX-UHPLC was able to separate protein variants into fractions based on protein charge (or lack thereof). Charged protein variants were generally referred to as 'acidic species' or 'basic species' in comparison to the major species (i.e., major isoforms). CEX-UHPLC was performed to fractionate protein charge variants on a UHPLC system using a column composed of a cation-exchange resin and an ion-gradient eluent.

[0500]

[0501] Specifically, CEX-UHPLC was performed on formulations that were stressed at 40°C for 7 days, stressed at 40°C for 21 days, or not subjected to temperature stress ( FIGS. 5a to 5f The initial major isoform content of formulations 1 to 4 was similar to the benchmark formulation ( Fig. 5a )(Difference within ±2% method variability). Formulations 1 to 4 also exhibited stability similar to the benchmark formulation ( Fig. 5a )(Difference within ±2% method variability). Glycine-based formulations showed a higher tendency for major isoform degradation compared to other test formulations, but this effect was within method variability (Fig. 5a and 5d All formulations showed similar acid species content compared to the benchmark formulation ( Figures 5b and 5e )(Difference within ±2% method variability). Glycine-based formulations showed a tendency toward higher acidic species content compared to other test formulations. Mannitol-based and proline-based formulations showed a tendency toward lower acidic species formation over incubation time compared to the benchmark formulation. All formulations showed similar but higher basic species content compared to the benchmark formulation ( Figs. 5c and 5f )(Difference within ±2% method variability). The mannitol-based formulation already showed a tendency toward higher basic species content compared to other test formulations at t0. The glycine-based formulation showed a higher increase in basic species content over time. The acidic and basic species measured in the formulations indicated that formulations 1 to 4 had stability similar to the benchmark formulation.

[0502] Invisible particles (SVP) were measured in all formulations by total holographic analysis. All tested formulations showed low particle concentrations. No significant changes were observed over a storage period of 21 days. Fig. 43 ).

[0503] Table 5 Is Provides a summary of the evaluation of four 180 mg / ml risankizumab formulations in terms of safety.

[0504]

[0505] Additional experiments were performed to measure the viscosity of formulations having a pH of 5.7 or 5.5. The viscosity of different batches of the formulations was measured using the falling ball method disclosed herein, and the results are as follows. Table 6 It was shown in.

[0506]

[0507] Surprisingly, pH 5.5 helped to significantly reduce the viscosity of the 180 mg / ml formulation by about 4 mPa.s, bringing the viscosity of the 180 mg / ml formulation closer to the established 150 mg / ml formulation while maintaining the stability of the 180 mg / ml formulation.

[0508] 6.3 Example 3: Risankizumab Particle Study

[0509] The process 4 DS described in Example 6 was formulated to produce a finished pharmaceutical product (DP4) containing 180 mg / mL risankizumab used in Example 3.

[0510] To understand the effects of temperature, mechanical stress, and high protein concentrations on the stability and particle formation of risankizumab (Risa) drug substance (DS) from a process perspective, several worst-case scenario experiments were designed and executed at the laboratory scale, including slow and fast freeze-thaw (FT), -20 °C va-Q-tainer transport simulations, and final bulk DS mixing studies. Particle formation was verified using micro-flow imaging (MFI) and turbidity measurements, and representative product quality (PQ) analyses were also performed. Full-scale transport studies were conducted to examine the full scale of particle formation A scaled-down model (SDM) representing the process was validated. The power input, mixing rotational speed, and filling of full-scale BDS tanks and single-use mixers (SUM) used at different manufacturing sites were verified. A mathematical model regarding the relationship between volumes was developed to establish a safety margin for unit operations of BDS mixing. The mathematical model for the accuracy of predicting the risk level of particle formation was further validated using historical full-scale mixing data.

[0511] 6.3.1 Materials and Methods

[0512] Table 7 As shown in [figure], three batches of 180 mg / mL risankizumab (Risa 180) ultra-high concentration (UHC) DS and two batches of 150 mg / mL risankizumab (Risa 150) control DS in the corresponding formulation buffer were used in this study.

[0513]

[0514] Laboratory-scale storage containers Celsius for FT and transport simulation SDM ® Pak-30 mL bags were purchased from Sartorius Stedim. The 30 mL bags were production-scale Celsius ® It was manufactured with the same film material as the flexible freeze-thaw (FFT) bag, ethylene-vinyl acetate (EVA), and the same thickness.

[0515] DS characteristics

[0516] Low temperature pH measurement.The pH values ​​of Risa DS were measured during the FT process using a low-temperature InLab® Cool Pro-ISM pH electrode connected to a SevenExcellence pH meter S400 with an operating temperature of -30 ℃ to 80 ℃. pH as a function of temperature was measured during this FT process for both the ABC SUL Risa 180 UHC and the BI GMP Risa 150 control.

[0517] DS density measurement. The densities of all batches of Risa 180 UHC and Risa 150 control were measured at temperatures of 2 ℃, 4 ℃, 5 ℃, 10 ℃, 15 ℃, 20 ℃, and 25 ℃ using a densimeter, model DMA 4501.

[0518] DS viscosity measurement. Apparent viscosity was measured using a VROC initium viscometer. The viscosities of Risa 180 UHC and the Risa 150 control were measured at different temperatures ranging from 4 °C to 25 °C. The experimental data Equation 1 It was fitted to the Arrhenius model for low viscosity, and

[0519]

[0520] Equation 1

[0521] Among the above equations η is an arbitrary given temperature T It is the viscosity of the mAb solution at, and A is a total number factor, and E a ε is the activation energy for viscous flow, and R is the ideal gas constant.

[0522] Furthermore, the Risa UHC ABC GMP batch was also diluted with its formulation buffer to obtain viscosity measurements as a function of protein concentrations from 30 to 180 mg / mL, and Equation 2 It was fitted to the simple exponential mAb concentration viscosity model described by,

[0523]

[0524] Equation 2

[0525] Among the above equations η is an arbitrary given mAb concentration at a fixed temperature c It is the viscosity of the mAb solution at, and η 0 is the viscosity of the solution at infinite dilution of mAb, and k is the exponential factor for the viscosity-concentration profile at a fixed temperature. Viscosity data for Risa GMP BDS (concentration 85 mg / mL) were also measured for model fitting.

[0526] SDM for DS storage and transport

[0527] Temperature-controlled instrument with 30 mL Celsius® Pak bag Celsius ® S 3 The effect of full-scale BDS storage and transport process conditions on particle formation was evaluated using a benchtop system. Production-scale Risa BDS was stored in 6 L Celsius® FFT bags. Small bags were stored in a BD Luer-Lok inside a biosafety cabinet (BSC). TM 27.5 mL of Risa DS was filled using a 30 mL sterile disposable syringe with a tip to maintain the same filling volume ratio as a 6 L full-scale bag filled with 5.5 L BDS.

[0528] FT SDM

[0529] To understand the influence of different freeze-thaw rate equipment in the manufacturing process, worst-case scenarios of low-speed FT and high-speed FT SDM were evaluated for the stability of Risa BDS. Three cycles of low-speed freeze-low-speed thaw and high-speed freeze-high-speed thaw were performed on Risa 180 UHC and Risa 150 control DS using 30 mL Celsius bags equipped with a controlled Sartorius FT system. Samples were analyzed for MFI, turbidity, and PQ at the starting point (cycle 0), FT cycle 1, cycle 2, and cycle 3.

[0530] The low-speed FT temperature profile is Fig. 6a This is shown in [figure]. The worst-case slow-freeze temperature profile was obtained from the longest record (76 hours) of the process of freezing a full-scale bag containing a Risa DS substitute in an upright -80 °C freezer during DS manufacturing. The worst-case slow-thaw temperature profile was traced from a 5.5 L full-scale Risa DS bag frozen on a laboratory benchtop at room temperature, and the thaw time was 27 hours.

[0531] The high-speed FT temperature profile is Fig. 6b It is shown in [Figure]. The worst-case fast-freezing temperature profile was obtained from the shortest recorded time (5 hours) during the process of freezing a 6 L Celsius bag containing 0.5 M sodium chloride in a -80 °C rapid freezer for DS manufacturing. The worst-case fast-thawing profile was based on the forced-air convection chamber (FACC) thawing process. Since full-scale bag thawing data using FACC was not available at the time the laboratory-scale particle formation study was conducted, the FACC thawing temperature profile was estimated using the heat transfer modeling described in detail below. The modeled temperature profile for a thawing time of 9 hours is Fig. 6b It was shown in.

[0532] To further evaluate the effect of storage temperature on Risa stability and particle formation, storage time studies for Risa 180 UHC DS were conducted at room temperature (approx. 21.5 ℃), in a refrigerator (4 ℃), and in a freezer (-20 ℃), as described in detail below.

[0533] FT SDM FACC Thawing Temperature Profile Modeling

[0534] The time for heating the frozen solid or the liquid solution was as follows,

[0535]

[0536] Equation S1

[0537] Among the above equations T(t) is the temperature of DS at time t.

[0538] Heating of a frozen state and a liquid state follows the same physical laws. Therefore, Equation S1 It was used to model the heating interval stages of solid and liquid solutions frozen during thawing, but different physical properties (heat capacity, density) were used for solids and liquids.

[0539] The time for the phase transition (melting) was calculated using the following heat balance equation.

[0540]

[0541] Equation S2

[0542] Therefore, the total thawing time was as follows.

[0543]

[0544] Equation S3

[0545] The input parameters used in the heat transfer model to predict thawing kinetics Table 8This is shown in [Figure]. Risa DS was a water-based formulation solution, and therefore, the heat capacity and density of water were used to simplify the modeling. In this study, the model predicted the thermodynamics when applied to FACC thawing for a 6 L Celsius bag filled with 5.5 L of water. The modeled thawing time was 9 hours.

[0546]

[0547] Risa UHC Storage Time Study

[0548] To further evaluate the effect of temperature on Risa stability and particle formation, laboratory-scale Risa UHC PUR DS was stored at room temperature (approx. 21.5 °C) and in a refrigerator (4 °C) for up to 10 days, and the samples were Table 9 As shown in [the table], it was tested using PQ assay, PS-20, turbidity, and MFI. Novelly generated 185 mg / mL Risa UHC DS in 10 mM acetate, 185 mM trehalose, 0.2 g / L PS-20, and pH 5.5 was used for storage time studies of 0, 1, 2, 3, 5, 7, and 10 days in two replicates (1 mL in a cryovial) at 4 ℃, room temperature, and approximately 21.5 ℃. Dispenses were taken at each interval and immediately stored in a -80 ℃ freezer to enable batch-by-batch analysis testing for PQ.

[0549] To evaluate whether prolonged -20°C from transport conditions could affect protein stability and lead to particle formation, Risa UHC DS was dispensed into three small PETG bottles each containing 6.5 mL of DS and three cryovials each containing 1.5 mL of DS, and Table 9As shown in [Figure], they were labeled T0, T2, and T4, respectively. The T0 sample was immediately frozen at -80°C. The remaining two bottles and cryovials were kept in a -20°C freezer for the corresponding periods. After 2 and 4 weeks, the bottles and cryovials were transferred to a -80°C freezer, respectively. All samples were submitted at once for MFI, PS-20, and PQ tests.

[0550]

[0551] Transport at -20 ℃ SDM

[0552] Risa 180 UHC BDS was scheduled to be transported at -20°C using a va-Q-tainer. To understand whether gradual temperature changes during long-term transport could lead to the formation of protein aggregates and SVP, and S with a 30 mL Celsius bag 3 To verify whether the Benchtop System SDM is a platform representative of full-scale Celsius bag particle formation studies, a 9-day extended full-scale transport study of -20 ℃ va-Q-tainers via air and land transport from the DS site to the DP site was conducted alongside the SDM. Additionally, to further evaluate the potential risks of particle formation, a previously recorded worst-case transport temperature profile of 12 days at -20 ℃ was also studied in the SDM.

[0553] Fig. 6cAs shown in [figure], the -20 ℃ va-Q-tainer transport temperature profile over 9 days monitored from a full-scale 6L bag transport study was applied to Risa 180 UHC and Risa 150 control groups using SDM with 30 mL bags in a controlled FT system. The full-scale transport study included five process steps: (1) freezing Risa DS to -70 ℃ using the slowest rapid freezer freezing speed of 8.1 hours, which is the worst-case full-scale condition; (2) equilibrating from -70 ℃ to -25 ℃ and storing for 12 hours to simulate the waiting time for the transport consignor; (3) increasing the transport temperature from -25 ℃ to -2 ℃ over 9.4 days of full-scale va-Q-tainer transport; (4) freezing from -2 ℃ to -70 ℃ for 11 hours to simulate full-scale freezing at the DP receiving site; and (5) thawing to 21 ℃ using the DP site FACC system for sampling for analysis tests. In total, considering one cycle of DS FT, one SDM transport cycle took 11 days (271 hours).

[0554] Two extended cycles of historical worst-case transport at -20°C over 12 days were also performed on the SDM platform for Risa 180 UHC and Risa 150 control subjects. One cycle of the transport temperature profile is Fig. 6dThis is shown in [figure]. One cycle of the worst-case transport process included five steps: (1) freezing Risa DS to -70 ℃ using the slowest freezing of 76 hours, which is the worst-case full-scale condition; (2) equilibrating from -70 ℃ to -40 ℃ and storing for 12 hours to simulate the waiting time of the transport consignor; (3) increasing the transport temperature from -40 ℃ to -10 ℃ over 12 days of worst-case va-Q-tainer transport; (4) freezing from -10 ℃ to -70 ℃ for 40 hours using the slowest full-scale freezing to simulate the DP site receiving the transported BDS bag; and (5) further thawing to 20 ℃ using 27 hours of thawing under worst-case conditions for sampling for analysis tests. Considering one cycle of the worst-case DS low-speed FT, one worst-case transport cycle took 19 days (450 hours).

[0555] BDS mixed SDM

[0556] In addition to evaluating the effect of temperature, the influence of mechanical stress during BDS mixing on Risa DS particle formation was also investigated. For the process development and manufacturing of Risa 180 UHC, a 200 L stainless steel (SS) tank equipped with a bottom-mounted mixer and a 50 L Cytiva Xcellerex single-use mixer (XDM) (SUM) will be used at the manufacturing site for final BDS mixing. Mechanical power input and shear stress from GMP BDS mixing were applied to the SDM mixing conditions. GMP process parameters such as impeller speed and mixing time were justified using a Mettler Toledo EasyMax 402 system utilizing a 100 mL mixing vessel equipped with crescent-shaped agitator blades. The agitator shaft was adjusted to a zero-mark line on the magnetic drive so that the crescent-shaped agitator blades were positioned at the bottom of the vessel, thereby mimicking a bottom-mounted mixer for both the tank and the SUM. Approximately 32 mL of liquid could be submerged in the EasyMax 100 mL mixing vessel; therefore, 40 mL of Risa DS was used for all mixing SDM experiments. The experiments were temperature-controlled at room temperature of 21.5 °C to mimic the GMP BDS mixing environment. The power input per unit volume and the shear rate of the SDM mixing were maintained to match full-scale mixing.

[0557] Since mixing requires the transfer of mechanical energy and thus mixes fluids within the container, the level of exposure to mechanical energy for mixing is the average power input per unit volume ( P / V It can be quantified as ), which is defined as follows, and

[0558]

[0559] Equation 3

[0560] Among the above equations P is the mixer power input, and Vis the total fluid volume within the container, and N p is the impeller power number, and N is the impeller rotation speed, and D is the impeller diameter, and ρ is the density of the fluid.

[0561] The purpose of the mixing operation was to ensure homogeneity after processing. The blend time required to reach 95% homogeneity for the single-phase fluid in the GMP mixing vessel was further calculated using the Dynochem Mixing and Heat Transfer Modeling Toolbox. Furthermore, a mathematical model was developed regarding the relationship between power input per unit volume, mixing rate, and filling volume for the BDS SS tank and BDS SUM to evaluate the risk level of particle formation. The experimental conditions for SDM mixing are described in detail below.

[0562] The average turbulent shear rate of the mixing vessel was calculated using the following experimental formula, and

[0563]

[0564] Equation S4

[0565] In the above equation, μ is the viscosity of the fluid.

[0566] The SDM mixing experimental conditions are Equations 3 and S4 It was calculated based on Table 10 It was summarized in. Laboratory-scale DS mixing experiments were Table 10 It was performed according to the mixing parameters listed in numbers 1 to 4. Samples were taken before and after mixing for analysis.

[0567]

[0568] Analysis method

[0569] Risa DS was sampled from the BSC into sterile PETG bottles or sterile cryovials. For all particle formation analyses, a 6.5 mL sample was taken from a 30 mL PETG bottle for MFI particle count measurement, and a 15 mL sample was taken from a 30 mL PETG bottle for turbidity measurement. All samples were analyzed in a fresh state without additional FTs. For representative PQ tests, all three 1.5 mL samples were taken from a 2 mL cryovial, and the cryovial samples were frozen in a -80 ℃ freezer until the time of testing.

[0570] MFI. SVP concentrations in the range of 2 to 300 μm were quantified using a Protein Simple MFI 5100 instrument (Bio-techne). Each Risa sample was measured in triplicate, and the average particle number values ​​were reported.

[0571] Turbidity. Turbidity measurements for the DS samples were performed using a 2100Q portable turbidimeter (Hach).

[0572] PQ was confirmed by performing SEC, cation exchange chromatography (CEX), and non-reducing capillary electrophoresis sodium-dodecyl-sulfate (CE-SDS-NR) analysis tests. The polysorbate-20 (PS-20) content was also tested to verify that the surfactant had not degraded.

[0573] SEC.Risa DS monomers and soluble aggregates were quantified using an analytical SEC with an Acquity UPLC protein BEH SEC 200 column (4.6 x 300 mm, 1.7 μm particle size) on a 1260 Infinity HPLC system. The mobile phase consisted of 200 mM L-arginine, 120 mM ammonium sulfate, 10% isopropyl alcohol, and pH 7.3, with a flow rate of 0.2 mL / min. The sample injection volume was 6 μL, the target load was 30 μg, and the run time was 25 min. The percentage distributions of high molecular weight species (HMW), monomeric species, and low molecular weight species (LMW) were calculated using peak areas.

[0574] CEX. The Risa DS charge variant is ProPac using the above HPLC instrument. TM Detection was performed using an analytical CEX equipped with a WCX-10 HPLC non-porous column (4 x 250 mm, 10 μm particle size). Mobile phase A was 20 mM (4-morpholino) ethanesulfonic acid monohydrate (MES) at pH 6.5, and mobile phase B was 20 mM MES, 500 mM sodium chloride, at pH 6.5. The sample was diluted to 1 mg / mL with mobile phase A, and the injection volume was 100 μL. Salt gradient elution of 7 to 100 %B was performed at a flow rate of 1 mL / min, and the run time was 50 minutes. The percentage distributions of basic charge variants, major peaks, and acid charge variants were calculated using peak areas.

[0575] CE-SDS-NR.The purity of Risa DS was determined using a bare fused silica capillary (50 μm x 30.5 cm length, SCIEX). 100 μL of CE sample (5 μL of 10 mg / mL Risa mixed with 95 μL of SDS matrix) was used per measurement. Electrophoretic separation was achieved over a distance of 10 cm, and subsequent detection was performed at 214 nm using a PA800 Plus CE system equipped with a UV detector. The main peak, the sum of the LMW forms, and the sum of the HMW forms were reported as the percentage calibration peak area (% CPA), which was defined as the CPA for the sum of all CPAs.

[0576] PS-20 content test. The PS-20 content in the final DS was determined using the above HPLC coupled with a Corona Veo RS charged aerosol detector with an Oasis MAX online column (2.1 x 20 mm, 30 μm particle size, 8 nm pore size, Waters).

[0577] Background imaging (BMI) and fluorescence microscopy (FMM).

[0578] To determine the protein composition of particles invisible to the naked eye, Risa DS samples from the worst-case mixed conditions were loaded onto fresh black membrane plates, washed with water (injection water clean level), and dried under vacuum. The dried membrane plates were imaged for BMI using an Aura system. After BMI measurement, the membrane plates were labeled with 10 mM aqueous thioflavin T, dried again under vacuum, and applied for FMM measurement using a Halo Aura system. The dye thioflavin was able to selectively label the β-sheets of the proteinaceous particles. The relative fraction of proteinaceous particles was determined from the ratio of fluorescence to the total number of particles.

[0579] V max Filtration study on

[0580] Filtration experiments were performed at a constant target pressure of 15 psi using a PendoTech filter screening and pressure monitoring system. Filtration volume was monitored using a Mettler Toledo XS 205 analytical balance. Risa DS was 3.5 cm³ until filter clogging was achieved or all DS samples were consumed. 2 It was loaded sequentially onto an Optiscale 25 Durapore PVDF 0.22 μm monolayer sterile filter. V for each batch of DS was calculated using a flux decay model. max The announced volumetric load capacity was calculated as. This V max It was the maximum liquid volume per unit area and was correlated with the corresponding MFI data.

[0581] 6.3.2 Results

[0582] Risa DS characteristics

[0583] The effect of low temperature on Risa DS pH shift during FT FIGS. 7a to 7b This is shown in [figure]. During the freezing process, the pH values ​​for both Risa 180 UHC and the Risa 150 control increased as the temperature decreased to -30 °C. During the thawing process, the pH for Risa DS decreased as the temperature increased, dropping to its starting pH value when the temperature returned to the starting point of room temperature. For the freezing process Fig. 7a The pH shift for Risa 180 UHC from 5.5 to 6.5, Fig. 7b It was observed that the pH shift from 5.7 to 6.4 for the Risa 150 control group was greater than that of the control group.

[0584] The effects of concentration and temperature on Risa DS density Fig. 8aThis was shown in [figure]. The density of the Risa DS solution increased as the concentration of mAb increased. Additionally, increasing the temperature of the Risa DS solution led to a decrease in density. The degree of density change as a function of temperature did not differ between the Risa 180 UHC and Risa 150 control groups.

[0585] The effects of concentration and temperature on the apparent viscosity of Risa DS Figures 8b and 8c It is shown in [figure]. The viscosity of Risa DS decreases as the temperature decreases ( Fig. 8b ) and as the concentration increases ( Fig. 8c ) increased exponentially. The viscosity of Risa 180 UHC was twice that of the Risa 150 control viscosity at room temperature, and this difference became more pronounced as the temperature decreased.

[0586] DS Storage and Transport SDM

[0587] In this study, a temperature-controlled Celsius® S that enables heat transfer rates equivalent to those of full-scale storage and transport processes 3 The SDM platform was used. The SDM exhibited the same kinetics as the full-scale process at the freeze or thaw front. The risk of particle formation during the Risa DS FT process and transport was estimated using the SDM. Additionally, a full-scale transport study was conducted, and the results of protein stability and particle formation assessments at both scales were demonstrated by comparing them with parallel SDM.

[0588] FT SDM under worst-case conditions

[0589] To understand the effects of different freeze-thaw rates on the stability and particle formation of Risa BDS in the GMP manufacturing process, worst-case scenarios of low-speed and high-speed FT temperature profiles of a nominal 6 L full-scale Celsius BDS bag were used. ® S 3It was applied to a 30 mL laboratory-scale Celsius bag using a benchtop FT system. Figures 6a and 6b As shown in [figure], the low-speed to low-speed FT cycle took 76 hours to freeze to -70°C and 27 hours to thaw to 18°C, representing the lowest FT rate of a typical upright -80°C freezer freezing and RT benchtop thawing process. In comparison, the high-speed to high-speed FT cycle took 5 hours to freeze to -70°C and 9 hours to thaw to 18°C, representing the highest FT rate of a -80°C rapid freezer freezing and FACC thawing process.

[0590] To confirm particle formation, the Risa BDS of each FT cycle was measured by MFI and turbidity, and the results FIGS. 9a to 9d, 10a and 10b It was shown in. Fig. 9a From, for Risa 180 UHC, an 8-fold increase in SVP was observed after the first cycle of low-speed FT, but no significant change was observed until the third cycle. Fig. 9b From, the first of the low-speed FT No increase in SVP was observed in the Risa 150 control group after the first cycle, but an increase was detected after the second cycle. In comparison, Fig. 9c From, while an increase in SVP was observed for Risa 180 UHC after the second cycle of high-speed FT, Fig. 9d From this, no increase in SVP was detected for the Risa 150 control group after three cycles of high-speed FT. In summary, changing the FT rate from low to high resulted in a decrease in SVP for both the Risa 180 UHC and 150 control groups. The potential for particle formation by FT increased with increasing Risa concentration.

[0591] Figures 10a and 10bAs shown in [figure], after 3 cycles of low-speed or high-speed FT, turbidity did not show a significant change for both Risa 180 UHC and Risa 150 control (3.9 to 4.1 NTU).

[0592] SEC / CEX / CE-SDS-NR analysis was also performed to evaluate the effect of temperature on the PQ of Risa BDS. FIGS. 11a to 11d As shown in [figure], the monomer content determined by SEC, the charged variants by HMW and CEX, and the purity and low molecular weight (LMW) species by CE-SDS-NR remained unchanged for both low-speed and high-speed FT for Risa 180 UHC and Risa 150 control after up to 3 cycles.

[0593] also, Table 11 As shown in [figure], PS-20 levels for Risa 180 UHC remained the same for all three cycles under worst-case conditions of low-speed and high-speed FT, confirming that there was no degradation of the surfactant polysorbate. Overall, high-speed FT was recommended for Risa 180 UHC in a rapid freezer-FACC within the GMP manufacturing process.

[0594]

[0595] To further evaluate the storage temperature conditions of Risa UHC BDS for Risa stability and quality, the final Risa UHC DS was stored at room temperature (21.5 °C) and in a cold refrigerator (4 °C) for up to 10 days, respectively. Additionally, the BDS was stored at -20 °C for an extended period of up to 4 weeks to ensure protein stability under transport conditions.

[0596] MFI and turbidity results for particle identification from storage time studies and PS-20 levels for Risa 180 UHC Table 12This is shown in [figure]. Not only did the particle count from MFI and turbidity measurements show no change, but the PS-20 levels also remained constant for all three constant temperature storage conditions. The constant PS-20 levels confirmed that there was no polysorbate decomposition.

[0597] Furthermore, PQ data from retention time studies by SEC / CEX / CE-SDS FIGS. 13a to 13c This is shown in [Figure]. The number of HMW species increased with increasing storage time at room temperature. By day 5, % HMW was 0.6%, which was double the initial storage time value of 0.3%. Additionally, basic charge variants increased with increasing storage time at room temperature. By day 5, % basics was 7.1%, which was an increase compared to the starting storage time value of 6.3%. All remaining PQ data for the storage time study were consistent with the starting point at the corresponding temperature. It was found that low-temperature storage in a 4°C refrigerator could maintain Risa more stably than storage at room temperature. Furthermore, no negative effects were found for extended studies at -20°C for up to one month. Therefore, low-temperature storage was recommended for Risa 180 UHC BDS.

[0598]

[0599] Full-scale study of transport at -20 ℃ and laboratory-scale simulation

[0600] Previously, Risa 150 BDS was transported on dry ice (nominal -78.5 °C) or at -20 °C using va-q-tainers. Va-q-tainers were qualified to maintain a constant temperature (-50 °C to 20 °C) for over 120 hours without an external energy supply. The film material in production-scale 6 L Celsius bags for Risa BDS storage is T-40 °C to -20 °C. g It was an EVA having. T gThe start was the same -24°C for both Risa 180 UHC and Risa 150 BDS (data not shown, measured by differential scanning calorimetry at the DP site). Therefore, to ensure the integrity of the bag material, -20°C was selected initially.

[0601] T of Risa BDS experienced during transport from the DS manufacturing site to the DP site g To further understand whether gradual temperature changes exceeding [value] can affect protein stability and lead to particle formation, and to use Celsius, equipped with a laboratory-scale bag SDM, as a representative platform for particle research evaluation, ® S 3 To validate the benchtop system, a 9-day full-scale transport study and parallel SDM were conducted for air and ground transport of a -20 ℃ va-Q-tainer from the DS site to the DP site. The temperature profile records from the full-scale transport study are Fig. 6c It was presented in and applied to SDM.

[0602] MFI for full-scale study transport of Risa 180 UHC before and after transport at -20 ℃ Fig. 17a ) and turbidity readings( Fig. 10c The number of SVPs detected by ) was similar, indicating that there is no concern regarding transport-induced particle formation. In addition, Figs. 17a and 10c In this case, the number of particles and turbidity in the SDM transport simulation were similar to these measurements from full-scale transport studies on Risa 180 UHC DS, confirming that SDM represents the full-scale process particle formation evaluation.

[0603] In addition, to further evaluate the potential risk of particle formation from the transport process, the previously recorded worst-case 12-day -20°C transport temperature profile for Risa BDS was also studied in SDM. The transport simulation also considered the worst-case freezing process at the DS site and the worst-case thawing process at the DP site, and one cycle of the transport temperature profile is Fig. 6d It was shown in. The transport of two extended cycles was also performed in SDM.

[0604] The MFI and turbidity results for particle verification from the worst-case transport simulation are Figs. 17c, 17d and 10d It was shown in. Fig. 17c represents the increase in SVP for Risa 180 UHC after Transport Cycle 1. Although Risa 180 UHC from different batches was manufactured with different starting material particle numbers, Fig. 9a An eightfold increase in SVP after transport cycle 2 was observed, consistent with the previous Risa 180 UHC subjected to stress through two cycles of low-speed FT under worst-case conditions. This indicates that the gradual temperature of -20 ℃ transport itself did not induce particle formation. Compared to this, Fig. 17d As shown in [figure], there was no increase in SVP for the Risa 150 control group after transport cycle 1. Similar to the turbidity readings from the FT study, Fig. 10d This indicates that there was no significant change in turbidity for both Risa 180 UHC and the Risa 150 control group after two transport cycles. Therefore, high levels of particles were not formed for the Risa 180 UHC and 150 control group after two cycles of transport at -20 ℃ under worst-case conditions, which indicates that Risa T g Concerns that transport exceeding [amount] induces particle formation have been ruled out.

[0605] In addition, the -20 ℃ transport full-scale study and Risa DS's PQ data from SDM FIGS. 12a to 12c As shown in [figure], it was detected to be constant before and after transport using SEC, CEX, and CE-SDS-NR tests. Furthermore, Table 11 As shown in [figure], PS-20 levels for Risa 180 UHC did not show any change during the full-scale transport study and up to two worst-case SDM transport cycles, which confirms that there is no polysorbate degradation during these -20°C transport conditions.

[0606] Miniaturized BDS Mixing Study

[0607] In addition to evaluating the effect of temperature, the influence of mechanical stress during BDS mixing on Risa DS particle formation was also investigated. Final BDS mixing is a critical unit operation in the production of Risa UHC DS. Final BDS mixing equipment consisting of a 200 L SS tank and a 50 L Cytiva XDM SUM was designed to mix Risa 180 UHC in the DS production process at different sites. During the mixing unit operation, the mechanical energy per unit volume and the average shear rate of the BDS mixing vessel were, respectively Equations 3 and S4 It was calculated as follows. In order for the SDM mix to represent the full-scale process, Table 10 As shown in [figure], the mechanical power input and shear stress from the GMP BDS tank were matched to the SDM mixing conditions.

[0608] Particle identification of Risa BDS mixed SDM by MFI and turbidity is FIGS. 18a to 18c This was shown in [figure]. Risa 180 UHC was more sensitive to mechanical stress caused by mechanical energy input compared to the Risa 150 control group. Fig. 18a As shown in [figure], the number of SVPs increased as the power input / volume (P / V) increased for the Risa UHC. Fig. 18bThere was no significant change in particle size for the Risa 150 control group. Worst-case conditions for Risa UHC P / V >1000 W / m 3 Is Fig. 18a A significant increase in SVP with a reading of 14,818 particles / mL (> 2 μm) by MFI at and Fig. 18c This resulted in a high turbidity reading of 5.7 NTU. Some Risa BDS batches faced filtration issues at the DP site, and investigations revealed that high levels of SVP from the BDS tank mixing unit operation caused filter clogging.

[0609] Correlation between Risa BDS filtration efficiency and particle data ( Fig. 14 From (as shown in), an MFI SVP (> 2 μm) count of > 10,000 particles / mL indicates low filtration efficiency (<150 L / m²). 2 It was correlated with ), which had the potential to clog the sterile filter (0.22 μm) used in the DP manufacturing process. Using these criteria defining a high-risk level, process parameters P / V >1000 W / m 3 It was found to have a high risk of clogging the sterile filters used in the DP manufacturing process. 517 W / m² 3 The mixing condition caused an 8-fold increase in SVP for Risa UHC, and therefore, process parameter 500 ≤ P / V ≤1000 W / m 3 It posed a moderate risk regarding the filter's low filtration capacity as it was on the safety edge. Process parameter P / V <500 W / m² 3 It must be safe, not cause concern regarding the low filtration efficiency of the sterile filter, and have a low risk of Risa UHC.

[0610] SEC / CEX / CE-SDS-NR was also tested to evaluate the effect of mechanical stress on the PQ of Risa BDS. Figures 15a and 15b As shown in [figure], the PQ properties remained unchanged for all mixing conditions. The mixture contained particles but appeared not to affect the PQ of Risa BDS. In addition, the PS-20 levels for Risa 180 UHC were Table 10 As shown in [figure], the same was maintained for the selected worst-case SDM mixing conditions, which confirms that there is no polysorbate degradation.

[0611] To further reduce the risk of particle formation, it is also important to understand the potential mechanisms associated with the content of formed SVP and the manipulation of mixing units. Worst-case mixing P / V >1000 W / m 3 The SVP formed in [location] was further analyzed using background membrane imaging (BMI) and fluorescence membrane microscopy (FMM) (described in detail above) to understand the particle composition. The relative fraction of proteinaceous particles was determined from the ratio of fluorescent particles to the total number of particles, which came out to about 98%, suggesting that most of the SVP in these samples is essentially proteinaceous.

[0612] Furthermore, in order to establish full-scale process mixing parameters to reduce the risk of particle formation, Figures 19a and 19b As shown in [figure], a mathematical model was developed regarding the relationship between power input per unit volume, mixing rate, and filling volume of BDS tanks and BDS SUM to evaluate the risk level for particle formation. The shear rate model Figures 16a and 16b It was shown in. Figures 19a and 19bAs shown in [figure], zones 0 to 500 indicated a low risk of particle formation using corresponding combinations of filling volume and rotation speed under mixing of less than 30 minutes. In contrast, zones 500 to 1000 showed a moderate risk, and zones 1000 to 1500 showed a high risk of SVP formation. To further validate the mathematical model for precision in predicting particle formation risk levels, the Risa final BDS tank mixing model Fig. 20 This is shown in [Figure]. The circles on the modeling map represent full-scale Risa batch mixing data, which correlated with significantly high levels of SVP (> 2 μm) counts (> 10,000 particles / mL). Of the six DS batches corresponding to DP bioload filtration clogging, five fell within the high-risk 1000 to 1500 mixing zone. One batch used mixing parameters from the medium-risk blue zone. The modeling for predicting the risk of significant SVP formation agreed very well with full-scale GMP data, which further validated the accuracy of the mathematical modeling.

[0613] Finally, during manufacturing, it is necessary to blend the final Risa BDS before bagging to ensure post-processing homogeneity. The blend time required to reach 95% homogeneity for the single-phase fluid in the GMP mixing vessel was further calculated using the Dynochem Mixing and Heat Transfer Modeling Toolbox. For the worst-case mixing scenario (largest filling volume and lowest rotation speed corresponding to 100% yield), the model estimated that it takes 46.6 seconds for the BDS tank to reach homogeneity and 9.0 seconds for the BDS SUM to reach homogeneity. Therefore, for the Risa UHC BDS GMP mixing vessel, a blending time of 5 minutes ≤ 30 minutes was recommended.

[0614] 6.4 Example 4: Particles observed upon dilution of risankizumab finished products DP1 and DP2

[0615] Risankizumab finished product 1 (DP1) was developed using the process and formulation described in international application PCT / US2013 / 038109. Risankizumab finished product 2 (DP2) was subsequently developed and approved by the FDA.

[0616] Risankizumab formulations DP1 and DP2 contain highly purified risankizumab APIs and are stable. However, Table 13 As shown in [figure], particles containing free fatty acids (FFA) were unexpectedly observed in DP1 and DP2, particularly when DP2 was diluted.

[0617]

[0618] 6.5 Example 5: Proteomic analysis identified candidate hitchhiker proteins

[0619] The inventors hypothesized that particle formation within the risankizumab product is attributed to the degradation of the surfactant PS20, which could lead to antibody aggregation. They hypothesized that the degradation of PS20 is caused by residual CHO cell esterase co-purified with the risankizumab bulk active ingredient. To identify the putative hitchhiker protein (HP) that may have led to the degradation of polysorbate-20 within the risankizumab finished product, host cell protein (HCP) from DP1 and DP2 bulk active ingredients (BDS) samples was concentrated and then analyzed using LC-MS / MS.

[0620] Multiple risankizumab BDS samples from each DS process were pooled to generate representative BDS materials for HCP enrichment and verification. The potential HP identified in the pooled risankizumab BDS samples was

[0621] · Estimated phospholipase B-like 2

[0622] · Sanceramidase-like

[0623] · Isoamyl acetate-hydrolyzed esterase 1 homologue

[0624] · Sphingomyelin phosphodiesterase

[0625] · Sialate O-acetylesterase-like

[0626] · Hepatic carboxyesterase-like isoform 1

[0627] · Hepatic carboxyesterase 4-like

[0628] · Ester hydrolase C11orf54 homologue isoform 1

[0629] · Lipoprotein lipase

[0630] · Calcineurin-like phosphoesterase domain-containing protein 1-like

[0631] · Contains partially peroxiredoxin-6 analogs.

[0632] By affinity purification, various hitchhiker proteins including putative phospholipase B-like 2 (PLBL2), acid ceramidase, isoamyl acetate-hydrolyzing esterase 1, sphingomyelin phosphodiesterase, hepatic carboxyesterase-like isoform 1 (CES1), hepatic carboxyesterase 4, ester hydrolase C11orf54 homologue isoform 1, sialate O-acetylesterase-like (SIAE), calcineurin-like phosphoesterase domain-containing protein 1, and peroxiredoxin-6-like, partial (Prdx6) were identified in pooled risankizumab BDS samples ( Fig. 21 ). Some of these proteins, for example, PLBL2 and sanceramidase, have been previously reported to be present in other antibody raw materials (Literature [Graf et al. (2021) J. Pharm. Sci. 110:3358-3567]).

[0633] 6.6 Example 6: Development of a Risankizumab Finished Drug with Improved Stability

[0634] The inventors hypothesized that the presence of a specific type of hitchhiker protein (e.g., host cell lipase) from host cells causes particle formation in diluted risankizumab finished pharmaceutical products. To address this problem and to improve the stability and shelf life of risankizumab products, as well as to further improve their quality, a novel risankizumab active pharmaceutical ingredient, Process 4 DS, was developed. An exemplary purification process for producing Process 4 DS is described below.

[0635] CHO cells expressing risankizumab were thawed and cultured in increasing volumes in shaking flasks, cell bags, and seed bioreactor stages to provide sufficient cells for inoculation into a production bioreactor. The cell culture medium was collected by centrifugation and filtration to efficiently remove cells, providing a purified collection for further purification of the product. The purified collection was subsequently processed through a series of chromatography steps, virus inactivation, virus filtration, concentration by tangential flow filtration, buffer exchange, and final formulation. The purification process was developed to reduce host cell lipase by screening various reagents and conditions, including but not limited to, protein A chromatography washing methods and wash buffers, deep filters, chromatography column resins (e.g., AEX resin, CEX resin, MM resin and / or HIC resin), and / or conditions for ultrafiltration and dilution filtration (UF / DF) processes. Two optimized purification processes, referred to herein as Process 3 and Process 4, were established by adjusting reagents and conditions to effectively reduce specific host cell lipase levels in purified risankizumab active pharmaceutical ingredient (DS) as measured by ELISA at an acceptable yield trade-off. In addition to the purification processes, the upstream cell culture process of Process 4 was further modified to improve culture life, productivity, and robustness.

[0636] A general overview of the purification process in Process 4 is Fig. 22 It is shown in [figure]. Specifically, the cell culture medium was collected and purified by centrifugation and deep filtration using an X0HC deep filter. The purified collectible was first [examined] MabSelect SuRe TM Purification was performed using affinity chromatography with protein A resin. The eluent was subjected to low pH inactivation using phosphoric acid and then deep filtered through X0HC and D0HC deep filters. Subsequently, the risankizumab samples were Capto TM The permeate was purified by Adhere mixed-mode chromatography. Poros TM It was further purified by cation exchange chromatography using XS resin. The eluent was subjected to viral filtration. Subsequently, the load was directly spiked with a high-salt solution, followed by ultrafiltration / determinate filtration (UF / DF) using salt-free 8 DV. Subsequently, the purified bulk active pharmaceutical ingredient (BDS) was formulated and stored appropriately.

[0637] The detailed parameters of the different steps of process 4 are listed below.

[0638] Exemplary Process A

[0639] In one exemplary process, the parameters of the different steps of process 4 are as follows.

[0640] 1st Retrieval: Cells and cell debris were removed from the production bioreactor tanks using primary recovery by centrifugation and deep filtration. The 4000 L production bioreactor served as a feed tank for the 510 Alfa Laval centrifuge. The centrifuge was run at a setpoint of 5130 rpm, a feed rate of 20 L / min, and discharge intervals of 171 seconds, 181 seconds, 179 seconds, and 181 seconds for GMP1 to GMP4, respectively. The centrifuge liquid was subsequently transferred to 24 1.1 m³ 2Following the Millipore X0HC media Pod unit, two Sartopore 2 30-inch (1.8 m each) 2 ) passed through a filter train consisting of 0.45 μm / 0.2 μm capsule filters.

[0641] The bioreactor effluent was centrifuged and deep-filtered, and the filter was flushed with a target weight of 528 kg of 50 mM sodium acetate, pH 5.5 buffer. Centrifugation and filtration of the effluent were performed as a single unit operation. Filtration was performed in the fermentation chamber at ambient temperature (18 to 25 °C). The temperature of the effluent was cooled to 18 to 22 °C with a set point of 20 °C prior to filtration. The filtrate was collected in a 3000 L collection tank, cooled to 2 to 8 °C, and could be stored for up to 3 days.

[0642] MabSelect SuRe Protein A Chromatography : MabSelect SuRe Protein A chromatography was used to capture risankizumab process 4 DS from the purified collection and reduce the amount of process-associated impurities. The MabSelect SuRe Self-Pack column (GE Healthcare) had a diameter of 60 cm and a target volume of approximately 62.0 L (bed height of 21 to 23 cm). The operation was as follows: Table 14A It was carried out in a fermentation chamber at ambient temperature (18 to 25 ℃) with the process parameters shown in [figure].

[0643] The MabSelect SuRe column was operated in binding and elution modes. Three cycles of MabSelect SuRe chromatography were required to process each batch. After equilibrating the column to 50 mM sodium acetate at pH 5.5, it was loaded with 13 to 35 g of risankizumab per L of resin. Three washing steps were performed after loading. Wash solution 1 was 50 mM sodium acetate at pH 5.5. Wash solution 2 was 50 mM Tris, 1 M arginine, and pH 8.0, and wash solution 3 was 50 mM sodium acetate at pH 5.5. Elution was performed with 50 mM sodium acetate at pH 3.5. Subsequently, the column was regenerated with 0.2 M sodium hydroxide and re-equilibrated with equilibration buffer before the next cycle loading.

[0644] The loading material (2 to 8 °C) was not heated prior to loading onto the column. Eluent peak collection started at a 0.2 OD rise and ended at a 0.2 OD fall (280 nm wavelength, 1 mm path length). The eluent was fed into a 500 L single-use mixing (SUM) system and passed offline through a 0.45 / 0.2 μm filter into the collection vessel. For eluent filtration, one Sartopore 2 30-inch (1.8 m each) was used. 2 ) 0.45 μm / 0.2 μm capsule filters were used. Each eluent filter was used for three cycles of MabSelect SuRe chromatography daily. The MabSelect SuRe eluent was collected in a 1000 L single-use mixed (SUM) system and could be stored at 9 to 25 °C for up to 36 hours or cooled to 2 to 8 °C for up to 92 hours before proceeding with low pH inactivation.

[0645]

[0646] pH inactivation and POD filtrationThe purpose of the pH inactivation step was to inactivate any potential foreign viruses. The pH inactivation step was performed in a fermentation chamber at ambient temperature (18 to 25 °C). The pH of the Protein A eluent was adjusted to 3.50 ± 0.10 (measured at 18 to 25 °C) using 0.5 M phosphoric acid. After a storage time of 60 to 90 minutes, the inactivated material was neutralized to a pH of 8.0 ± 0.1 (measured at 18 to 25 °C) using 2.0 M Tris. The conductivity of the material had to be within the range of 3.6 to 4.8 mS / cm (measured at 24 to 26 °C) for subsequent filtration; accordingly, dilution was not required prior to POD filtration.

[0647] Deep filtration was used to remove particles within the process stream and reduce impurity levels. The filter train consisted of two 1.1 m units. 2 Following the Millipore D0HC media Pod unit, 5 1.1 m 2 Millipore X0HC media pod unit and Sartopore 2 30-inch (1.8 m each) 2 It was composed of 0.45 μm / 0.2 μm capsule filters. The filter train was approximately 37.5 L / m² 2 After equilibrating with 25 mM Tris, 25 mM sodium chloride, and pH 8.0, the contents of the feed tank were filtered. The filter train was subsequently processed at approximately 20.0 L / m³ 2 The solution was rinsed with 25 mM Tris, 25 mM sodium chloride, pH 8.0. The filtrate was collected in a 1000 L SUM system and subjected to Capto Adhere chromatography on the same day.

[0648] Capto Adhere chromatography: The impurity levels in the process stream were reduced using a Capto Adhere chromatography step. The column packed with Capto Adhere resin had a diameter of 45 cm and a target volume of 25.4 L (bed height of 16 cm). The operation was as follows: Table 14B-1 The process was performed in a purification chamber at ambient temperature (18 to 25 °C) with the process parameters shown in [figure]. The Capto Adhere column was operated in pass-through mode. One cycle of Capto Adhere chromatography was required to process each batch. The column was first pre-equilibrated with 2 M sodium chloride, and then equilibrated with 25 mM Tris, 25 mM sodium chloride, and pH 8.0. The column was loaded with 150 to 300 g of risankizumab per L of resin and then washed with 260 mM Tris, pH 8.0. The column was regenerated with 0.1 M acetic acid, pH 2.9, and 2 M sodium chloride. After each batch, the column was sterilized with 1 M sodium hydroxide and stored in 0.1 M sodium hydroxide.

[0649] The load material was maintained at 18 to 25 °C before loading onto the column. During loading, product pass-through collection started at 1 OD of the peak front and terminated at 5 OD of the peak tail during washing (280 nm wavelength, 1 mm path length). Capto Adhere pass-through was collected in a 1000 L SUM system.

[0650] Capto Adhere FTW was adjusted to a target pH of 5.25 on the day of Capto Adhere chromatography. To prepare Poros XS rods, the Capto Adhere FTW material was titrated with 2 M acetic acid to a pH of 5.25 ± 0.1 (measured at 18 to 25 °C), and conductivity was adjusted to 4.5 to 7.5 mS / cm using WFI if necessary. The adjusted Capto Adhere FTW was placed in one Sartopore 2 30-inch (1.8 m) 2 ) It was filtered with a 0.45 μm / 0.2 μm capsule filter. The filtered and adjusted Capto Adhere FTW can be maintained at 9 to 25 °C for up to 56 hours or cooled to 2 to 8 °C for up to 135 hours before proceeding to Poros XS chromatography.

[0651]

[0652] Poros XS Chromatography : A Poros XS chromatography step was used to reduce process-related impurities such as basic species and 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 as follows: Table 14C-1The process was performed in a purification chamber at ambient temperature (18 to 25 °C) with the process parameters shown in [figure]. The Poros XS column was operated in binding and elution modes. Two cycles of Poros XS chromatography were required to process each batch. The column was equilibrated with 50 mM sodium acetate, 31 mM sodium chloride, and pH 5.25. The loading range for the column was 30 to 60 g of risankizumab per liter of resin. The washing step was 50 mM sodium acetate, 31 mM sodium chloride, and pH 5.25. Elution was performed with 50 mM sodium acetate, 181 mM sodium chloride, and pH 5.25. The column was regenerated with 25 mM Tris, 3 M sodium chloride, and pH 8.5 before the next cycle. Finally, the column was sterilized with 1.0 M sodium hydroxide and stored in 0.1 M sodium hydroxide. The columns were sterilized and stored at the end of the last cycle of each batch execution.

[0653] Poros XS loads on the day of Poros XS chromatography in one Sartopore 2 30-inch (1.8 m each) 2 ) It was filtered through 0.45 μm / 0.2 μm capsule filters. Eluent peak collection started at 1 OD of the peak front during elution and terminated at 5 OD of the peak tail (280 nm wavelength, 1 mm path length). For eluent filtration as the eluent exited the chromatography skid and entered the collection vessel, the eluent was filtered through two Sartopore 2 30-inch (1.8 m each) tubes. 2) Passed through a 0.45 / 0.2 μm filter using a 0.45 μm / 0.2 μm capsule filter. The filter was used for two cycles of Poros XS chromatography on the day of the process. The Poros XS eluent was collected in a 500 L SUM system and could be stored for 54 hours at 9 to 25 °C or cooled to 2 to 8 °C for up to 132 hours before proceeding with nanofiltration.

[0654]

[0655] Virus filtration Virus filtration provided the ability to remove exotic viruses larger than 20 nm in size. The process was performed in a purification room at ambient temperature (18 to 25 °C). The virus filtration filter train consisted of one Millipore Virosolve Pro Magnus 2.2 Shield (1.53 m each) 2 ) 0.1 μm capsule filter, two parallel Millipore Virosolve Pro Magnus 2.1 filters (0.51 m each) 2 ) and a single Sartopore 2 30 inches (1.8 m) 2 It consisted of 0.45 μm / 0.2 μm capsule filters. Before filtration of the product, each pretreatment filter and Virosolve nanofilter was flushed with more than 52 L of WFI, followed by flushing with more than 51 L containing 50 mM sodium acetate, 181 mM sodium chloride, pH 5.25. Filtration of the product was performed using a quattroflow pump, with a target nanofilter pressure of 23 psig and an upper limit of 32 psig. Post-filtration flushing was performed with 31 L of 50 mM sodium acetate, 181 mM sodium chloride, pH 5.25. The viral filtrate can be stored for up to 56 hours at 9 to 25 °C or cooled to 2 to 8 °C for up to 133 hours.

[0656] UF / DFThe product was concentrated using the UF / DF step and filtered into the desired volume using the appropriate buffer. The process utilized a 30 kD Millipore Pellicon3 Biomax UF module and D-screen, and the following Table 14D-1 The process was carried out in a purification chamber at ambient temperature (18 to 25 °C) with the process parameters shown in [Figure]. The loading material was diluted 10-fold with 5 M sodium chloride (9 parts of nanofiltrate to 1 part of 5 M sodium chloride), and the pH of the loading material was adjusted to 5.25 ± 0.1 (measured at 18 to 25 °C) using 2 M sodium acetate. Before loading the adjusted loading material onto the UF / DF membrane, a single Sartopore 2 30-inch (1.8 m²) was transferred into a recirculation tank. 2 It was filtered using a 0.45 μm / 0.2 μm capsule filter.

[0657] The total UF / DF is 11.4 m 2 10 1.14 m² for the membrane area 2 The procedure was performed on a Z-2300 skid using a membrane. The UF / DF load was concentrated to a target of 50 g / L, then filtered in a fixed volume with an 87 g / L trehalose dihydrate solution and concentrated to 235 g / L. As the retentate was removed from the ultrafiltration membrane and system and fed into the collection vessel, a 100 L Impulse Mixer system, the retentate was filtered through a single Sartopore 2 10-inch 0.45 / 0.2 μm (0.45 m) filter. 2 ) was passed through a sterile filter. The ultrafiltration system was rinsed with approximately 7 kg of 70 g / L trehalose dihydrate rinse solution to recover the product retained in the system. Both Rinse Solution 1 and Rinse Solution 2 were tested using the same Sartopore 2 10-inch 0.45 / 0.2 μm (0.45 m) filter. 2The rinse solution was individually transferred to the rinse solution 1 collection bag and the rinse solution 2 collection bag through a sterile filter. After recovering the rinse solution, the concentrate was diluted with appropriate amounts of rinse solution 1 and rinse solution 2 to achieve a concentration target of 200 g / L.

[0658] The concentrate pool was formulated with the addition of a 20x formulation buffer consisting of 200 mM acetate, 185 mM trehalose, 0.4% Tween 20, pH 5.50. The final bulk active pharmaceutical ingredient was diluted with 180 g / L risankizumab in a 1x formulation buffer consisting of 10 mM acetate, 185 mM trehalose, 0.02% Tween 20, pH 5.5. Subsequently, the formulated UF / DF concentrate was filtered through a 0.22 μm Millipak 200 sterile filter (0.1 m 2 The final formulated UF / DF concentrate can be stored at 9 to 25°C for up to 24 hours or cooled to 2 to 8°C for up to 120 hours before proceeding to the final bagging step.

[0659]

[0660] bagging The purpose of bagging was to package and store the final bulk active pharmaceutical ingredient. The operation was performed in a purification room at ambient temperature (18 to 25 °C). The filtered and formulated UF / DF concentrate was pumped into a sterile 6 L Celsius FFT bag. The bag was filled to a volume of approximately 6 kg. The Process 4 parameter objectives are as follows: Table 14E-1 It was summarized in.

[0661]

[0662]

[0663] Risankizumab produced by processes 3 and 4 was formulated to produce finished product 3 (DP3) and finished product 4 (DP4), respectively, both having a PS20 of 0.20 mg / mL. No visible or glittering particles were observed in the DP3 form during 24 months of stability at 4°C. Another form further confirmed the increased stability of DP3 compared to DP2. Specifically, no visible product-related particles were observed in this DP3 form at 6 months, whereas glittering particles were observed in a similar DP2 form at 3 months. Similarly, no visible or glittering particles were observed in the DP4 vial form during 18 months of stability at 4°C.

[0664] Exemplary Process B

[0665] Alternatively, in another exemplary process, the parameters of the different steps of process 4 are as follows.

[0666] 1st Retrieval: Cells and cell debris were removed from the production bioreactor tanks using primary recovery by centrifugation and deep filtration. The 3000 L production bioreactor served as a feed tank for the 710 Alpha Laval centrifuge. The centrifuge was run at a feed rate of 30 L / min at a setpoint of 5555 rpm, with discharge intervals of 215 seconds for GMP1 to GMP3 and 252 seconds for GMP4, respectively. The centrifuges were subsequently transferred to 18 1.1 m³ 2 Following the Millipore X0HC media pod unit, two Sartopore 2 30-inch (1.8 m each) units 2 ) passed through a filter train consisting of 0.45 μm / 0.2 μm capsule filters.

[0667] The bioreactor effluent was centrifuged and deep-filtered, and the filter was flushed with a target weight of 396 kg of 50 mM sodium acetate, pH 5.5 buffer. Centrifugation and filtration of the effluent were performed as a single unit operation. Filtration was performed in the fermentation chamber at ambient temperature (18 to 25 °C). The temperature of the effluent was cooled to 18 to 22 °C with a set point of 20 °C prior to filtration. The filtrate was collected in a 3000 L collection tank, cooled to 2 to 8 °C, and could be stored for up to 3 days.

[0668] MabSelect SuRe Protein A Chromatography : MabSelect SuRe Protein A chromatography was used to capture risankizumab process 4 DS from the purified collection and reduce the amount of process-associated impurities. The MabSelect SuRe Self-Pack column (GE Healthcare) had a diameter of 60 cm and a target volume of approximately 62.0 L (bed height of 21 to 23 cm). The operation was as follows: Table 14A-2 It was carried out in a fermentation chamber at ambient temperature (18 to 25 ℃) with the process parameters shown in [figure].

[0669] The MabSelect SuRe column was operated in binding and elution modes. Three cycles of MabSelect SuRe chromatography were required to process each batch. After equilibrating the column to 50 mM sodium acetate at pH 5.5, it was loaded with 13 to 35 g of risankizumab per liter of resin. Three washing steps were performed after loading. Wash solution 1 was 50 mM sodium acetate at pH 5.5. Wash solution 2 was 50 mM Tris, 1 M arginine, at pH 8.0, and wash solution 3 was 50 mM sodium acetate at pH 5.5. Elution was performed with 50 mM sodium acetate at pH 3.5. Subsequently, the column was regenerated with 0.2 M sodium hydroxide and re-equilibrated with equilibration buffer before the next cycle loading.

[0670] The loading material (2 to 8 °C) was not heated prior to loading onto the column. Eluent peak collection started at a 0.2 OD rise and ended at a 0.2 OD fall (280 nm wavelength, 1 mm path length). The eluent was fed into a 300 L portable stainless steel tank or a 500 L single-use mixing (SUM) system, passed offline through a 0.45 / 0.2 μm filter, and then introduced into the collection vessel. For eluent filtration, one Sartopore 2 30-inch (1.8 m each) was used. 2 ) 0.45 μm / 0.2 μm capsule filters were used. Each eluent filter was used daily for three cycles of MabSelect SuRe chromatography. MabSelect SuRe eluents were collected in a 700 L portable stainless-steel tank or a 1000 L single-use mixing (SUM) system and could be stored at 9 to 25 °C for up to 1 day or cooled to 2 to 8 °C for up to 3 days before proceeding with low pH inactivation.

[0671]

[0672] pH inactivation and POD filtration The purpose of the pH inactivation step was to inactivate any potential foreign viruses. The pH inactivation step was performed in a fermentation chamber at ambient temperature (18 to 25 °C). The pH of the Protein A eluent was adjusted to 3.5 ± 0.1 (measured at 18 to 25 °C) using 0.5 M phosphoric acid. After a storage period of 60 to 90 minutes, the inactivated material was neutralized to a pH of 8.0 ± 0.1 (measured at 18 to 25 °C) using 2.0 M Tris. The conductivity of the material had to be within the range of 3.8 to 4.8 mS / cm (measured at 24 to 26 °C) for subsequent filtration; accordingly, dilution was not required prior to POD filtration.

[0673] Deep filtration was used to remove particles within the process stream and reduce impurity levels. The filter train consisted of two 1.1 m filters at GMP1 and GMP2. 2 Following the Millipore D0HC media Pod unit, 5 1.1 m 2 Millipore X0HC media pod unit and Sartopore 2 30-inch (1.8 m each) 2 It consisted of 0.45 μm / 0.2 μm capsule filters. To increase the stage yield, the number of D0HC and X0HC filters was reduced to 1 and 4 in GMP3 and GMP4, respectively. The filter train was approximately 37.5 L / m² 2 After equilibrating with 25 mM Tris, 25 mM sodium chloride, and pH 8.0, the contents of the feed tank were filtered. The filter train was subsequently processed at approximately 20.0 L / m³ 2 The solution was rinsed with 25 mM Tris, 25 mM sodium chloride, 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.

[0674] Capto Adhere chromatography : The impurity levels in the process stream were reduced using a Capto Adhere chromatography step. 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 as follows: Table 14B-2The process was performed in a purification chamber at ambient temperature (18 to 25 °C) with the process parameters shown in [figure]. The Capto Adhere column was operated in pass-through mode. One cycle of Capto Adhere chromatography was required to process each batch. The column was first pre-equilibrated with 2 M sodium chloride, and then equilibrated with 25 mM Tris, 25 mM sodium chloride, and pH 8.0. The column was loaded with 150 to 300 g of risankizumab per L of resin and then washed with 260 mM Tris, pH 8.0. The column was regenerated with 0.1 M acetic acid, pH 2.9, and 2 M sodium chloride. After each batch, the column was sterilized with 1 M sodium hydroxide and stored in 0.1 M sodium hydroxide.

[0675] The load material was maintained at 18 to 25 °C before loading onto the column. During loading, product pass-through collection started at 1 OD of the peak front and terminated at 5 OD of the peak tail during washing (280 nm wavelength, 1 mm path length). Capto Adhere pass-throughs were collected in a 1000 L portable stainless-steel tank or a 1000 L SUM system.

[0676] Capto Adhere FTW was adjusted to a target pH of 5.25 on the day of Capto Adhere chromatography. To prepare Poros XS rods, the Capto Adhere FTW material was titrated with 2 M acetic acid to a pH of 5.25 ± 0.1 (measured at 18 to 25 °C), and conductivity was adjusted to 4.5 to 7.5 mS / cm using WFI if necessary. The adjusted Capto Adhere FTW was placed in one Sartopore 2 30-inch (1.8 m) 2) Filtered with a 0.45 μm / 0.2 μm capsule filter. The filtered and adjusted Capto Adhere FTW can be stored at 9 to 25 °C for up to 1 day or cooled to 2 to 8 °C for up to 3 days before proceeding with Poros XS chromatography.

[0677]

[0678] Poros XS Chromatography : Process-related impurities such as basic species and host cell proteins and leached protein A were reduced using a Poros XS chromatography step. Poros XS The resin-packed column had a diameter of 60 cm and a target volume of 56.5 L (bed height of 20 cm). The operation was as follows: Table 14C-2The process was carried out in a purification chamber at ambient temperature (18 to 25 °C) with the process parameters shown in [figure]. The Poros XS column was operated in binding and elution modes. Two cycles of Poros XS chromatography were required to process each batch. The column was equilibrated with 50 mM sodium acetate, 31 mM sodium chloride, and pH 5.25. The loading range for the column was 25 to 50 g of risankizumab per liter of resin. The washing step was 50 mM sodium acetate, 31 mM sodium chloride, and pH 5.25. Elution was performed with 50 mM sodium acetate, 181 mM sodium chloride, and pH 5.25. While the elution buffer pH for GMP1 to GMP3 was close to the target value, the elution buffer pH for GMP4 was adjusted to 5.34, the higher end of the elution pH batch record range (5.15 to 5.35), using 5 M sodium hydroxide to improve the Poros XS step yield. The column was regenerated with 25 mM Tris, 3 M sodium chloride, and pH 8.5 before the next cycle. Finally, the column was sterilized with 1.0 M sodium hydroxide and stored in 0.1 M sodium hydroxide. The column was sterilized and stored at the end of the last cycle of each batch run.

[0679] Poros XS loads on the day of Poros XS chromatography in one Sartopore 2 30-inch (1.8 m each) 2 ) It was filtered through 0.45 μm / 0.2 μm capsule filters. Eluent peak collection started at 1 OD of the peak front during elution and terminated at 5 OD of the peak tail (280 nm wavelength, 1 mm path length). For eluent filtration as the eluent exited the chromatography skid and entered the collection vessel, the eluent was filtered through two Sartopore 2 30-inch (1.8 m each) tubes. 2) Passed through a 0.45 / 0.2 μm filter using a 0.45 μm / 0.2 μm capsule filter. The filter was used for two cycles of Poros XS chromatography on the day of the process. The Poros XS eluent was collected in a 500 L portable stainless-steel tank or a 1000 L SUM system and could be stored at 2 to 25 °C for up to 5 days before proceeding to nanofiltration.

[0680]

[0681] Virus filtration Virus filtration provided the ability to remove exotic viruses larger than 20 nm in size. The process was performed in a purification room at ambient temperature (18 to 25 °C). The virus filtration filter train consisted of two parallel Millipore Virosolve Pro Magnus 2.1 Shields (0.51 m each). 2 ) 0.1 μm capsule filter, two parallel Millipore Virosolve Pro Magnus 2.1 filters (0.51 m each) 2 ) and a single Sartopore 2 30 inches (1.8 m) 2 It consisted of 0.45 μm / 0.2 μm capsule filters. Before filtration of the product, each pretreatment filter and Virosolve nanofilter was flushed with more than 102 L of WFI, followed by flushing with more than 26 L of 50 mM sodium acetate, 181 mM sodium chloride, pH 5.25. Filtration of the product was performed using a quattroflow pump, with a target nanofilter pressure of 23 psig and an upper limit of 32 psig. Post-filtration flushing was performed with 20 L of 50 mM sodium acetate, 181 mM sodium chloride, pH 5.25. The virus filtrate can be stored at 2 to 25 °C for up to 5 days.

[0682] UF / DFThe product was concentrated using the UF / DF step and filtered into the final formulation buffer. The process utilized a 30 kD Millipore Pellicon3 Biomax UF module and D-screen, and the following Table 14D-2 The process was carried out in a purification chamber at ambient temperature (18 to 25 °C) with the process parameters shown in [Figure]. The loading material was diluted 10-fold with 5 M sodium chloride (9 parts of nanofiltrate to 1 part of 5 M sodium chloride), and the pH of the loading material was adjusted to 5.45 ± 0.1 (measured at 18 to 25 °C) using 2 M sodium acetate. Before loading the adjusted loading material onto the UF / DF membrane, a single Sartopore 2 30-inch (1.8 m²) was transferred into a recirculation tank. 2 It was filtered using a 0.45 μm / 0.2 μm capsule filter.

[0683] The total UF / DF is 9.12 m 2 8 1.14 m² for the membrane area 2 The procedure was performed on a Z-2300 skid using a membrane. The UF / DF load was concentrated to a target of 50 g / L, then filtered in 0.002% (w / v) sodium chloride and concentrated to 235 g / L. When the concentrate was removed from the ultrafiltration membrane and system and fed into the collection vessel, a 100 L Impulse Mixer system, the concentrate was filtered through a single Sartopore 2 10-inch 0.45 / 0.2 μm (0.45 m) 2 ) was passed through a sterile filter. The ultrafiltration system was rinsed with approximately 5 kg of 0.002% sodium chloride to recover the product stored in the system. Both Rinse Solution 1 and Rinse Solution 2 were tested using the same Sartopore 2 10-inch 0.45 / 0.2 μm (0.45 m) filter. 2The rinse solution was individually transferred to the rinse solution 1 collection bag and the rinse solution 2 collection bag through a sterile filter. After recovering the rinse solution, the concentrate was diluted with appropriate amounts of rinse solution 1 and rinse solution 2 to achieve a target concentration of 200 g / L.

[0684] The concentrate pool was formulated with the addition of 5X formulation buffer, consisting of 50 mM acetate, 925 mM trehalose, 0.1% Tween 20, pH 5.70. The final bulk active pharmaceutical ingredient was diluted with 150 g / L risankizumab in 1X formulation buffer, consisting of 10 mM acetate, 185 mM trehalose, 0.02% Tween 20, pH 5.70. Subsequently, the formulated UF / DF concentrate was filtered through a 0.22 μm Millipak 200 sterile filter (0.1 m 2 It was filtered through ). The final formulated UF / DF concentrate can be stored at 9 to 25°C for up to 1 day before proceeding to the final bagging step, and then cooled at 2 to 8°C for up to 5 days.

[0685]

[0686] bagging The purpose of bagging was to package and store the final bulk active pharmaceutical ingredient. The operation was performed in a purification room at ambient temperature (18 to 25 °C). The filtered and formulated UF / DF concentrate was pumped into a sterile 6 L Celsius FFT bag. The bag was filled to a volume of approximately 6 kg. The Process 4 parameter objectives are as follows: Table 14E-2 It was summarized in.

[0687]

[0688]

[0689]

[0690] Risankizumab produced by processes 3 and 4 was formulated to produce finished product 3 (DP3) and finished product 4 (DP4), respectively, both having a PS20 of 0.20 mg / mL. No visible or glittering particles were observed in the DP3 form during 24 months of stability at 4°C. Another form further confirmed the increased stability of DP3 compared to DP2. Specifically, no visible product-related particles were observed in this DP3 form at 6 months, whereas glittering particles were observed in a similar DP2 form at 3 months. Similarly, no visible or glittering particles were observed in the DP4 vial form during 18 months of stability at 4°C.

[0691] 6.7 Example 7: Confirmation of PLA2 by LC-MS Proteomics

[0692] Several types of phospholipases, including phospholipase A2 group XV (PLA2 G15), were identified as potential factors contributing to PS20 degradation. These lipases may be present in very low abundance within DS, which can complicate detection by LC-MS / MS. Concentrating lipase levels through immunoaffinity purification using immobilized antibodies against specific lipases can increase the abundance levels required for detection by LC-MS / MS. In this example, PLA2 within DP1, DP2, DP3, and DP4 DS was concentrated using immunoaffinity purification. The concentrated DS were analyzed by enzyme-linked immunoassay (ELISA) and LC-MS / MS.

[0693] PLA2 ELISA : PLA2 concentration was measured using the ELISA described in other embodiments disclosed herein. The results indicate that PLA2 was concentrated 92-fold in DP1, 57-fold in DP2, and 80-fold in DP3. No PLA2 was detected in the DP4 sample. Table 15 ).

[0694]

[0695] LC-MS / MS: Table 16 The nanoLC-MS / MS analysis shown in [figure] confirmed PLA2G15 (UniProt ID: G3HKV9) in the eluent of concentrated DS of DP1 and DP2 (two or more unique peptides for protein identification).

[0696]

[0697] In this study, the PLA2G15 protein was successfully concentrated by immunoaffinity purification and subsequently detected in DP1 and DP2 DS using LC-MS / MS. A single PLA2G15 intrinsic peptide was detected in DP3, which was below the threshold required for protein identification, while no PLA2G15 intrinsic peptide was detected in DP4 DS. These findings were consistent with PS20 degradation observed during long-term storage or under accelerated stability conditions in DP1 and DP2 DS, whereas no PS20 degradation was observed in DP3 and DP4.

[0698] 6.8 Example 8: Confirmation of PLA2 by Western Blot Analysis

[0699] Phospholipase A2 group XV (PLA2G15) levels in different risankizumab DS samples were also evaluated by Western blot.

[0700] Risankizumab BDS samples were obtained by pooling batches of risankizumab DS generated by the same risankizumab process. Risankizumab in the samples was significantly depleted by ultrafiltration using an Amicon filter with a molecular weight cutoff of 100 kDa. 200 microliters of the pooled BDS samples were added to the Amicon filter and centrifuged until 100 microliters of filtrate were obtained. The filtrate containing some residual risankizumab as well as proteins with a molecular weight of less than 100 kDa was evaluated by Western blot analysis.

[0701] Risankizumab samples and CHO PLA2G15 proteins were analyzed on 4 to 12% SDS-PAGE. Proteins were transferred to a PVDF membrane and probed with a rabbit anti-PLA2G15 antibody. An anti-rabbit antibody conjugated to horseradish peroxidase was used as the detection reagent. Color development was completed using a chromogenic TMB substrate. On Western blot, the PLA2G15 band at a molecular weight of 47 kDa was observed in all risankizumab BDS batches except DP4, with the exception that it was observed less frequently in the DP3 batch ( Fig. 23 ).

[0702] 6.9 Example 9: Knockout cell line data indicated that PLBL2 is not a problematic hitchhiker protein

[0703] To identify specific hitchhiker proteins that led to the degradation of PS20, CHO cell lines depleted of specific hitchhiker proteins PLBL2, PLA2, or LPL were generated and characterized.

[0704] The results showed no effect on process performance and product quality across all tested conditions. Observations at 3 months (5 ℃, 25 ℃, and 40 ℃) and 6 months (storage conditions of 5 ℃, 25 ℃, and 40 ℃) compared to the start of the study (0 months) indicated that the absence of PLBL2 in BDS did not improve PS20 stability compared to the relevant process control conditions. FIGS. 24a to 24d This trend was observed in both CAD and FFA test results reported by the analysis development group ( FIGS. 24a to 24d These data indicated that PLBL2 is unlikely to be the key hitchhiker protein causing PS20 degradation in risankizumab formulations.

[0705]

[0706] 6.10 Example 10: Hitchhiker protein spiking study revealed PLA2 as a problematic hitchhiker protein.

[0707] PS20 is primarily composed of polyoxyethylene sorbitan laurate acid esters. However, due to the characteristics 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. (Reference [Ayorinde et al. (2000) Rapid Commun. Mass Spectrometer 14:2116-2124; Li et al. (2014) Anal. Chem . 86:5150-5157], [Martos et al. (2017) J. Pharm. Sci [., 106:1722-1735]). Commercial PS20 lots typically contain more than 3,000 different chemical components.

[0708] Enzymes are one of the HPs present in active pharmaceutical ingredients, such as lipases and esterases. The active site of an enzyme consists of a combination of amino acid residues with a specific structure, which varies depending on the different type of enzyme. Consequently, enzymes generally exhibit different activity and specificity toward different substrates. Since PS20 is a chemical mixture, different enzymes may have different degradation rates for the various components within PS20. Therefore, different enzymes can lead to different PS20 degradation patterns (profiles). Thus, degradation pattern analysis can provide useful information for identifying specific enzyme groups as potential root causes of PS20 degradation.

[0709] PLA2G15 spiking study in DP3 substance and risankizumab buffer

[0710] The PS20-CAD subspecies method was used. This method was originally developed to qualitatively determine the subspecies composition and to quantify PS20 subspecies relative to PS20 manufacturing standards. The HPLC system used in this study was an Agilent 1260 II infinity HPLC equipped with a four-way pump, mobile phase degassing unit, refrigerated autosampler, temperature-controlled column compartment, and a Thermo Scientific charged aerosol detector (CAD). Data were collected using a Waters Empower acquisition system. The degradation profile of PS20 in PLA2G15-spiketed DP3 was consistent with the PS20 degradation pattern (profile) observed in diluted DP2 material. Fig. 25a ).

[0711] PLA2G15 spiking study at various spiking levels in DP4 material

[0712] The PS20-CAD subspecies method described above was used. DP4 solutions spiked with PLA2G15 at different concentrations were analyzed. PS20 subspecies data were collected from samples incubated at 25 °C on days T0, Day 1, Day 4, and Day 11. During data collection, all samples were maintained at 5 °C in an autosampler. PS20 concentration data from T0 to 1 month (1M) were also collected. The PLA2G15 spike concentration-dependent PS20 degradation rates were observed. The PLA2G15-induced PS20 degradation patterns were very similar across all PLA2G15 spike levels ( Fig. 25b ).

[0713] PLBL2 Spiking Research

[0714] Phospholipase B-like protein 2 (PLBL2) was detected in the DP2 material. Early literature reported that PLBL2 can induce PS20 degradation in antibody formulations. However, very high concentrations were used in the study (literature [Dixit, et al, Journal of pharmaceutical science[ , 2016, 105:1657-1666]). Recent publications have suggested that PLBL2 is unlikely to be the cause of PS20 degradation in antibody formulations due to its low activity (Reference [Zhang et al. Journal of pharmaceutical science , 2020, 109: 2710-2718]).

[0715] The PS20-CAD subspecies method described above was used. The DP3 drug solution was spiked with 5 μg / mL PLBL2 enzyme. The solution was mixed and tested. After incubation at 25°C for approximately 30 hours, no detectable PS20 degradation was observed. Typical results Fig. 26 This was shown in [figure]. These results confirmed the low activity of PLBL2 against PS20 degradation. The spiking level (5 μg / mL) was much higher than the PLBL2 concentration detected in the DP2 material by the PLBL2 ELISA method. The absence of detectable PS20 degradation after about 30 hours of incubation at 25°C indicates that PLBL2 is unlikely to be the primary underlying cause of PS20 degradation in the DP2 material.

[0716] CES Spiking Research

[0717] Carboxyl esterase 1 (CES 1) was detected in the DP2 material. The literature has reported that CES 1 can induce PS20 degradation in antibody formulations (Literature [Zhang et al. Pharmaceutical Research [ , 2022, 39:75-87]). The PS20-CAD subspecies method described above was used. The DP3 drug solution was spiked with 5 μg / mL CES 1 enzyme. The solution was mixed and tested.

[0718] Significant PS20 degradation was observed after only a few hours of incubation at 25°C. The PS20 degradation patterns (profiles) were similar at different incubation times (although the degradation levels differed). CES 1 can induce significant degradation of PS20 within risankizumab formulations. However, the PS20 degradation pattern induced by CES 1 within risankizumab formulations was significantly different from the PS20 degradation pattern / profile observed in the DP2 material. The major PS20 peak (polyoxyethylene sorbitan mono-laurate) was significantly degraded by CES 1, while the PS20 subspecies region with a retention time exceeding 50 minutes showed only minimal degradation. Typical results Fig. 27a The overlay of the PS20 degradation pattern (profile) observed in DP2 and the PS20 degradation pattern (profile) induced by CES 1 is shown in [figure]. Fig. 27b This was shown in [figure], and they were very different. Therefore, it was unlikely that CES 1 was the major contributor to PS20 degradation in DP2 material.

[0719] Sialate O-acetylesterase spiking study

[0720] Sialate O-acetylesterase (SIAE) was detected in the DP2 material. The literature reported that SIAE could induce PS20 degradation in antibody formulations, and moderate PS20 degradation was observed after 5 days of incubation at 45°C, but noted that high enzyme concentrations (5 μg / mL) were used in the study (Literature [Zhang et al. Journal of pharmaceutical sciences The reported PS20 degradation pattern differed from the pattern observed in DP2 materials. In the literature, SIAE caused a signifi...

Claims

Claim 1 A liquid aqueous pharmaceutical formulation suitable for subcutaneous injection comprising (i) risankizumab at a concentration of about 180 mg / ml, (ii) water, and (iii) a surfactant, having a pH of 5.0 to 6.

5. Claim 2 A liquid aqueous pharmaceutical formulation comprising risankizumab at a concentration of about 180 mg / ml, comprising 10,000 or fewer invisible particles (SVP) per ml, wherein the SVP have a diameter greater than 2 μm. Claim 3 A liquid aqueous pharmaceutical formulation comprising risankizumab at a concentration of about 180 mg / ml, wherein the liquid aqueous pharmaceutical formulation comprises less than 250 pg of phospholipase A2 (PLA2) per mg of said risankizumab. Claim 4 A liquid aqueous pharmaceutical formulation comprising risankizumab at a concentration of about 180 mg / ml, comprising 10,000 or fewer invisible particles (SVP) per ml and less than 250 pg of phospholipase A2 (PLA2) per mg of said risankizumab, wherein said SVP has a diameter greater than 2 μm. Claim 5 A formulation according to claim 3 or 4, wherein the PLA2 is PLA2G15. Claim 6 A formulation according to any one of claims 3 to 5, comprising less than 9 pg of PLA2 per mg of risankizumab. Claim 7 A formulation according to any one of claims 3 to 6, wherein the level of PLA2 is determined by an enzyme-linked immunoassay (ELISA). Claim 8 A formulation according to any one of paragraphs 3 to 7, wherein no visible or glittering particles are observed for 24 months at 4 ℃. Claim 9 A formulation according to any one of claims 3 to 8, comprising polysorbate 20 (PS20), wherein the concentration of said PS20 in said formulation after storage at 5°C for 6 months is at least 80% of the concentration of said PS20 in said formulation before storage at 5°C for 6 months. Claim 10 A formulation according to claim 9, wherein the concentration of PS20 in the formulation after storage at 25°C for 6 months is at least 60% of the concentration of PS20 in the formulation before storage at 25°C for 6 months. Claim 11 A formulation according to claim 9 or 10, wherein the concentration of PS20 in the formulation after storage at 40°C for 6 months is at least 40% of the concentration of PS20 in the formulation before storage at 4°C for 6 months. Claim 12 A formulation according to any one of claims 9 to 11, wherein the concentration of PS20 is measured using a high-performance liquid chromatography-charged aerosol detector (HPLC-CAD). Claim 13 A formulation according to any one of claims 3 to 12, wherein the incidence of anti-drug antibodies (ADA) occurring after treatment is less than 4.7% after administering a single subcutaneous dose of 150 mg of the formulation to a human subject. Claim 14 In Clause 13, a formulation that does not result in the occurrence of ADA after treatment. Claim 15 A formulation according to claim 13 or 14, wherein the presence of ADA is determined using a verified potency-based cross-linked electrochemiluminescence immunoassay. Claim 16 A formulation according to any one of claims 3 to 12, wherein at least 99.1% of the risankizumab species in the formulation exists as a monomer. Claim 17 A formulation according to claim 16, wherein the percentage of the risankizumab species present as monomers is determined by ultra-high performance size exclusion chromatography (UP-SEC). Claim 18 A formulation according to any one of claims 3 to 12, wherein 0.4% or less of the risankizumab species in the formulation is present as a high molecular weight (HMW) species, and the HMW species is a species having a higher molecular weight than the monomer. Claim 19 In paragraph 18, the percentage of the risankizumab species present as an HMW species is determined by UP-SEC, in the formulation. Claim 20 A formulation according to any one of claims 3 to 12, wherein more than 97.5% of the risankizumab species in the formulation is present as a major peak measured by capillary gel electrophoresis (CGE-NR) under non-reducing conditions. Claim 21 A formulation according to any one of claims 3 to 12, wherein less than 2.2% of the risankizumab species in the formulation is present as low molecular weight (LMW) species as measured by CGE-NR, and said LMW species are species having a retention time shorter than the main peak. Claim 22 A formulation according to any one of paragraphs 2 and 4 through 21, wherein the size of the SVP is measured by micro-flow imaging. Claim 23 A formulation according to any one of paragraphs 2 and 4 through 22, wherein the number of SVPs in the formulation is measured by micro-flow imaging. Claim 24 A formulation according to any one of paragraphs 2 and 4 through 23, comprising 9,000 or fewer SVPs per ml, 8,000 or fewer SVPs per ml, 7,000 or fewer SVPs per ml, 6,000 or fewer SVPs per ml, 5,000 or fewer SVPs per ml, 4,000 or fewer SVPs per ml, 3,000 or fewer SVPs per ml, 2,000 or fewer SVPs per ml, 1,000 or fewer SVPs per ml, 800 or fewer SVPs per ml, 600 or fewer SVPs per ml, 400 or fewer SVPs per ml, or 200 or fewer SVPs per ml. Claim 25 A method for reducing the formation of invisible particles (SVP) in a liquid aqueous pharmaceutical formulation containing risankizumab at a concentration of about 180 mg / ml, comprising a freeze-thaw process (F / T), wherein the F / T comprises freezing said formulation from about 18°C ​​to about -70°C for 10 hours or less, and then thawing said formulation from about -70°C to about 18°C ​​for 15 hours or less. Claim 26 A method according to claim 25, wherein the F / T comprises freezing the formulation from about 18°C ​​to about -70°C within about 5 hours, and then thawing the formulation from about -70°C to about 18°C ​​within about 9 hours. Claim 27 A method according to claim 25 or 26, wherein the F / T comprises three or fewer, two or fewer, or one or fewer cycles of freezing and thawing of the formulation. Claim 28 A method for reducing the formation of invisible particles (SVP) in a liquid aqueous pharmaceutical formulation containing risankizumab at a concentration of approximately 180 mg / ml, comprising a mixing process, wherein the power per volume (P / V) of the mixing process is 1000 W / m² 3 A method wherein the mixing time of the above mixing process is 30 minutes or less. Claim 29 A method for reducing the formation of invisible particles (SVP) in a liquid aqueous pharmaceutical formulation containing risankizumab at a concentration of about 180 mg / ml, comprising (i) a freeze-thaw process (F / T) and (ii) a mixing process, wherein (i) the F / T comprises freezing the formulation from about 18 °C to about -70 °C for 10 hours or less, and then thawing the formulation from about -70 °C to about 18 °C for 15 hours or less, and (ii) the power per volume (P / V) of the mixing process is 1000 W / m² 3 A method wherein the mixing time of the above mixing process is 30 minutes or less. Claim 30 A method according to claim 29, wherein the F / T comprises freezing the formulation from about 18°C ​​to about -70°C within about 5 hours, and then thawing the formulation from about -70°C to about 18°C ​​within about 9 hours. Claim 31 A method according to claim 29 or 30, wherein the F / T comprises three or fewer, two or fewer, or one or fewer cycles of freezing and thawing of the formulation. Claim 32 In any one of claims 28 to 31, the P / V of the mixing process is 500 W / m 3 Lee Ha-in, method. Claim 33 A method according to any one of claims 28 to 32, wherein the mixing time of the mixing process is 5 minutes to 30 minutes. Claim 34 A liquid aqueous pharmaceutical formulation produced by the method of any one of paragraphs 25 to 33. Claim 35 A formulation of any one of claims 2 to 24 and 34 or a method of any one of claims 25 to 33, wherein the formulation further comprises water and a surfactant, and the formulation has a pH of 5.0 to 6.5 and is suitable for subcutaneous injection. Claim 36 A formulation or method of claim 1 or 35, wherein the surfactant is PS20. Claim 37 A formulation or method of claim 36, wherein the PS20 is at a concentration of about 0.2 mg / ml. Claim 38 A formulation of any one of claims 1 to 24 and 34 to 37 or a method of any one of claims 25 to 33 and 35 to 37, wherein the viscosity of the formulation is less than 20.0 mPa·s at 20 ℃. Claim 39 A formulation or method according to claim 38, wherein the viscosity of the formulation is 14.0 mPa.s to 20.0 mPa.s at 20 ℃. Claim 40 A formulation or method of claim 39, wherein the viscosity of the formulation is about 15 mPa.s, about 17 mPa.s, about 17 mPa.s, or about 19 mPa.s at 20 ℃. Claim 41 A formulation or method according to any one of claims 38 to 40, wherein the viscosity of the formulation is measured by an ALP method or a falling ball viscometer. Claim 42 A formulation of any one of claims 1 to 24 and 34 to 41 or a method of any one of claims 25 to 33 and 35 to 41, wherein the formulation or method further comprises (iv) an isotonic agent, (v) a buffer and / or (vi) a solubilizing agent. Claim 43 A formulation or method of paragraph 42, wherein the formulation comprises an isotonic agent. Claim 44 A formulation or method of claim 43, wherein the formulation comprises (a) trehalose or (b) mannitol. Claim 45 A formulation or method according to any one of paragraphs 42 to 44, wherein the formulation comprises a buffer solution. Claim 46 A formulation or method according to claim 45, wherein the formulation comprises an acetate buffer. Claim 47 A formulation or method of claim 46, wherein the acetate buffer is at a concentration of about 10 mM or about 9.5 mM. Claim 48 A formulation or method of claim 46 or 47, wherein the formulation comprises (a) sodium acetate at a concentration of about 9.1 mM and acetic acid at a concentration of about 0.4 mM, or (b) sodium acetate at a concentration of about 9.1 mM and acetic acid at a concentration of about 0.9 mM, or (c) sodium acetate at a concentration of about 8.7 mM and acetic acid at a concentration of about 1.3 mM. Claim 49 A formulation of any one of claims 1 to 24 and 34 to 44 or a method of any one of claims 25 to 33 and 35 to 44, wherein the formulation does not contain a buffer. Claim 50 A formulation or method according to any one of claims 42 to 49, wherein the formulation comprises a solubilizing agent. Claim 51 A formulation or method of claim 50, wherein the formulation comprises (a) proline at a concentration of about 225 mM or (b) glycine at a concentration of about 225 mM. Claim 52 A formulation of any one of claims 1 to 24 and 34 to 51 or a method of any one of claims 25 to 33 and 35 to 51, wherein the formulation has a pH of about 5.2, about 5.5, about 5.7, or about 6.

2. Claim 53 A formulation or method of claim 52, wherein the formulation has a pH of about 5.

5. Claim 54 A formulation of any one of claims 1 to 24 and 34 to 53 or a method of any one of claims 25 to 33 and 35 to 53, wherein the formulation has an osmotic concentration of 320 mOsmol / kg to 350 mOsmol / kg. Claim 55 A formulation of any one of claims 1 to 24 and 34 to 41 or a method of any one of claims 25 to 33 and 35 to 41, comprising (i) PS20 at a concentration of about 0.2 mg / ml, (ii) trehalose at a concentration of about 185 mM, and (iii) acetate buffer at a concentration of 9.5 mM, wherein the formulation has a pH of about 5.

7. Claim 56 A formulation of any one of claims 1 to 24 and 34 to 41 or a method of any one of claims 25 to 33 and 35 to 41, comprising (i) PS20 at a concentration of about 0.2 mg / ml, (ii) trehalose at a concentration of about 185 mM, and (iii) acetate buffer at a concentration of 9.5 mM, wherein the formulation has a pH of about 5.

5. Claim 57 A formulation of any one of claims 1 to 24 and 34 to 41 or a method of any one of claims 25 to 33 and 35 to 41, comprising (i) PS20 at a concentration of about 0.2 mg / ml, (ii) trehalose at a concentration of about 185 mM, and (iii) sodium acetate at a concentration of about 9.1 mM and acetic acid at a concentration of about 0.4 mM, wherein the formulation has a pH of about 5.

7. Claim 58 A formulation of any one of claims 1 to 24 and 34 to 41 or a method of any one of claims 25 to 33 and 35 to 41, comprising (i) PS20 at a concentration of about 0.2 mg / ml, (ii) trehalose at a concentration of about 185 mM, and (iii) sodium acetate at a concentration of about 9.1 mM and acetic acid at a concentration of about 0.4 mM, wherein the formulation has a pH of about 5.

5. Claim 59 A formulation of any one of claims 1 to 24 and 34 to 41 or a method of any one of claims 25 to 33 and 35 to 41, comprising (i) PS20 at a concentration of about 0.2 mg / ml, (ii) trehalose at a concentration of about 185 mM, and (iii) acetate buffer at a concentration of 10 mM, wherein the formulation has a pH of about 5.

7. Claim 60 A formulation of any one of claims 1 to 24 and 34 to 41 or a method of any one of claims 25 to 33 and 35 to 41, comprising (i) PS20 at a concentration of about 0.2 mg / ml, (ii) trehalose at a concentration of about 185 mM, and (iii) acetate buffer at a concentration of 10 mM, wherein the formulation has a pH of about 5.

5. Claim 61 A formulation of any one of claims 1 to 24 and 34 to 41 or a method of any one of claims 25 to 33 and 35 to 41, comprising (i) PS20 at a concentration of about 0.2 mg / ml, (ii) trehalose at a concentration of about 185 mM, and (iii) sodium acetate at a concentration of about 9.1 mM and acetic acid at a concentration of about 0.9 mM, wherein the formulation has a pH of about 5.

7. Claim 62 A formulation of any one of claims 1 to 24 and 34 to 41 or a method of any one of claims 25 to 33 and 35 to 41, comprising (i) PS20 at a concentration of about 0.2 mg / ml, (ii) trehalose at a concentration of about 185 mM, and (iii) sodium acetate at a concentration of about 8.7 mM and acetic acid at a concentration of about 1.3 mM, wherein the formulation has a pH of about 5.

5. Claim 63 A formulation of any one of claims 1 to 24 and 34 to 41 or a method of any one of claims 25 to 33 and 35 to 41, comprising (i) PS20 at a concentration of about 0.2 mg / ml and (ii) mannitol at a concentration of about 190 mM, wherein the formulation has a pH of about 5.

7. Claim 64 A formulation of any one of claims 1 to 24 and 34 to 41 or a method of any one of claims 25 to 33 and 35 to 41, comprising (i) PS20 at a concentration of about 0.2 mg / ml and (ii) glycine at a concentration of about 225 mM, wherein the formulation has a pH of about 5.

7. Claim 65 A formulation of any one of claims 1 to 24 and 34 to 41 or a method of any one of claims 25 to 33 and 35 to 41, comprising (i) PS20 at a concentration of about 0.2 mg / ml and (ii) proline at a concentration of about 225 mM, wherein the formulation has a pH of about 5.7.

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