Preparation of antibody drug

Abstract

The invention relates to a preparation of an antibody drug, which belongs to the field of biological medicines, is used for a preparation of a high-concentration antibody drug with an anti-oxidation requirement, and also has an anti-degradation capability. The preparation contains 20 mmol / L to 30 mmol / L of L-methionine and 200 mmol / L of trehalose, and is particularly suitable for being used for preparing Jukoulikumab, omaszumab and cetuximab. The preparation not only has a better anti-oxidation effect, but also can realize a synergistic stabilization effect by combining high-concentration L-Met with 200 mM trehalose, has anti-degradation ability, is widely applicable to various therapeutic antibodies, and provides an extensible solution for improving the stability and shelf life of a high-concentration biological preparation.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a formulation of a monoclonal antibody drug. Background Technology

[0002] Monoclonal antibodies (mAbs) have revolutionized the treatment of complex diseases such as cancer, autoimmune diseases, and infectious diseases due to their high specificity, potency, and good safety profile, becoming an emerging field of biological drugs.

[0003] Antibody drugs have traditionally been administered primarily via intravenous infusion, which has drawbacks such as long administration time, high risk, and low convenience. In recent years, subcutaneous injection has emerged as a new route of administration for antibody drugs. Because subcutaneous injection involves volumes less than 2 mL, it is relatively rapid, safe, and convenient, leading to a growing preference for subcutaneous injection (SC). Therefore, the development of high-concentration mAb formulations suitable for subcutaneous injection is imperative.

[0004] However, increased protein concentration exacerbates physical and chemical instability, including aggregation, conformational changes, and oxidation, thereby affecting efficacy and safety. Among these challenges, oxidation is a key degradation pathway that targets redox-sensitive residues such as methionine (Met), cysteine ​​(Cys), and tryptophan (Trp), inducing structural modifications that impair antibody function. For example, the therapeutic anti-IL-17A monoclonal antibody Secukinumab contains a surface-exposed free cysteine ​​residue (Cys97) in its complementarity-determining region (CDR), making it highly susceptible to oxidation and inactivation. This vulnerability underscores the urgent need for robust antioxidant strategies in high-concentration mAb formulations.

[0005] Traditional methods for mitigating oxidation include lyophilization and protein engineering, but these methods face limitations in terms of cost, scalability, and clinical applicability. Various antioxidants have been investigated, including reducing agents, metal ion chelators, natural antioxidants, and synthetic antioxidants. However, while the addition of antioxidants such as L-Met is common, current research has primarily focused on low concentrations (typically less than 10 mM), with insufficient exploration of the potential of high-concentration antioxidants. Despite the inclusion of antioxidant stabilizers in various formulations, oxidation problems persist, especially during long-term storage and use. Therefore, developing a formulation that effectively prevents the oxidation of high-concentration antibody drugs, while systematically evaluating its antioxidant properties, is of significant scientific and practical importance for ensuring the long-term stability and efficacy of therapeutic drugs. Summary of the Invention

[0006] This invention provides a high-concentration antibody drug formulation with antioxidant and anti-degradation capabilities, and is also applicable to low-concentration antibody drugs. The technical solution is as follows: An antibody drug formulation is an aqueous solution containing 20 mmol / L to 30 mmol / L L-methionine (L-Met) and 200 mmol / L trehalose.

[0007] Preferably, the concentration of L-methionine in the formulation is 25 mmol / L.

[0008] The antibody drug mentioned is secukinumab, omalizumab, or cetuximab.

[0009] The concentration of the antibody drug in the formulation is from 10 mg / mL to 150 mg / mL.

[0010] Preferably, the concentration of secukinumab and omalizumab in the formulation is 150 mg / mL.

[0011] Preferably, the secukinumab formulation further comprises polysorbate 80 (PS80) and histidine / histidine hydrochloride, wherein the concentration of polysorbate 80 is 0.2 g / L, and the concentration of histidine / histidine hydrochloride is 20 mmol / L, wherein histidine is 0.75 mg / mL (equivalent to 4.83 mmol / L), and histidine hydrochloride monohydrate is 3.18 mg / mL (equivalent to 15.17 mmol / L), and the pH of the formulation is 5.7 to 5.9. Preferably, the pH of the formulation is 5.8, as shown in Table 1.

[0012] Table 1. Formulation of a secukinumab formulation

[0013]

[0014] The omalizumab formulation also contains histidine 1.37 mg / mL (equivalent to 8.83 mmol / L), L-histidine monohydrate hydrochloride 2.34 mg / mL (equivalent to 11.16 mmol / L), and polysorbate 20 0.4 mg / mL, as shown in Table 2.

[0015] Table 2. Formulation of an omalizumab preparation

[0016]

[0017] Preferably, the concentration of cetuximab in the formulation is 10 mg / mL.

[0018] The cetuximab formulation also contains sodium chloride 8.0 mg / mL (equivalent to 136.9 mmol / L), sodium dihydrogen phosphate monohydrate 0.41 mg / mL (equivalent to 2.97 mmol / L), disodium hydrogen phosphate dodecahydrate 1.25 mg / mL (equivalent to 3.49 mmol / L), and polysorbate 80 0.07 mg / mL, as shown in Table 3.

[0019] Table 3. Formulation of a cetuximab preparation

[0020]

[0021] This formulation contains a high concentration of L-Met, which not only exhibits superior antioxidant activity compared to formulations containing low concentrations of L-Met, but also achieves synergistic stabilization through the combination of high-concentration L-Met and 200 mM trehalose. It possesses anti-degradation capabilities and effectively preserves the integrity of monoclonal antibody molecules in high-temperature and light-induced experiments. It is widely applicable to various therapeutic antibodies (such as Secukinumab, Omalizumab, and Cetuximab), providing a scalable solution for improving the stability and shelf life of high-concentration biological agents. Attached Figure Description

[0022] Figure 1 Results of RP-HPLC analysis of the oxidation rate of antibody preparations using different antioxidants during 30-day storage.

[0023] Figure 2 Results of RP-HPLC analysis of the main peak ratio of antibody preparations using different antioxidants during 30-day storage.

[0024] Figure 3 Results of SEC-HPLC analysis of antibody preparations using different antioxidants during a 30-day storage period.

[0025] Figure 4 Results of RP-HPLC analysis of the oxidation rate of antibody formulations using different combinations of antioxidants during 30-day storage.

[0026] Figure 5 Results of RP-HPLC analysis of the main peak ratio of antibody preparations using different combinations of antioxidants during 30-day storage.

[0027] Figure 6Results of SEC-HPLC analysis of antibody formulations using different combinations of antioxidants during a 30-day storage period.

[0028] Figure 7 The results of RP-HPLC analysis of the oxidation ratio of antibody preparations with different concentrations of L-Met during 30 days of storage.

[0029] Figure 8 Results of RP-HPLC analysis of the main peak ratio using antibody preparations with different concentrations of L-Met during 30-day storage.

[0030] Figure 9 Results of SEC-HPLC analysis of aggregate proportions using antibody preparations with different concentrations of L-Met during a 30-day storage period.

[0031] Figure 10 Results of SEC-HPLC analysis of monomer ratios from antibody preparations with different concentrations of L-Met during a 30-day storage period.

[0032] Figure 11 The results of RP-HPLC analysis of the oxidation ratio of antibody preparations with different high concentrations of L-Met during 30 days of storage.

[0033] Figure 12 Results of RP-HPLC analysis of the main peak ratio using antibody preparations with different high concentrations of L-Met during 30-day storage.

[0034] Figure 13 Results of SEC-HPLC analysis of aggregate proportions using antibody formulations with different high concentrations of L-Met during a 30-day storage period.

[0035] Figure 14 Results of SEC-HPLC analysis of monomer ratios from antibody preparations with different high concentrations of L-Met during a 30-day storage period.

[0036] Figure 15 Results of RP-HPLC analysis of the oxidation ratio of antibody preparations using different polyols during a 30-day storage period.

[0037] Figure 16 Results of RP-HPLC analysis of the main peak ratio using antibody preparations with different polyols during a 30-day storage period.

[0038] Figure 17 Results of SEC-HPLC analysis of aggregate proportions using antibody formulations with different polyols during a 30-day storage period.

[0039] Figure 18Results of SEC-HPLC analysis of monomer ratios from antibody formulations using different polyols during a 30-day storage period.

[0040] Figure 19 Results of RP-HPLC analysis of the oxidation ratio of antibody preparations using different concentrations of trehalose during 30-day storage.

[0041] Figure 20 Results of RP-HPLC analysis of the main peak ratio using antibody preparations with different concentrations of trehalose during 30-day storage.

[0042] Figure 21 Results of SEC-HPLC analysis of aggregate proportions from antibody preparations using different concentrations of trehalose during a 30-day storage period.

[0043] Figure 22 Results of SEC-HPLC analysis of monomer ratios in antibody preparations with different concentrations of trehalose during a 30-day storage period.

[0044] Figure 23 The results of charge heterogeneity detection using IEX-HPLC on antibody preparations with high and low concentrations of L-Met and trehalose after 30 days of storage.

[0045] Figure 24 The purity of antibody preparations with high and low concentrations of L-Met and trehalose was determined by non-reducing CE-SDS after 30 days of storage.

[0046] Figure 25 The purity of antibody preparations with high and low concentrations of L-Met and trehalose was determined by CE-SDS analysis after 30 days of storage.

[0047] Figure 26 The oxidation ratio of antibody preparations with high and low concentrations of L-Met and trehalose was determined by RP-HPLC during a 14-day simulated light exposure period.

[0048] Figure 27 Results of RP-HPLC analysis of the main peak ratio of antibody preparations with high and low concentrations of L-Met and trehalose during a 14-day simulated light exposure period.

[0049] Figure 28 Results of SEC-HPLC analysis of aggregate ratios in antibody formulations with high and low concentrations of L-Met and trehalose during a 14-day simulated light exposure period.

[0050] Figure 29 Results of SEC-HPLC analysis of monomer ratios in antibody formulations with high and low concentrations of L-Met and trehalose during a 14-day simulated light exposure period.

[0051] Figure 30 The results of IEX-HPLC analysis of the proportion of acidic peaks in antibody preparations with high and low concentrations of L-Met and trehalose during a 14-day simulated light exposure period.

[0052] Figure 31 The results of IEX-HPLC analysis of the main peak ratio of antibody preparations with high and low concentrations of L-Met and trehalose during a 14-day simulated light exposure period. Detailed Implementation

[0053] To prepare and test the performance indicators of different high-concentration antibody formulations, we used the following instruments, equipment, reagents, and consumables:

[0054] Instruments and equipment: The Agilent 1260 chromatograph (Agilent Technologies, Inc., USA) is equipped with a Sepax Bio reversed-phase column (2.1 × 150 mm, 3 μm). An Agilent 1290 chromatograph (Agilent Technologies, USA), equipped with an ACQUITY UPLC Protein BEH SEC column, 200 μm (4.6 × 150 mm, 1.7 μm). The Agilent 1260 Infinity HPLC system is equipped with a Thermo MabPac SCX-10 BioLC™ cation exchange column (4 × 150 mm, 10 μm). PA800 plus drug analysis system (Sciex Corporation, USA); Uncle All-in-One Protein Stability Analyzer (Unchained Labs, USA); The Waters 2695 HPLC system was equipped with a 2489 UV detector, a TOSOH TSK-GEL BioAssist G3SWXL (300 mm × 7.8 mm, 5 μm) column, and a Dionex ProPac WCX-10 column. Waters Acquity UPLC ultra-high performance liquid chromatography system, equipped with Aeris WIDEPORE C4 series chromatographic columns; The Beckman PA800 plus capillary electrophoresis system is equipped with uncoated capillaries with an inner diameter of 50 μm, a total length of 31 cm, and an effective length of 21 cm. Illuminated incubator (Memmert, Germany), HPP260.

[0055] Reagents: The monoclonal antibodies Secukinumab (with the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:2 for the heavy and light chains, respectively), Omalizumab (with the amino acid sequences of SEQ ID NO:3 and SEQ ID NO:4 for the heavy and light chains, respectively), and Cetuximab (with the amino acid sequences of SEQ ID NO:5 and SEQ ID NO:6 for the heavy and light chains, respectively) were provided by Taizhou Mabtech Pharmaceutical Co., Ltd., and all were expressed using Chinese hamster ovary (CHO) cells. The antibody preparation method is as follows: DNA coding sequences are designed according to the amino acid sequences of the antibody heavy and light chains and the codon preferences of CHO cells, inserted into a eukaryotic expression vector, and transfected into Chinese hamster ovary (CHO) cells, specifically CHO-K1 cells. Cell lines that stably express monoclonal antibodies are screened, monoclonalized and amplified, and a cell bank is established. The expression cell lines are then subjected to large-scale fermentation culture in serum-free medium. The cell culture medium containing monoclonal antibodies is purified and devirulentized through a series of steps such as liquid chromatography and filtration to obtain a solution containing monoclonal antibodies. The monoclonal antibodies are then replaced into a solution prepared according to the formulation by means of filtration or dialysis.

[0056] N-acetyl-DL-tryptophan (abbreviated as NAT, CAS No. 87-32-1), N-acetyl-L-cysteine ​​(abbreviated as NAC, CAS No. 616-91-1) and L-methionine (L-Met, CAS No. 63-68-3) were purchased from Sigma-Aldrich.

[0057] Histidine, histidine hydrochloride, trehalose dihydrate, sucrose, mannitol and sorbitol were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China).

[0058] Polysorbate 80 (PS80) and polysorbate 20 (PS20) were purchased from Nanjing Well Pharmaceutical Group Co., Ltd. (Nanjing, China).

[0059] Citric acid, anhydrous sodium citrate, sodium dihydrogen phosphate monohydrate, disodium hydrogen phosphate dodecahydrate, arginine hydrochloride, glutathione (reduced form), sodium bisulfite, trifluoroacetic acid, acetonitrile, sodium phosphate, 2-mercaptoethanol, and iodoacetamide were purchased from Sinopharm Chemical Reagent Company.

[0060] All reagents were of analytical grade or higher, and all experiments used ultrapure water (resistivity ≥18.2 MΩ-cm).

[0061] Example 1: Antioxidant Screening

[0062] To establish a high-concentration antibody formulation with antioxidant effects, we used Secukinumab (150 mg / mL) as a model and compared its antioxidant effects with those of traditional antioxidants N-acetyl-DL-tryptophan (NAT), N-acetyl-L-cysteine ​​(NAC), glutathione (GSH), and sodium bisulfite (SHS) against L-methionine (L-Met).

[0063] The study was conducted in two groups: one group compared the antioxidant effects of L-Met with NAT and NAC, and the other group compared the antioxidant effects of L-Met with glutathione and sodium bisulfite.

[0064] The specific method is as follows:

[0065] 1.1 Comparison of antioxidant effects of L-Met with NAT and NAC

[0066] 1.1.1 Sample Preparation

[0067] Secukinumab was prepared according to the formulation in the table below, and the solution was prepared with an antibody concentration of 150 mg / mL.

[0068] Table 4. Formulations of each group in the comparison experiment of L-Met with NAT and NAC.

[0069]

[0070] After preparation, the antibody formulation was aliquoted into 2 mL sterile glass vials, sealed, and subjected to an accelerated stability study at 40°C for 30 days. Oxidation and aggregation analyses were performed on the samples at predetermined time intervals (days 0, 10, 20, and 30).

[0071] 1.1.2 Detection

[0072] The detection method described in the reference "Regl, C., et al., A Generic HPLC Method for Absolute Quantification of Oxidation in Monoclonal Antibodies and Fc-Fusion Proteins Using UV and MS Detection. Anal Chem, 2017. 89(16): p. 8391-8398." established a detection method, and used RP-HPLC and SEC-UPLC techniques to quantify the antibody oxidation level and molecular aggregation profile, respectively.

[0073] 1.1.2.1. Reversed-phase high-performance liquid chromatography (RP-HPLC)

[0074] Reversed-phase high-performance liquid chromatography (RP-HPLC) was performed using an Agilent 1260 chromatograph (Agilent Technologies, USA) equipped with a Sepax Bio column (2.1 × 150 mm 3 μm). Mobile phase A was 0.1% trifluoroacetic acid (TFA) aqueous solution, and mobile phase B was 0.1% trifluoroacetic acid (TFA) acetonitrile solution. The gradient elution program was as follows: column temperature maintained at 40 °C, and detection wavelength at 280 nm. The antibody in the sample was diluted to 5 mg / mL with ultrapure water, centrifuged at 17,000 g for 5 min, and 10 μL of supernatant was injected. The antibody oxidation level was assessed based on peak area.

[0075] 1.1.2.2. Size Exclusion Ultra-High Performance Liquid Chromatography (SEC-UPLC)

[0076] SEC-UPLC analysis was performed using an Agilent 1290 chromatograph (Agilent Technologies, USA) equipped with an ACQUITY UPLC @Protein BEH SEC 200 Å column (4.6 × 150 mm 1.7 μm). The mobile phase was 0.1 M sodium phosphate buffer (pH 6.8), the flow rate was 0.2 mL / min, and the column temperature was maintained at 25 °C. Samples were diluted to 10 mg / mL with the mobile phase, centrifuged at 17,000 g for 3 min to remove insoluble particles, and 10 μL of the supernatant was loaded onto the sample. Proteins were detected at 280 nm, and monomer content and polymer ratio were calculated based on peak area.

[0077] 1.1.3 Results

[0078] RP-HPLC analysis revealed different oxidation trends in the tested formulations (Figure 1). After 30 days of storage, neither NAC nor NAT showed significant antioxidant effects. Notably, compared to the control group (without antioxidant), the NAC formulation showed an increase in oxidation-related pre-peak impurities, suggesting a possible pro-oxidation effect under high temperature and pressure. Similarly, NAT exhibited limited antioxidant capacity, with oxidation levels comparable to the control group. In contrast, L-Met significantly inhibited oxidation and reduced pre-peak impurities compared to the control group. Notably, the oxidation rate in the L-Met group slowed significantly after day 10, highlighting its sustained antioxidant activity.

[0079] These findings were further confirmed by antibody purity assessment using RP-HPLC (Figure 2). At day 30, the L-Met formulation exhibited the highest antibody purity with the slowest rate of decline. In contrast, the NAC and NAT formulations showed a faster decline in purity, highlighting the superior protective effect of L-Met.

[0080] SEC-UPLC analysis of aggregation behavior revealed key differences among the different antioxidants (Figure 3). The L-Met formulation retained 97.3% monomer content with only 2.0% aggregates, superior to NAT (96.8% monomers, 2.4% aggregates) and NAC (96.9% monomers, 3.3% aggregates). Notably, the NAC-treated sample showed an increase in high molecular weight species (HMWS) at day 30, suggesting the potential formation of incompatible or reactive byproducts under stress conditions. These results collectively demonstrate that low concentrations of L-Met not only mitigate oxidation but also effectively inhibit aggregation, making it a potent stabilizer for high-concentration mAb formulations.

[0081] 1.2 Comparison of the antioxidant effects of L-Met, glutathione, and sodium bisulfite

[0082] 1.2.1 Sample Preparation

[0083] Prepare the Seukinumab formulation according to the formulation in the table below, and then conduct stability tests under high temperature and light conditions.

[0084] Table 5. Formulations of each group in the comparative experiment of L-Met with glutathione and sodium bisulfite.

[0085]

[0086] Note: mM = mmol / L, the same applies below.

[0087] High-temperature test protocol: Place the prepared antibody preparation in a Memmert high-temperature incubator at 40°C and 60% humidity, and take samples periodically; Light test protocol: Place the prepared antibody preparation in a Memmert light incubator at 2~8°C and 8000 lx light, and take samples periodically.

[0088] 1.2.2 Detection

[0089] The periodically collected samples were analyzed using methods including SEC-UPLC (size exclusion liquid chromatography), RP-HPLC (reversed-phase liquid chromatography), IEX-HPLC (ion exchange chromatography), and CE-SDS (sodium dodecyl sulfate capillary gel electrophoresis). The sample detection methods are as follows:

[0090] 1.2.2.1 SEC-UPLC

[0091] The Waters 2695 HPLC system, 2489 UV detector, and TOSOH TSK-GEL BioAssist G3SWXL (300 mm × 7.8 mm, 5 μm) column were used for detection. Samples were diluted with blank reagent to a concentration of 10 mg / mL before loading. The flow rate was 0.7 mL / min; the injection volume was 20 μL; the column temperature was room temperature; the main peak was detected at 280 nm, and other peaks were detected at 214 nm. The peak area at 214 nm was corrected to the peak area at 280 nm using the wavelength ratio factor, and the concentration of all peaks was calculated at 280 nm using the area normalization method.

[0092] 1.2.2.2 RP-HPLC

[0093] A Waters Acquity UPLC system was used with an Aeris Widepore C4 column. The mobile phase consisted of 0.1% trifluoroacetic acid-water solution and 0.1% trifluoroacetic acid-acetonitrile solution. Flow rate: 1 mL / min; wavelength: 280 nm; column temperature: 70℃; sample chamber temperature: 8℃. A working solution of 5 mg / mL was prepared from the sample using ultrapure water, and 5 μL was loaded. The content of the main peak and related proteins in the sample was calculated using the area normalization method.

[0094] 1.2.2.3 IEX-HPLC

[0095] A Waters 2695 HPLC system, a 2489 UV detector, and a Dionex ProPac WCX-10 column were used for detection. Mobile phase A was 10 mM phosphate (pH 6.0), and mobile phase B was 10 mM phosphate-500 mM sodium chloride (pH 6.0). Gradient elution was used (0 min, mobile phase B 8%; 0–30 min, mobile phase B increased to 100%; 30–60 min, mobile phase B maintained at 100%; 40–60 min, mobile phase B 8%), at a flow rate of 1 mL / min. The sample was diluted to 1.0 mg / mL, and 100 μL was loaded. The sample temperature was 2–8 ℃, the column temperature was 35 ℃, the detection wavelength was 280 nm, and the detection time was 60 min. The peak area percentages of the main peak, acidic region, and basic region were calculated using the area normalization method.

[0096] 1.2.2.4 CE-SDS

[0097] A Beckman PA800 plus capillary electrophoresis system was used, employing an uncoated capillary (50 μm inner diameter, 31 cm total length, 21 cm effective length). Detection conditions were: separation voltage 15 kV, capillary temperature 30 ℃, sample chamber temperature 15 ℃, and detection wavelength 220 nm. The percentage of the sample's light chain peak + heavy chain peak (reduced form) or main peak (non-reduced form) peak area was calculated.

[0098] 1.2.3 Results

[0099] The results of SEC-HPLC, RP-HPLC, IEX-HPLC, and CE-SDS analysis of the samples are as follows:

[0100] Table 6. High-Temperature Experiment SEC-HPLC Results Comparison of L-Met with Glutathione and Sodium Bisulfite

[0101]

[0102] Table 7. High-Temperature RP-HPLC Results Comparison of L-Met with Glutathione and Sodium Bisulfite

[0103]

[0104] Table 8. High-Temperature Experiment IEX-HPLC Results Comparison of L-Met with Glutathione and Sodium Bisulfite

[0105]

[0106] Table 9. High-Temperature Experiment CE-SDS Results Comparing L-Met with Glutathione and Sodium Bisulfite

[0107]

[0108] Table Notes: NGHC: Non-Glycosylated Heavy Chain; R-CE: Reduced CE-SDS; NR-CE: Non-Reduced CE-SDS. The same applies below.

[0109] Table 10. Comparison of L-Met with glutathione and sodium bisulfite under light irradiation: SEC-HPLC results

[0110]

[0111] Table 11. Comparison of L-Met with glutathione and sodium bisulfite under light irradiation: RP-HPLC results

[0112]

[0113] Table 12 Comparison of L-Met with glutathione and sodium bisulfite under light irradiation: IEX-HPLC results

[0114]

[0115] Table 13. Comparison of L-Met with glutathione and sodium bisulfite in CE-SDS light irradiation experiments.

[0116]

[0117] Experimental results comparing L-Met with antioxidants such as glutathione and sodium bisulfite showed that the Met-M group (i.e., the group with 25 mmol / L L-Met added as an antioxidant) had higher purity values ​​(R-CE) for the SEC-HPLC main peak, RP-HPLC main peak, IEX-HPLC main peak, and CE-SDS on day 30 of the high-temperature experiment and day 14 of the light-induced experiment. This indicates that the formulation with 25 mmol / L L-Met added better preserved the integrity of the monoclonal antibody and had a better effect.

[0118] Example 2: Synergistic effect of antioxidant combination

[0119] Given the excellent antioxidant and anti-aggregation properties of low-concentration L-Met, we further investigated whether combining L-Met with traditional antioxidants—NAT and NAC—could provide synergistic stabilizing effects for high-concentration Sekunumab (150 mg / mL). Although combination antioxidant strategies are rarely used in biologics, their potential in high-concentration biologics remains largely untapped. To fill this gap, we conducted accelerated stability studies (40 °C, 30 days) combined with RP-HPLC and SEC-UPLC analyses to assess the oxidation and aggregation trends of paired formulations containing L-Met and either NAT or NAC.

[0120] The specific method is as follows:

[0121] 2.1 Sample Preparation

[0122] Secukinumab was formulated according to the formulation in the table below to prepare a solution with an antibody concentration of 150 mg / mL. After preparation, the antibody formulation was aliquoted into 2 mL sterile glass vials, sealed, and subjected to an accelerated stability study at 40°C for 30 days. Oxidation and aggregation analyses were performed on the samples at predetermined time intervals (days 0, 10, 20, and 30).

[0123] Table 14 Formulations for each group in the experiment on the synergistic effect of L-Met with other antioxidants.

[0124]

[0125] 2.2 Detection

[0126] The sample was tested according to the methods described in "1.1.2.1. Reversed-phase high performance liquid chromatography (RP-HPLC)" and "1.1.2.2. Size exclusion ultra-high performance liquid chromatography (SEC-UPLC)" in Example 1.

[0127] 2.3 Results

[0128] RP-HPLC analysis revealed different oxidation dynamics in different test combinations (Figure 4). The L-Met + NAC group exhibited accelerated oxidation kinetics, with pre-peak impurities exceeding those of the control group, suggesting potential incompatibility or pro-oxidation under pressure conditions. Conversely, the L-Met + NAT formulation initially showed a slight reduction in oxidation-related pre-peak impurities, but this effect gradually diminished over time, reaching oxidation levels exceeding those of the L-Met-only group by day 30. Notably, the pure L-Met formulation consistently exhibited the lowest oxidation levels. RP-HPLC purity assessment confirmed these trends. Figure 5 ).

[0129] SEC analysis further clarifies the aggregation behavior (Figure 6). The L-Met + NAT group retained 96.9% of the monomers (2.2% aggregates), the L-Met + NAC group retained 95.5% of the monomers (3.3% aggregates), while the L-Met alone group outperformed both, retaining 97.3% of the monomers and only 2.0% of the aggregates. No synergistic effects were observed in these combinations, consistent with conventional antioxidant strategies that typically use a single antioxidant in formulations.

[0130] The results showed that the combined antioxidant strategy failed to improve stability, while the NAC combination introduced additional risks of oxidative degradation. These findings challenge the assumption that multiple antioxidants can produce a synergistic effect in high-concentration systems and highlight the necessity of optimizing specific molecules. Therefore, L-Met was selected as a standalone antioxidant for further formulation development.

[0131] Example 3: Optimization of L-Met Concentration

[0132] To further evaluate the concentration-dependent antioxidant effect of L-Met, secukinumab (150 mg / mL) was formulated with low concentrations (2 mM, 5 mM) and high concentrations (25 mM) of L-Met, and accelerated stability tests were performed (40 °C, 30 days). The antioxidant properties of these formulations were evaluated using RP-HPLC and SEC. The specific methods are as follows:

[0133] 3.1 Sample Preparation

[0134] Secukinumab was formulated according to the formulation in the table below to prepare a solution with an antibody concentration of 150 mg / mL. After preparation, the antibody formulation was aliquoted into 2 mL sterile glass vials, sealed, and subjected to an accelerated stability study at 40°C for 30 days. Oxidation and aggregation analyses were performed on the samples at predetermined time intervals (days 0, 10, 20, and 30).

[0135] Table 15 Formulations of each group in the comparative experiment of the effects of different concentrations of L-Met

[0136]

[0137] 3.2 Detection

[0138] The sample was tested according to the methods described in "1.1.2.1. Reversed-phase high performance liquid chromatography (RP-HPLC)" and "1.1.2.2. Size exclusion ultra-high performance liquid chromatography (SEC-UPLC)" in Example 1.

[0139] 3.3 Results

[0140] RP-HPLC analysis revealed significant differences in oxidation kinetics among different test concentrations (Figure 7). The low-concentration L-Met formulations (2 mM and 5 mM groups) showed moderate antioxidant effects, while the high-concentration L-Met formulation (25 mM group) exhibited enhanced antioxidant effects, significantly reducing oxidation levels during the test.

[0141] Purity quantification by RP-HPLC further highlighted the superiority of high-concentration L-Met (Figure 8). SEC analysis further elucidated the anti-aggregation advantage of high-concentration L-Met (Figure 9). The 25 mM L-Met formulation showed the lowest polymer content. This indicates that higher concentrations of L-Met not only enhance antioxidant capacity but also significantly inhibit protein aggregation. However, the monomer purity remained comparable across groups, suggesting that high-concentration L-Met preferentially inhibits aggregate formation without altering the intrinsic stability of the monomers (Figure 10).

[0142] These results established the critical threshold for the efficacy of L-Met: higher concentrations significantly slowed oxidative kinetics and inhibited aggregation, while lower concentrations provided only partial protection.

[0143] Example 4: Threshold Concentration Analysis

[0144] To determine the minimum effective concentration of L-Met for stabilizing high-concentration mAb formulations, we evaluated three high-concentration L-Met conditions (20 mM, 25 mM, and 30 mM) using Sekunumab (150 mg / mL) in accelerated stability testing (40 °C, 30 days). RP-HPLC and SEC analyses were used to assess oxidation-related pre-peak impurities, purity, and aggregation, aiming to determine a threshold above which further increases in L-Met concentration would result in diminishing returns. The specific methods are as follows:

[0145] 4.1 Sample Preparation

[0146] Secukinumab was formulated according to the formulation in the table below to prepare a solution with an antibody concentration of 150 mg / mL. After preparation, the antibody formulation was aliquoted into 2 mL sterile glass vials, sealed, and subjected to an accelerated stability study at 40°C for 30 days. Oxidation and aggregation analyses were performed on the samples at predetermined time intervals (days 0, 10, 20, and 30).

[0147] Table 16 Formulations of each group in the comparative experiment of the effects of different high concentrations of L-Met

[0148]

[0149] 4.2 Detection

[0150] The sample was tested according to the methods described in "1.1.2.1. Reversed-phase high performance liquid chromatography (RP-HPLC)" and "1.1.2.2. Size exclusion ultra-high performance liquid chromatography (SEC-UPLC)" in Example 1.

[0151] 4.3 Results

[0152] RP-HPLC results showed that the oxidation trends of all three formulations were almost identical. Figure 11 RP-HPLC purity also showed a similar rate of decline ( Figure 12 ).

[0153] SEC analysis further confirmed this threshold effect. Aggregation levels remained consistently low across all high-concentration L-Met groups. Figure 13The change in SEC purity is also negligible. Figure 14 These results further confirm that high concentrations of L-Met (>20 mM) can effectively stabilize antibodies and inhibit oxidation and aggregation. Based on these experimental results, this study selected 25 mM L-Met as the optimal antioxidant concentration. This saturation effect highlights a key inflection point in the L-Met protective mechanism, namely, the peak efficiency of molecular shielding and redox scavenging.

[0154] Based on these findings, 25 mM L-Met was selected as the optimal concentration for high-concentration mAb formulations. This choice balances potent stabilizing effects against oxidation and aggregation with practical considerations, including formulation compatibility (such as osmotic pressure and viscosity) and cost-effectiveness. The absence of adverse reactions such as aggregation or chemical interference caused by excipients further validates that this threshold represents a strategic balance between therapeutic efficacy and manufacturability.

[0155] Example 5: Screening of polyols and their synergistic effect with L-Met

[0156] We evaluated the synergistic stabilizing effects between polyols such as trehalose, sucrose, sorbitol, and mannitol and high concentrations of L-Met (25 mM).

[0157] 5.1 Comparison of trehalose, sucrose, and combinations of trehalose and sorbitol

[0158] 5.1.1 Comparison of trehalose, sucrose, and combinations of trehalose and sorbitol

[0159] We used RP-HPLC and SEC-UPLC to screen for trehalose (200 mM Trehalose), sucrose (200 mM Sucrose), and the combination of trehalose and sorbitol (200 mM Trehalose + 5 mM sobitol). The specific methods are as follows:

[0160] 5.1.1.1 Sample Preparation

[0161] Secukinumab was formulated according to the formulation in the table below to prepare a solution with an antibody concentration of 150 mg / mL. After preparation, the antibody formulation was aliquoted into 2 mL sterile glass vials, sealed, and subjected to an accelerated stability study at 40°C for 30 days. Oxidation and aggregation analyses were performed on the samples at predetermined time intervals (days 0, 10, 20, and 30).

[0162] Table 17 Formulations of each group in the comparative experiment of the effects of different polyols

[0163]

[0164] 5.1.1.2 Detection

[0165] The sample was tested according to the methods described in "1.1.2.1. Reversed-phase high performance liquid chromatography (RP-HPLC)" and "1.1.2.2. Size exclusion ultra-high performance liquid chromatography (SEC-UPLC)" in Example 1.

[0166] 5.1.1.3 Results

[0167] Under accelerated stabilization conditions (40 °C, 30 days), all three polyol formulations exhibited considerable antioxidant and anti-aggregation capabilities (Figure 15). Figure 16 , Figure 17 , Figure 18 However, trehalose appears to have a significant advantage in inhibiting oxidation. RP-HPLC analysis showed that, at day 10, the trehalose-treated sample had lower levels of oxidation-related pre-peak impurities compared to the sucrose and trehalose-sorbitol combination. Figure 15 These observations position trehalose as the preferred polyol for synergistic stabilization with L-Met.

[0168] 5.1.2 Optimization of Trehalose Concentration

[0169] To determine the minimum effective trehalose concentration for optimal synergistic effect with L-Met, we evaluated formulations containing 0 mM, 100 mM, and 200 mM trehalose, along with an optimized L-Met concentration (25 mM). The specific methods are as follows:

[0170] 5.1.2.1 Sample Preparation

[0171] Secukinumab was formulated according to the formulation in the table below to prepare a solution with an antibody concentration of 150 mg / mL. After preparation, the antibody formulation was aliquoted into 2 mL sterile glass vials, sealed, and subjected to an accelerated stability study at 40°C for 30 days. Samples were taken at predetermined time intervals (days 0, 10, 20, and 30).

[0172] Table 18 Formulations of each group in the comparative experiment of the effects of different concentrations of trehalose

[0173]

[0174] 5.1.2.2 Detection

[0175] The sample was tested according to the methods described in "1.1.2.1. Reversed-phase high performance liquid chromatography (RP-HPLC)" and "1.1.2.2. Size exclusion ultra-high performance liquid chromatography (SEC-UPLC)" in Example 1.

[0176] 5.1.2.3 Results

[0177] RP-HPLC analysis showed that the antioxidant effect was concentration-dependent; compared with 100 mM trehalose, 200 mM trehalose reduced oxidation-related impurities by day 30. Figure 19 , Figure 20 This aligns with the known ability of trehalose, at higher concentrations, to form a hydration layer around proteins, thereby protecting redox-sensitive residues from reactive oxygen species.

[0178] See SEC-UPLC results Figure 21 , Figure 22 This study showed significant differences in the anti-aggregation efficacy of trehalose at different concentrations. Although both 100 mM and 200 mM concentrations maintained monomer content, the 200 mM trehalose formulation had a lower HMWS (High Molecular Weight Species) content. Figure 21 This indicates that trehalose has an aggregation-inhibiting effect that varies with concentration.

[0179] 5.2 Comparison of Trehalose, Sucrose, Sorbitol, and Mannitol

[0180] We also compared the effects of monoclonal antibody formulations containing different sugars such as trehalose, sorbitol, sucrose, and mannitol in high-temperature and light-induced tests.

[0181] 5.2.1 Sample Preparation

[0182] The formulations of Secukinumab using different sugars are as follows:

[0183] Table 19 Formulations of each group in the comparative experiment of trehalose, sucrose, sorbitol, and mannitol.

[0184]

[0185] Note: mM = mmol / L.

[0186] The formulation was prepared according to the above formula, and then high temperature test and light irradiation test were carried out according to the method in Example 1.2.1, and samples were taken for testing.

[0187] 5.2.2 Detection

[0188] The samples taken from the high-temperature test and the light irradiation test were analyzed by SEC-HPLC, RP-HPLC, IEX-HPLC and CE-SDS according to the method in Example 1.2.2.

[0189] 5.2.3 Results

[0190] The test results are as follows:

[0191] Table 20 Comparative SEC-HPLC results of trehalose, sucrose, sorbitol, and mannitol at high temperatures

[0192]

[0193] Table 21 Comparative RP-HPLC results of trehalose, sucrose, sorbitol, and mannitol at high temperatures

[0194]

[0195] Table 22 Comparative high-temperature IEX-HPLC results of trehalose, sucrose, sorbitol, and mannitol

[0196]

[0197] Table 23 Comparative CE-SDS results of high-temperature experiments for trehalose, sucrose, sorbitol, and mannitol

[0198]

[0199] Table 24. SEC-HPLC Results of Comparative Light Irradiation Experiments for Trehalose, Sucrose, Sorbitol, and Mannitol

[0200]

[0201] Table 25 RP-HPLC Results of Comparative Light Irradiation Experiments for Trehalose, Sucrose, Sorbitol, and Mannitol

[0202]

[0203] Table 26. IEX-HPLC Results of Comparative Light Irradiation Experiments for Trehalose, Sucrose, Sorbitol, and Mannitol

[0204]

[0205] Table 27 CE-SDS Results of Comparative Light Irradiation Experiments for Trehalose, Sucrose, Sorbitol, and Mannitol

[0206]

[0207] Experimental results showed that the purity (R-CE) values ​​of the SEC-HPLC main peak, RP-HPLC main peak, IEX-HPLC main peak, and CE-SDS of the group with added 200 mM trehalose were higher on day 30 of the high temperature experiment and day 14 of the light experiment. This indicates that the formulation with added 200 mM trehalose better preserved the integrity of the monoclonal antibody and had a better effect.

[0208] Example 6: Comparison of different buffer solutions and pH values

[0209] We compared the effects of histidine buffer (HBS), citrate buffer (CBS), and phosphate buffer (PBS) at different pH levels under high temperature and light exposure conditions.

[0210] 6.1 Sample Preparation

[0211] Secukinumab was formulated according to the formulation in the table below, and then stability tests were conducted under high temperature and light conditions.

[0212] Table 28 Formulations of each group in the comparative experiments with different buffer solutions and pH values

[0213]

[0214] Note: mM = mmol / L, the same applies below.

[0215] The formulation was prepared according to the above formula, and then high temperature test and light irradiation test were carried out according to the method in Example 1.2.1, and samples were taken for testing.

[0216] 6.2 Testing

[0217] The samples taken from the high-temperature test and the light irradiation test were analyzed by SEC-HPLC, RP-HPLC, IEX-HPLC and CE-SDS according to the method in Example 1.2.2.

[0218] 6.3 Results

[0219] The detection results of samples under different buffer solutions and pH values ​​are as follows:

[0220] Table 29. SEC-HPLC results of high-temperature experiments with different buffer solutions and pH values.

[0221]

[0222] Table 30. High-temperature RP-HPLC results comparing different buffer solutions and pH values.

[0223]

[0224] Table 31. High-Temperature Experiment IEX-HPLC Results Comparing Different Buffers and pH Values

[0225]

[0226] Table 32 CE-SDS Results of High-Temperature Experiments with Different Buffers and pH Values

[0227]

[0228] Note: NGHC represents sugar-free heavy chain, R-CE represents reduced SDS, NR-CE represents non-reduced SDS, and so on.

[0229] Table 33 SEC-HPLC Results of Light Irradiation Experiments with Different Buffers and pH

[0230]

[0231] Table 34. RP-HPLC results of light-irradiation experiments with different buffer solutions and pH values.

[0232]

[0233] Table 35. IEX-HPLC Results of Light Irradiation Experiments with Different Buffers and pH Levels

[0234]

[0235] Table 36. CE-SDS Results of Light Irradiation Experiments with Different Buffers and pH Levels

[0236]

[0237] Experimental results showed that the purity (R-CE) values ​​of the SEC-HPLC main peak, RP-HPLC main peak, IEX-HPLC main peak, and CE-SDS of the 20 mmol / L histidine buffer at pH 5.8 were high on day 30 of the high-temperature experiment and day 14 of the light-induced experiment, indicating that the integrity of the monoclonal antibody was well preserved. The formulation using the 20 mmol / L histidine buffer at pH 5.8 had a better effect.

[0238] Example 7: Comprehensive Stability Assessment

[0239] 7.1 Charge heterogeneity and purity assessment

[0240] To further validate the stability of the optimized formulation (25 mM L-Met + 200 mM trehalose), charge heterogeneity and purity were rigorously assessed using ion-exchange chromatography (IEX-HPLC) and capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) under both reducing and non-reducing conditions. These analyses are crucial for assessing the long-term stability risks associated with chemical modifications such as deamidation, oxidation, and fragmentation, which can be exacerbated by high concentrations of the antibody formulation. The specific methods are as follows:

[0241] 7.1.1 Sample Preparation

[0242] Prepare the formulation according to the following formula.

[0243] Experimental formulation:

[0244] Table 37 Optimized Seukinumab formulation (experimental)

[0245]

[0246] Reference standard formulation:

[0247] Table 38 Secukinumab control formulation (reference standard)

[0248]

[0249] After preparation, the antibody preparation was aliquoted into 2 mL sterile glass vials, sealed, and subjected to an accelerated stability study at 40°C for 30 days. The samples on day 30 were then subjected to the following tests.

[0250] 7.1.2 Detection

[0251] 7.1.2.1 Ion exchange chromatography (IEX-HPLC)

[0252] IEX-HPLC analysis was performed on an Agilent 1260 Infinity HPLC system equipped with a MabPac SCX-10 BioLC™ column (4 × 150 mm 10 μm). Mobile phase A was 10 mM sodium phosphate buffer (pH 7.8), and mobile phase B was 10 mM sodium phosphate buffer (pH 7.8) containing 200 mM NaCl. The gradient elution program was as follows: column temperature was maintained at 30 °C, flow rate at 1.0 mL / min, and detection wavelength at 280 nm. Samples were diluted to 1 mg / mL with PBS, centrifuged at 13,200 rpm for 2 min, and 10 μL of the supernatant was injected. Changes in the acidic peak group (APG) were used to assess the effect of thermal stress on the antibody.

[0253] 7.1.2.2 Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS)

[0254] CE-SDS analysis was performed under both reducing and non-reducing conditions using a PA800 plus pharmaceutical analysis system (Sciex, USA). Under reducing conditions, the sample was mixed with a 10 kDa internal standard and 2-mercaptoethanol, heated at 70 °C for 10 min, and then injected at 5 kV. Under non-reducing conditions, iodoacetamide was used instead of 2-mercaptoethanol, and the sample was injected at 10 kV. The separation voltage was 15 kV, the column temperature was 25 °C (reducing) and 20 °C (non-reducing), and the detection wavelength was 220 nm. Antibody purity and impurity content were calculated based on peak area.

[0255] 7.1.3 Results

[0256] IEX-HPLC separates charge variants based on electrostatic interactions. After storage at 40°C for 30 days, the optimized formulation showed minimal differences in acidity, alkalinity, and main peak distribution compared to the control group (5 mM L-Met + 200 mM trehalose). Figure 23 The optimized formulation showed an acidic peak of 24.2% and a main peak of 54.7%, which is very close to the control group (acidic peak: 23.8%; main peak: 53.1%). This consistency indicates that high concentrations of L-Met do not induce additional charge variations or accelerate deamidation.

[0257] CE-SDS analysis under both reducing and non-reducing conditions further confirmed the structural integrity, and the purity curves of the two formulations were comparable. Figure 24 , Figure 25 These data collectively demonstrate that high concentrations of L-Met can synergistically work with trehalose to maintain charge uniformity and structural purity, thus solving the critical stability issue in high-concentration mAb formulations.

[0258] 7.2 Differential Scanning Fluorescence (DSF)

[0259] To assess the conformational stability of the optimized formulation (25 mM L-Met + 200 mM trehalose), the experimental and control formulations prepared in 7.1.1 were stored at 40°C for 30 days and then analyzed using differential scanning fluorometry (DSF). DSF monitors intrinsic fluorescence changes during thermal denaturation, thereby accurately determining the thermal denaturation temperature (Tm) and initial aggregation temperature (Tagg)—key parameters for assessing structural integrity.

[0260] 7.2.1 Sample Preparation

[0261] Same as 7.1.1.

[0262] 7.2.2 Detection

[0263] Thermal stability was assessed using the Uncle system (Unchained Labs, USA). Melting temperature (Tm) was determined by monitoring intrinsic protein fluorescence (IPF, excitation wavelength 266 nm, emission scan wavelength 280–450 nm). The antibody solution (10 mg / mL) was heated from 25 °C to 95 °C at a rate of 1 °C / min, with equilibration for 1 minute before each measurement. Tm and Tagg were calculated using Uncle analysis software to assess thermal stability and aggregation behavior.

[0264] 7.2.3 Results

[0265] The DSF test results are shown in the table below.

[0266] Table 39 Thermal stability assessment of formulations

[0267]

[0268] The test results showed that the Tm values ​​of the two formulations were almost identical, with a difference (ΔTm) < 1 °C (Table 9). Similarly, the Tagg values ​​remained comparable, indicating that high concentrations of L-Met had no adverse effect on thermal decomposition or aggregation tendency. These results are consistent with industry standards for conformational stability, confirming that high concentrations of L-Met do not disrupt the stability of the antibody's tertiary structure or promote premature aggregation.

[0269] 7.3 Colloidal stability assessment of formulation

[0270] Colloidal stability is a key factor determining manufacturability and shelf life. DLS is used to measure the hydrodynamic diameter (rh) and polydispersity index (PDI) to assess colloidal stability. The specific method is as follows:

[0271] 7.3.1 Sample Preparation

[0272] Same as 7.1.1.

[0273] 7.3.2 Detection

[0274] DLS experiments were performed using an Uncle instrument (Unchained Labs, USA) to evaluate particle size distribution. Measurements were taken at a concentration of 150 mg / mL and a temperature of 25°C, with an equilibration time of 5 minutes. Data from multiple runs were averaged, and intensity distribution maps were analyzed to determine the hydrodynamic diameter (rh) and polydispersity index (PDI). A PDI value below 0.7 indicated good monodispersity of the sample.

[0275] 7.3.3 Results

[0276] The test results are shown in the table below.

[0277] Table 40 Colloidal stability assessment of formulations

[0278]

[0279] The results showed that, compared with the control group, the optimized formulation exhibited minimal changes in particle size and distribution, with PDI values ​​for both groups ≈ 0.2 (Table 10), indicating a monodisperse distribution. These findings confirm that high concentrations of L-Met do not cause particle aggregation or uneven distribution. This strong colloidal stability may be attributed to the antioxidant protective effect of L-Met and the preferential exclusion effect of trehalose, which together minimize protein-protein interactions.

[0280] 7.4 Evaluation of the photodegradation resistance of the formulation

[0281] Because antibodies contain photosensitive residues (such as tryptophan and tyrosine), they are prone to photodegradation. To evaluate the protective efficacy of high-concentration L-Met under light conditions, a simulated light-condition experiment was conducted on the optimized formulation. The specific method is as follows:

[0282] 7.4.1 Sample Preparation

[0283] The formulation is the same as in 7.1.1. After preparation, the antibody preparation is aliquoted into 2 mL sterile glass bottles, sealed, and placed in a Memmert light incubator at 2-8°C and 8000 lx light conditions for a 14-day simulated light condition test. Samples are taken on days 0, 7, and 14 for subsequent testing.

[0284] 7.4.2 Detection

[0285] The samples were analyzed according to the methods described in "1.1.2.1. Reversed-phase high-performance liquid chromatography (RP-HPLC)" and "1.1.2.2. Size exclusion ultra-high performance liquid chromatography (SEC-UPLC)" in Example 1 and "7.1.2.1 Ion exchange chromatography (IEX-HPLC)" in Example 7.

[0286] 7.4.3 Results

[0287] RP-HPLC analysis showed no significant difference in oxidation-related pre-peak impurities between high-concentration and low-concentration L-Met samples (Figure 26). Figure 27 This indicates that L-Met has strong antioxidant capabilities.

[0288] SEC further demonstrated that high concentrations of L-Met can inhibit photoinduced aggregation, maintaining monomer content above 97%. Figure 28 , Figure 29 ).

[0289] RP-HPLC analysis showed no significant difference in oxidation-related pre-peak impurities between high-concentration and low-concentration L-Met samples (Figure 26). Figure 27 This indicates that L-Met has strong antioxidant capabilities.

[0290] SEC further demonstrated that high concentrations of L-Met can inhibit photoinduced aggregation, maintaining monomer content above 97%. Figure 28 , Figure 29 ).

[0291] IEX-HPLC analysis confirmed that the charge heterogeneity in the optimized formulation was reduced (Figure 30). Figure 31 This is likely due to L-Met's ability to scavenge reactive oxygen species generated under light irradiation. These results highlight the dual role of high concentrations of L-Met in mitigating both oxidative and physical degradation pathways.

[0292] Example 8: Study on the wide applicability of the formulation

[0293] To validate the general applicability of the optimized high-concentration L-Met formulation (25 mM L-Met + 200 mM trehalose) to various antibody therapies, we expanded our evaluation to include omalizumab (anti-IgE) and cetuximab (anti-EGFR), two commercially available mAbs with different structural and functional characteristics. We systematically evaluated the thermal stability parameters of omalizumab and cetuximab formulated with high-concentration L-Met, comparing them with the original formulations, including melting temperatures (Tm1, Tm2) and Tagg, to elucidate the effects of the formulation on conformational and colloidal stability. The specific methods are as follows:

[0294] 8.1 Sample Preparation

[0295] Omalizumab (CMAB007) and cetuximab (CMAB009) were used as reference standards. 25 mmol / L L-Met was added to the reference standard formulation (antibody concentration 150 mg / mL) to create the +L-Met group. Furthermore, the sugar in the reference standard formulation was replaced with 200 mM trehalose to create the +L-Met +trehalose group. The specific formulations are as follows:

[0296] Table 41 Omalizumab formulation to be tested

[0297]

[0298] Table 42. Formulations of the cetuximab formulations to be tested

[0299]

[0300] Prepare the antibody preparation according to the above formula. After preparation, aliquot the antibody preparation into 2 mL sterile glass bottles, seal them, and conduct an accelerated stability study at 40°C for 30 days. Perform the following tests on the samples on day 30.

[0301] 8.2 Testing

[0302] The detection method is the same as that described in 7.2.2 of Example 7, and the thermal stability is evaluated using the Uncle System (Unchained Labs, USA).

[0303] 8.3 Results

[0304] The Uncle system test results are shown in the table below.

[0305] Table 43. Broad applicability of high-concentration L-Met in various therapeutic antibodies

[0306]

[0307] The results showed that for omalizumab, adding a high concentration of L-Met increased the second thermal transition temperature (Tm2) by 1.2 °C (from 81.9 °C to 83.1 °C) and the initial denaturation initiation temperature (Tonset) by 1.0 °C, while Tm1 remained unchanged (71.5 °C, compared to 71.4 °C in the control group) (Table 11). However, the Tagg values ​​observed at wavelengths of 266 nm and 473 nm were lower than those of the control formulation. These results indicate that high concentrations of L-Met can improve conformational stiffness without affecting intrinsic stability, which is a crucial advantage for subcutaneous formulations requiring long-term storage.

[0308] In contrast, the thermostability parameters of cetuximab when formulated with high concentrations of L-Met did not change significantly. Tm1 (69.0 °C vs. 69.5 °C), Tm2 (74.2 °C vs. 74.5 °C), and Tonset (63.4 °C vs. 62.6 °C) remained comparable to the control formulation. Similarly, the Tagg values ​​at 266 nm (71.4 °C) and 473 nm (71.4 °C) were close to those of the control group (70.5 °C and 70.7 °C, respectively), confirming that this formulation neither disrupts the stability of structurally different antibodies nor induces their aggregation.

[0309] These results highlight the broad applicability of high-concentration L-Met strategies in antibody subclasses and formulations. Improved thermostability of omalizumab and maintained stability of cetuximab were observed, demonstrating the versatility of formulations in mitigating degradation risks without requiring customization for specific molecules. This formulation approach, by synergistically addressing oxidation and aggregation issues, provides a transformable solution for stabilizing next-generation high-concentration biologics, regardless of target or structural complexity.

Claims

1. An antibody drug formulation, characterized in that it is... An aqueous solution of antibody drug containing 20 mmol / L to 30 mmol / L L-methionine and 200 mmol / L trehalose.

2. The antibody drug formulation according to claim 1, characterized in that, The L-methionine content is 25 mmol / L.

3. The antibody drug formulation according to claim 1, characterized in that, The concentration of the antibody drug in the formulation is from 10 mg / mL to 150 mg / mL.

4. The antibody drug formulation according to claim 1, characterized in that, The antibody drug mentioned is secukinumab.

5. The antibody drug formulation according to claim 1, characterized in that, The antibody drug mentioned is omalizumab.

6. The antibody drug formulation according to claim 1, characterized in that, The antibody drug mentioned is cetuximab.

7. The antibody drug formulation according to claim 4, characterized in that, The concentration of secukinumab in the formulation is 150 mg / mL.

8. The antibody drug formulation according to claim 5, characterized in that, The concentration of omalizumab in the formulation is 150 mg / mL.

9. The antibody drug formulation according to claim 6, characterized in that, The concentration of cetuximab in the formulation is 10 mg / mL.

10. The antibody drug formulation according to claim 7, characterized in that, The secukinumab formulation contains a total of 20 mmol / L of histidine and histidine hydrochloride, and 0.2 mg / mL of polysorbate 80, with a pH of 5.7 to 5.

9.

11. The antibody pharmaceutical formulation according to claim 10, characterized in that, The mass concentrations of histidine and histidine hydrochloride are 4.83 mmol / L for histidine and 15.17 mmol / L for histidine hydrochloride, respectively.

12. The antibody drug formulation according to claim 8, characterized in that, The omalizumab formulation contains histidine 8.83 mmol / L, histidine hydrochloride 11.16 mmol / L, and polysorbate 20 0.4 mg / mL.

13. The antibody drug formulation according to claim 9, characterized in that, The cetuximab formulation contains 136.9 mmol / L sodium chloride, 2.97 mmol / L sodium dihydrogen phosphate, 3.49 mmol / L disodium hydrogen phosphate, and 800.07 mg / mL polysorbate.

14. The antibody drug formulation according to claims 4, 7, 10, and 11, characterized in that, The formulation contains 150 mg / mL secukinumab, histidine 4.83 mmol / L, histidine hydrochloride 15.17 mmol / L, trehalose 200 mmol / L, methionine 25 mmol / L, polysorbate 80 0.2 g / L, and a pH of 5.7 to 5.9.

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