High-concentration antibody preparation and application thereof

Abstract

The invention discloses a high-concentration antibody preparation and application thereof, and belongs to the technical field of antibody pharmaceutical preparations, the preparation takes an antibody as an active ingredient, adopts glutamic acid and histidine hydrochloric acid buffer solution, is matched with non-ionic saccharide and sugar alcohol auxiliary materials, and uses a non-ionic buffer system to maintain the pH value of the system stable; and the stability risk caused by the ionic auxiliary material is reduced. The preparation can realize high-concentration preparation of more than 100mg / mL, obviously inhibits antibody aggregation, fragmentation and oxidative degradation, improves purity, stability and injection convenience, and is suitable for subcutaneous injection. The problems that a traditional high-concentration antibody preparation is prone to aggregation, high in viscosity, poor in stability and the like are solved, the prescription is scientific and controllable, and the method is suitable for clinical transformation and large-scale production.

Description

Technical Field

[0001] This invention belongs to the field of antibody drug formulation technology, specifically relating to a high-concentration antibody self-buffering formulation based on acidic amino acids and nonionic excipients. Background Technology

[0002] With the development of biopharmaceutical technology, antibody drugs, due to their strong targeting and significant efficacy, have been widely used in the treatment of autoimmune diseases, tumors, and other fields. To meet the demand for patient-friendly administration methods such as subcutaneous administration, high-concentration antibody formulations (≥100 mg / mL) have become a research hotspot. However, the development of high-concentration formulations faces many challenges: enhanced intermolecular interactions of antibodies can easily lead to aggregation, affecting efficacy and increasing the risk of immunogenicity; increased formulation viscosity reduces injection convenience; traditional formulations rely on exogenous buffers to maintain pH stability, which can easily cause ionic strength imbalance, thereby exacerbating protein instability. Summary of the Invention

[0003] This invention provides a high-concentration antibody formulation and its application. The invention uses a glutamate-histidine buffer solution as a buffer system to formulate a high-concentration antibody formulation. By adding non-ionic excipients, the stability and purity of the formulation are synergistically improved, constructing a stable buffer system suitable for high-concentration antibodies. This fundamentally solves the technical problems of molecular aggregation and fragmentation commonly found in existing high-concentration antibody formulations, enabling the preparation of high-concentration formulations with antibody concentrations exceeding 100 mg / mL, while significantly improving the long-term stability and purity of the formulation. Specifically, the high-concentration antibody formulation of this invention uses antibodies as the active ingredient and acidic amino acids or organic acids as the core components for pH adjustment and buffering, and may further contain at least one stabilizer. Through the synergistic effect of the above system, the aggregation and degradation reaction rates of antibodies can be controlled, antibody chemical stability can be improved, the shelf life of the formulation can be extended, and a stable antibody drug formulation suitable for subcutaneous injection can be obtained.

[0004] The acidic amino acids or organic acids include glutamic acid, acetic acid, and histidine / histidine hydrochloride. A stable formulation environment is provided by balancing the pH regulation capability of the antibody's intrinsic system.

[0005] The nonionic excipients include sugars and sugar alcohols, specifically one or more combinations of sucrose, trehalose, maltose, glucose, fructose, sorbitol, mannitol, xylitol, glycerol, propylene glycol, dextran, and hydroxypropyl-β-cyclodextrin. These excipients can play multiple roles: maintaining the osmotic pressure of the formulation system; reducing non-specific interactions between antibody molecules and minimizing protein self-aggregation; and inhibiting chemical instability pathways such as antibody oxidative degradation, further enhancing the overall stability of the formulation. Sucrose and sorbitol are preferred nonionic excipients.

[0006] The high-concentration antibody preparation provided by this invention mainly comprises the following components: Antibody: 100mg / mL~180mg / mL; Glutamate or glutamate-histidine buffer system: 5 mmol / L~50 mmol / L; Nonionic excipients: 100mmol / L~250mmol / L; Polysorbate 80: 0.01%~0.2% (w / v); The pH range of the formulation system is 5.0~6.7.

[0007] Furthermore, the high-concentration antibody preparation provided by the present invention mainly comprises the following components: Antibody: 100mg / mL~180mg / mL; Glutamate or glutamate-histidine buffer system: 5 mmol / L~50 mmol / L; Sucrose: 100mmol / L~250mmol / L; Polysorbate 80: 0.01%~0.2% (w / v); The pH range of the formulation system is 5.0~6.7.

[0008] Furthermore, the high-concentration antibody preparation provided by the present invention mainly comprises the following components: Antibody: 100mg / mL~180mg / mL; Glutamate or glutamate-histidine buffer system: 5 mmol / L~50 mmol / L; Sorbitol: 100 mmol / L~250 mmol / L; Polysorbate 80: 0.01%~0.2% (w / v); The pH range of the formulation system is 5.0~6.7.

[0009] The high-concentration antibody preparation described in this invention is used to prepare antibody drugs for subcutaneous injection, which are suitable for the treatment of autoimmune diseases, tumors and other diseases, and can meet the clinical drug delivery requirements of high drug loading, small volume and patient-friendly.

[0010] The beneficial effects of this invention are: This invention utilizes the self-buffering properties of antibodies to stabilize the formulation environment by leveraging the antibody's inherent pH-regulating ability. Combined with a glutamate and histidine buffer system, it reduces the need for exogenous ionic buffers, preventing ionic imbalances from exacerbating protein aggregation and fragmentation. The addition of non-ionic sugars and sugar alcohols such as sucrose and sorbitol inhibits antibody self-aggregation and oxidative degradation, improving formulation viscosity. The resulting formulation exhibits antibody concentrations exceeding 100 mg / mL, high purity retention under temperature and light stress, effective control of degradation and aggregation, significantly improved stability, purity, and injection compliance, and extended shelf life. This addresses the core challenges in developing high-concentration antibody formulations and is suitable for subcutaneous injection and industrial production. Detailed Implementation

[0011] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0012] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments used without a specified manufacturer are all conventional products that can be purchased through legitimate channels.

[0013] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0014] The target of the antibody described in this invention is IL-17A (which specifically binds to interleukin-17A), and its sequence is as follows: The light chain amino acid sequence is shown in SEQ ID NO:1, and the specific sequence is as follows: EIVLTQSPGTLSLSPGERATLSCRASQSISNNYLAWYQQKPGQAPRLLIYKASNLYSGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDYVPWTFGQGTRLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; The heavy chain amino acid sequence is shown in SEQ ID NO:2, and the specific sequence is as follows: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVASISSNGSYTYYADSVKGRFTISRDNAKNSLYLQMNSLRVEDTAVYCVRRGGAGWLSNSGGTGAG DYWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.

[0015] General sample testing methods: Average particle size distribution determination (DLS, dynamic light scattering): The average particle size (Z-average) of different formulations at 25°C was determined using the dynamic light scattering module of the Uncle multifunctional protein stability analyzer.

[0016] Insoluble particulate matter determination: The determination and judgment were performed using an insoluble particulate matter detector in accordance with the first method (light obscuration method) of General Chapter 0903 of the 2020 edition of the Pharmacopoeia of the People's Republic of China for the examination of insoluble particulate matter.

[0017] SEC-HPLC: An Agilent 1260 high-performance liquid chromatograph was used, with a TOSOH column (TSKgel G3000SWXL). The mobile phase consisted of 50 mmol / L phosphate buffer + 200 mmol / L NaCl solution, pH 6.7, a flow rate of 1 mL / min, and a detection wavelength of 280 nm.

[0018] Transfer 100 μL of the test sample, add 900 μL of the blank formulation solution of the test sample to dilute to 2 mg / mL, inject 20 μL, and run isocratic time for 15 minutes.

[0019] Purity was determined by high performance liquid chromatography according to General Chapter 0514 of the Pharmacopoeia of the People's Republic of China (2020 Edition, Part III), and the purity was calculated by the area normalization method.

[0020] CEX-HPLC: An Agilent 1260 high-performance liquid chromatograph was used, with a Thermo, ProPac column. TM WCX-10 analytical column (4mm×250mm)).

[0021] Mobile phase A: 30 mmol / L 2-morpholinoethanesulfonic acid (MES) + 20 mmol / L NaCl buffer solution, pH 6.0; Mobile phase B: 30 mmol / L MES + 500 mmol / L NaCl buffer solution, pH 6.0.

[0022] The flow rate was 0.8 mL / min, the detection wavelength was 280 nm, and the column temperature was 40 °C.

[0023] Transfer 100 μL of the test sample, add 900 μL of mobile phase A to dilute to 2 mg / mL, inject 10 μL, and run the gradient for 35 minutes. The mobile phase gradient is shown in Table 1 below.

[0024] Table 1. Mobile Phase Gradient Table

[0025] Purity was determined by high performance liquid chromatography according to General Chapter 0514 of the Pharmacopoeia of the People's Republic of China (2020 Edition, Part III), and the purity was calculated by the area normalization method.

[0026] Viscosity test: The viscosity of the sample was measured at 25°C using a Honeycomb viscosity instrument (Unchained Labs).

[0027] Example 1 Buffer system and pH screening: 1. The effect of pH on stability: This formulation is a subcutaneous formulation. The pre-designed formulation uses a platform formulation histidine-histidine hydrochloride buffer system. A suitable pH was screened within this system, with a pH range of 5.0 to 7.0. The testing conditions were: 25℃ for 2 weeks and 4 weeks, and 40℃ for 1 week and 2 weeks.

[0028] Preparation method: Using a Merk 30kD ultrafiltration membrane, the antibody buffer was transferred to 20mM histidine-histidine hydrochloride buffer containing sucrose at different pH values. Polysorbate 80 (PS80) was added to a final concentration of 0.02% w / v, and the protein concentration was adjusted to 150 mg / mL. The solution was then aliquoted into sterile vials (1.0 mL / vial) in a laminar flow hood to obtain the sample for formulation development. The 20mM histidine-histidine hydrochloride buffer was prepared as follows: a 20mM histidine solution was prepared by weighing solid histidine. A 20mM histidine hydrochloride solution was also prepared. The pH of the histidine solution was then adjusted to the target value using the histidine hydrochloride solution, maintaining the histidine concentration at 20mM.

[0029]

[0030] 1.1 Accelerating stability: The samples were stored under accelerated conditions at 25℃±2℃, and samples were taken within a set time. The stability was evaluated by SEC purity, and the test results are shown in Table 2.

[0031] Table 2 Accelerated stability test results

[0032] 1.2 Stability under heat and pressure: The samples were stored under accelerated conditions at 40℃±2℃, and samples were taken within a set time. The stability was evaluated by SEC purity. The test results are shown in Table 3.

[0033] Table 3 Results of hot-pressurization stability test

[0034] The stability results at 25℃ and 40℃ show that pH has a significant impact on purity, and lower pH is beneficial to antibody stability; further, it is speculated that the type of buffer system has a significant impact on stability.

[0035] 2. The impact of the buffer system on stability: Based on pH screening results, a low pH environment is more conducive to antibody stability. Two buffer systems were selected based on the buffering capacity of each buffer: glutamate-histidine hydrochloride buffer and acetate-sodium acetate buffer. Three formulations of the glutamate-histidine buffer were prepared at pH 5.0, pH 5.3, and pH 5.7, and two formulations of the acetate buffer were prepared at pH 5.0 and pH 5.5, for a total of five formulations. Screening was conducted for pH range and acid type. The protein concentration was 150 mg / mL. For example, the preparation method of a pH 5.3 glutamate-histidine solution includes: weighing 3.68 g of glutamate, 4.2 g of histidine, 60 g of sucrose, and 0.2 g of polysorbate 80, and adding purified water to a final volume of 1 L, resulting in a pH of 5.3.

[0036] Table 4 pH screening formulation composition

[0037] The stress test protocol included three forced degradation conditions: 40℃ for 4 weeks, 5 cycles of freeze-thaw cycles (-70℃ / room temperature), and 3 days of room temperature shaking. Changes in the purity of the SEC-HPLC main peak, CEX-HPLC main peak, particle size, and insoluble particles were investigated. The results are shown in Tables 5-7.

[0038] Table 5 Results of the 4-week observation at 40℃

[0039] Table 6 Results of repeated freeze-thaw cycles

[0040] Table 7 Results of room temperature oscillation

[0041] The test results showed that the quality of each formulation decreased to some extent under temperature and pressure conditions, with formulations 1-3 showing the least decrease, indicating that these formulations were the most stable, and they remained stable at pH 5.0-5.7.

[0042] Example 2 Effect of protein concentration on stability: 1. Viscosity evaluation: The antibody was replaced with a 30kD ultrafiltration membrane from Merk (Merk) to a pH 5.2 glutamate-histidine solution and a histidine-histidine hydrochloride buffer containing 6 wt.% sucrose, or the antibody was replaced with a pH 5.2 histidine-histidine hydrochloride buffer containing arginine or sodium chloride. The antibody was adjusted to different protein concentrations, and polysorbate 80 (PS80) was added to a final concentration of 0.02% w / v. The samples were aseptically aliquoted into vials (1.0 mL / vial) to obtain the samples. Note: All buffer solutions used were sterile water for injection. The viscosity of the samples was examined after 3 days of refrigeration, and the change in the purity of the SEC-HPLC main peak was examined after 4 weeks of storage at 40°C. The test results are shown in Table 8.

[0043] Table 8 Viscosity Evaluation Results

[0044] The results showed that arginine significantly reduced sample viscosity, but its addition also significantly decreased the purity of the SEC main peak. The glutamate-histidine hydrochloride buffer formulation maintained sufficient viscosity for high protein concentrations, while the glutamate-antibody buffer formulation exhibited the highest purity of the SEC main peak. Therefore, the glutamate-antibody buffer formulation was selected as the preferred choice.

[0045] 2. Protein concentration screening: Based on the evaluation results of pH, buffer system, and viscosity, the glutamate-histidine buffer showed the best overall performance. Antibodies were replaced with a glutamate-histidine solution containing 6 wt.% sucrose at pH 5.2 using a 30 kD ultrafiltration membrane from Merk. The antibodies were then concentrated to different concentrations according to Table 9, and polysorbate 80 (PS80) was added to a final concentration of 0.02% w / v.

[0046] Table 9 Protein concentration screening formulation composition

[0047] Table 10 Results after 4 weeks of storage at 40℃

[0048] The test results showed that the purity and viscosity of SEC in formulations 1-3 did not change significantly, indicating that the protein concentration was stable in the range of 100mg / mL to 180mg / mL.

[0049] Example 3 The effect of excipients on stability: Based on the screening results of the buffer system and pH, excipients were screened. A glutamate-antibody buffer system was selected, with four different types of excipients added: 6 wt.% sucrose, 4% sorbitol, 150 mM sodium chloride, and 200 mM arginine, at a pH of 5.2. Stability tests were conducted under different conditions. The preliminary screening formulation design and stability conditions are shown in Table 11 below.

[0050] Sample preparation: Antibodies were replaced using a Merk 30kD ultrafiltration membrane with a replacement volume of 6-8 CV. The antibodies were then transferred to a pH 5.2, 25mM glutamate-histidine hydrochloride solution. The PS80 concentration in the formulation was adjusted to 0.02% using 2% PS80 stock solution, and the protein concentration was adjusted to 150 mg / mL. After sterilization by filtration through a 0.22 μm filter, the samples were aliquoted into sterile vials.

[0051] Stability testing conditions: samples were placed at 25℃ and 40℃ for 2 weeks and 4 weeks, respectively; and exposed to 4500 Lux light for 5 days and 10 days, respectively. The stability of the samples was evaluated by purity testing, and the results are shown in Tables 12-13.

[0052] Table 11 Excipient Screening Formulation Composition

[0053] Table 12 Results of accelerated screening and temperature-pressure tests for excipients

[0054] Table 13 Results of Light Irradiation Test for Excipient Screening

[0055] Results: Under both temperature and light stability test conditions, sucrose and sorbitol were the optimal excipients, while sodium chloride and arginine were less effective, mainly because they are ionic excipients and not suitable for this system.

[0056] In summary, the antibody is best suited for a pH 5.0–5.7 glutamate-histidine buffer, preferably using carbohydrate excipients, and is suitable for protein concentrations of 100 mg / mL–180 mg / mL.

[0057] Those skilled in the art can obtain the target antibody using conventional genetic engineering methods based on the antibody sequence disclosed in this specification. Then, the antibody can be mixed with the selected buffer system according to conventional formulation preparation methods and aseptically filtered to prepare the high-concentration antibody formulation of the present invention, thereby achieving the expected technical effect of the present invention.

Claims

1. A high-concentration antibody preparation, characterized in that, The active ingredient is an antibody, and the buffer system is either glutamate or glutamate-histidine; the antibody concentration is ≥100mg / mL.

2. The high-concentration antibody preparation according to claim 1, characterized in that, The formulation comprises a nonionic excipient and polysorbate 80; wherein the nonionic excipient is selected from one or more combinations of sucrose, trehalose, maltose, glucose, fructose, sorbitol, mannitol, xylitol, glycerol, propylene glycol, dextran, and hydroxypropyl-β-cyclodextrin.

3. The high-concentration antibody preparation according to claim 2, characterized in that, The concentration ranges of each component are as follows: Antibody: 100mg / mL~180mg / mL; Glutamate or glutamate-histidine buffer system: 5 mmol / L~50 mmol / L; Nonionic excipients: 100mmol / L~250mmol / L; Polysorbate 80: 0.01%~0.2% (w / v); The pH value of the formulation system is controlled between 5.0 and 6.

7.

4. The high-concentration antibody preparation according to claim 3, characterized in that, The nonionic excipient is sucrose or sorbitol.

5. The high-concentration antibody preparation according to any one of claims 1-4, characterized in that, The concentration of the antibody is 100 mg / mL to 180 mg / mL, and the concentration of the glutamate or glutamate-histidine buffer system is 25 mmol / L.

6. The high-concentration antibody preparation according to any one of claims 1-4, characterized in that, The antibody target is IL-17A, and its sequence is as follows: Light chain: EIVLTQSPGTLSLSPGERATLSCRASQSISNNYLAWYQQKPGQAPRLLIYKASNLYSGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDYVPWTFGQGTRLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; Heavy chain: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVASISSNGSYTYYADSVKGRFTISRDNAKNSLYLQMNSLRVEDTAVYCVRRGGAGWLSNSGGTGAG DYWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.

7. The high-concentration antibody preparation according to any one of claims 1-4, characterized in that, The preparation is for subcutaneous injection.

8. The use of the high-concentration antibody preparation according to any one of claims 1-4 in the preparation of a medicament for treating autoimmune diseases and tumors.

9. The application according to claim 7, characterized in that, The drug is an antibody drug administered via subcutaneous injection, designed to meet the clinical needs for high drug loading, small volume, and patient-friendly administration.

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