High concentration compositions of pd-1 antibodies and methods of making the same

Abstract

The present disclosure provides a high-concentration composition of a PD-1 antibody and a method for preparing the same, which comprises pembrolizumab, wherein a portion of the pembrolizumab comprises a glucuronidation modification at the lysine at position 27 of the light chain of the amino acid sequence set forth in SEQ ID NO: 2, and wherein the content of the light chain of the pembrolizumab comprising the glucuronidation modification is less than or equal to 3% based on the percentage of the total content of the light chain of the pembrolizumab in the composition. The present disclosure also provides a pharmaceutical preparation comprising the above-mentioned composition. The present disclosure additionally provides a method for detecting the above-mentioned pembrolizumab comprising the glucuronidation modification and the use thereof in quality inspection or quality control of a product containing pembrolizumab.

Description

Technical Field

[0001] This disclosure relates to the field of pharmaceutical formulations. Specifically, this disclosure provides a high-concentration composition of PD-1 antibody and a method for preparing the same. Background Technology

[0002] With the continuous development of therapeutic recombinant protein drugs, the need for high-concentration subcutaneous administration is becoming increasingly urgent. Especially in the treatment of chronic diseases such as cancer, there is a pressing need to develop high-concentration, small-volume antibody formulations to improve patient convenience and outpatient treatment adherence. Therefore, high-concentration subcutaneous injection formulations have become one of the key development directions for the differentiated research and development of therapeutic recombinant protein drugs.

[0003] Under normal physiological conditions, programmed death receptor 1 (PD-1) and programmed death ligand 1 (PD-L1) are important negative immunomodulators that inhibit T cell activation and mediate the occurrence of infectious diseases, autoimmune diseases, and immune escape by tumor cells. However, during tumorigenesis, tumor cells abnormally express PD-L1, which binds to PD-1 on the surface of T cells, transmitting inhibitory signals that suppress T cell activation and proliferation. This allows tumor cells to evade the surveillance and attack of the immune system, achieving immune escape.

[0004] Pembrolizumab is a humanized monoclonal antibody that specifically binds to the PD-1 receptor on the surface of T cells, blocking the interaction between PD-1 and ligands such as PD-L1, thereby relieving the immunosuppression of T cells by tumor cells, restoring the anti-tumor activity of T cells, enhancing the body's anti-tumor immune response, and achieving the purpose of killing tumor cells.

[0005] Although high-concentration formulations have been developed for some antibody drugs, the production of high-concentration antibody formulations (e.g., ≥100 mg / mL) still faces significant challenges, including irreversible aggregation, irreversible precipitation and / or high viscosity issues, and the potential generation of new impurities. As a drug, pembrolizumab must maintain its stability and efficacy. Quality control of the pharmaceutical composition focuses primarily on controlling the content of the active ingredient and related substances (such as variants or impurities), especially ensuring that the content of related substances meets stringent pharmaceutical standards. Summary of the Invention

[0006] In a first aspect, this disclosure provides a composition comprising pembrolizumab, wherein a portion of the pembrolizumab contains a glucuronidation modification at the 27th lysine residue of its light chain as shown in SEQ ID NO:2, and wherein the light chain content of the pembrolizumab containing the glucuronidation modification is less than or equal to 3% as a percentage (e.g., calculated based on trypsin peptide mass fingerprinting analysis) of the total light chain content of the pembrolizumab containing the glucuronidation modification in the composition.

[0007] Secondly, this disclosure provides pharmaceutical formulations comprising the compositions described in the first aspect, and one or more pharmaceutically acceptable carriers.

[0008] Thirdly, this disclosure provides a method for detecting the presence of glucuronidated pembrolizumab in a composition or pharmaceutical formulation containing pembrolizumab, wherein the glucuronidation modification occurs at the 27th lysine residue of the light chain of pembrolizumab as shown in SEQ ID NO:2, the method comprising: The composition or pharmaceutical preparation is digested with trypsin to obtain the digested product; The enzyme digestion product is subjected to peptide mass fingerprint analysis, for example, using LC-MS / MS, to determine the presence or absence of the pembrolizumab containing glucuronidation modification based on the primary and secondary mass spectra.

[0009] Fourthly, this disclosure provides a method for quality inspection or quality control of products containing pembrolizumab, comprising detecting the content of glucuronidated pembrolizumab in the product containing pembrolizumab, wherein the glucuronidation modification occurs at the 27th lysine residue of the light chain of the amino acid sequence of pembrolizumab as shown in SEQ ID NO:2.

[0010] Fifthly, this disclosure provides the application of pembrolizumab with glucuronidation modification in the quality inspection or quality control of products containing pembrolizumab, wherein the glucuronidation modification occurs at the 27th lysine residue of the light chain of the amino acid sequence of pembrolizumab as shown in SEQ ID NO:2.

[0011] In some embodiments, the composition, pharmaceutical formulation, or product containing pembrolizumab comprises at least 100 mg / ml, at least 110 mg / ml, at least 120 mg / ml, at least 130 mg / ml, at least 140 mg / ml, at least 150 mg / ml, at least 160 mg / ml, at least 170 mg / ml, at least 180 mg / ml, at least 190 mg / ml, or at least 200 mg / ml of pembrolizumab.

[0012] Sequence Description SEQ ID NO:1 is the amino acid sequence of the two identical heavy chains of pembrolizumab, as shown below (Note: the underlined parts are CDR domains, and the CDR regions are annotated according to the Kabat coding rules): QVQLVQSGVEVKKPGASVKVSCKASGYTFT NYYMY WVRQAPGQGLEWMG GINPSNGGTNFNEKFKN RVTLTTDSSTTTAYMELKSLQFDDTAVYYCAR RDYRFDMGFDY WGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO:2 is the amino acid sequence of the two identical light chains of pembrolizumab, as shown below (Note: the underlined parts are CDR domains, and the CDR regions are annotated according to the Kabat coding rules): EIVLTQSPATLSLSPGERATLSC RASKGVSTSGYSYLH WYQQKPGQAPRLLIY LASYLES GVPARFSGSGSGTDFTLTISSLEPEDFAVYYC QHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC The term "pembrolizumab" refers to a compound formed by two identical heavy chains and two identical light chains linked by disulfide bonds, wherein the two identical heavy chain sequences are shown in SEQ ID NO:1 and the two identical light chain sequences are shown in SEQ ID NO:2.

[0013] In this disclosure, the term "about" means a range of values ​​that a person skilled in the art would consider equivalent to the listed values ​​(e.g., having the same function or result), such as + / -20% of the listed values.

[0014] In this disclosure, the terms “including,” “comprising,” and “containing” mean “including but not limited to” and are not intended to exclude other parts, additives, components, or steps.

[0015] The inventors of this disclosure unexpectedly discovered that modified pembrolizumab is produced during the preparation of pembrolizumab, such as pembrolizumab with glucuronidation modification, for example, said glucuronidation modification occurring at the 27th position of lysine (K) in the light chain of pembrolizumab. 27 (Located within the light chain CDR1 region).

[0016] In some embodiments, the light chain content of the pembrolizumab containing the glucuronidation modification being less than or equal to 3% of the total light chain content of pembrolizumab in the composition does not affect the binding activity and biological activity of the pembrolizumab sample. Therefore, in these embodiments, if the light chain content of the pembrolizumab containing the glucuronidation modification is assessed to be no higher than about 3% during the preparation of pembrolizumab, it is generally not necessary to perform operations to remove variants or impurities, which simplifies the production process and saves production costs to some extent.

[0017] In a first aspect, this disclosure provides compositions comprising pembrolizumab, wherein a portion of the pembrolizumab contains a glucuronidation modification at the 27th lysine residue of the light chain as shown in SEQ ID NO:2.

[0018] In some embodiments of the first aspect, the composition comprises at least 100 mg / ml, at least 110 mg / ml, at least 120 mg / ml, at least 130 mg / ml, at least 140 mg / ml, at least 150 mg / ml, at least 160 mg / ml, at least 170 mg / ml, at least 180 mg / ml, at least 190 mg / ml, or at least 200 mg / ml of pembrolizumab.

[0019] In some embodiments of the first aspect, based on trypsin-derived peptide mass fingerprinting analysis, the light chain content of the glucuronidated pembrolizumab in the composition is less than or equal to 3%, calculated as a percentage of the light chain content of the glucuronidated pembrolizumab in the composition relative to the total light chain content of pembrolizumab in the composition.

[0020] In some embodiments of the first aspect, the composition comprising the light chain content of the glucuronidated pembrolizumab as a percentage of the total light chain content of pembrolizumab in the composition is greater than or equal to 0.001%, greater than or equal to 0.01%, greater than or equal to 0.05%, greater than or equal to 0.1%, greater than or equal to 0.5%, greater than or equal to 1%, greater than or equal to 1.5%, greater than or equal to 2.0%, or greater than or equal to 2.5%.

[0021] In some embodiments of the first aspect, the composition comprising the light chain content of the glucuronidated pembrolizumab is less than or equal to 3.0%, less than or equal to 2.9%, less than or equal to 2.8%, less than or equal to 2.7%, less than or equal to 2.6%, less than or equal to 2.5%, less than or equal to 2.4%, less than or equal to 2.3%, less than or equal to 2.2%, less than or equal to 2.1%, less than or equal to 2.0%, and less than or equal to 2.0%. 1.9%, less than or equal to 1.8%, less than or equal to 1.7%, less than or equal to 1.6%, less than or equal to 1.5%, less than or equal to 1.4%, less than or equal to 1.3%, less than or equal to 1.2%, less than or equal to 1.1%, less than or equal to 1.0%, less than or equal to 0.9%, less than or equal to 0.8%, less than or equal to 0.7%, less than or equal to 0.6%, less than or equal to 0.5%, less than or equal to 0.4%, less than or equal to 0.3%, less than or equal to 0.2%, less than or equal to 0.1%, less than or equal to 0.05%, or less than or equal to 0.01%.

[0022] In some embodiments of the first aspect, the composition comprising the light chain content of the glucuronidated pembrolizumab is 0.001% to 3.0%, 0.01% to 3.0%, 0.05% to 3.0%, 0.1% to 3.0%, 0.2% to 3.0%, 0.3% to 3.0%, 0.4% to 3.0%, 0.5% to 3.0%, 0.6% to 3.0%, 0.7% to 3.0%, 0.8% to 3.0%, 0.9% to 3.0%, 1.0%, based on the percentage of the light chain content of the glucuronidated pembrolizumab in the total light chain content of pembrolizumab in the composition. % to 3.0%, 1.1% to 3.0%, 1.2% to 3.0%, 1.3% to 3.0%, 1.4% to 3.0%, 1.5% to 3.0%, 1.6% to 3.0%, 1.7% to 3.0%, 1.8% to 3.0%, 1.9% to 3.0%, 2.0% to 3.0%, 2.1% to 3.0%, 2.2% to 3.0%, 2.3% to 3.0%, 2.4% to 3.0%, 2.5% to 3.0%, 2.6% to 3.0%, 2.7% to 3.0%, 2.8% to 3.0%, 2.9% to 3.0%, or any interval within any of the foregoing ranges or any value within the intervals.

[0023] In some embodiments of the first aspect, the composition contains 0.08% to 2.59% light chain content of the glucuronidated pembrolizumab, for example, 0.08%, 0.67%, 1.30%, 2.59%, or any range or interval within the foregoing values ​​or ranges, based on the percentage of light chain content of the glucuronidated pembrolizumab in the total light chain content of pembrolizumab in the composition.

[0024] It should be understood that the above-mentioned content of pembrolizumab containing the glucuronidation modification is expressed as a percentage of the content of the light chain containing the modification to the total content of all light chains in the composition; however, this is merely one way of expressing it. Those skilled in the art will understand that the content of pembrolizumab containing the modification can also be expressed in various other ways, for example, as a percentage of the content of the glucuronidated peptide (which may be referred to as the "modified peptide") in the composition to the sum of the content of the modified peptide and the corresponding unmodified peptide (e.g., having the same or substantially the same amino acid sequence but not containing the modification).

[0025] Those skilled in the art can use various methods to measure and determine the content of the glucuronidated pembrolizumab in the composition. For example, in some embodiments, the content can be measured and determined by the following methods: (a) Take a certain amount of sample, add urea to a final concentration of 6M and TCEP to 10mM, incubate at 37℃ for 20min for denaturation and reduction, then add IAM to a final concentration of 20mM, incubate at 37℃ for 15min for alkylation treatment. (b) Then add 50mM ammonium bicarbonate solution to dilute the urea concentration to 1M, and then add sequencing grade trypsin at a protein:enzyme ratio of 10:1 (mass ratio), digest at 37℃ for 25min, and add formic acid to a final concentration of 1% to terminate the digestion. (c) After centrifugation, the samples were analyzed for peptide mass fingerprinting using liquid chromatography-tandem high-resolution mass spectrometry. Peptides were separated using a C18 column and then analyzed by primary and secondary mass spectrometry. Glucuronidated modified peptides (ASK) were then extracted. 27 The XIC values ​​of GVSTSGYSYLHWYQQKPGQAPR (SEQ ID NO:3), with mass-to-charge ratios of major ions of 743.86 and 595.29, and their corresponding unmodified peptides (GVSTSGYSYLHWYQQKPGQAPR (SEQ ID NO:4), with mass-to-charge ratios of major ions of 628.31 and 837.41), were calculated with a mass deviation of 20 ppm. The peak areas of each target component were obtained by integration, and the modification ratio was calculated according to the following formula: .

[0026] In a second aspect, this disclosure provides pharmaceutical formulations comprising the compositions described in the first aspect, and one or more pharmaceutically acceptable carriers.

[0027] Pharmaceutically acceptable carriers are non-toxic to the recipient at the dosage and concentration used. In some embodiments, pharmaceutically acceptable carriers include, for example: water; buffers such as phosphates, citrates, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives; hydrophilic polymers such as polyvinylpyrrolidone; chelating agents such as EDTA, etc.

[0028] In some embodiments, the pharmaceutical formulation may also contain one or more of the following: lubricants, such as talc, magnesium stearate and mineral oil; wetting agents; emulsifiers; suspending agents; preservatives, such as benzoic acid, sorbic acid and calcium propionate; sweeteners and / or flavoring agents, etc.

[0029] In some embodiments, the compositions, pharmaceutical formulations and / or products containing pembrolizumab in this disclosure may comprise one or more of histidine, sucrose, L-arginine, methionine, polysorbate 80, and hyaluronidase.

[0030] In some embodiments, the compositions, pharmaceutical preparations and / or products containing pembrolizumab of this disclosure may be formulated as tablets, pills, powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, suppositories or capsules.

[0031] In some embodiments, the compositions, pharmaceutical formulations and / or products containing pembrolizumab of this disclosure may be delivered using any physiologically acceptable route of administration, including but not limited to: oral administration, parenteral administration, nasal administration, rectal administration, intraperitoneal administration, intravascular injection, subcutaneous administration, transdermal administration, inhalation administration, etc.

[0032] In some implementations, the pharmaceutical formulation intended for in vivo administration must be sterile. This can be easily achieved by using sterile filter membranes.

[0033] In some implementations, a pharmaceutical preparation for therapeutic use can be formulated for storage by mixing reagents of the desired purity with, as appropriate, pharmaceutically acceptable carriers, excipients, etc., in the form of a lyophilized formulation or an aqueous solution.

[0034] In a third aspect, this disclosure provides a method for detecting pembrolizumab containing glucuronidated modification in a composition or pharmaceutical formulation containing pembrolizumab, wherein the glucuronidation modification occurs at the 27th lysine residue of the light chain of the amino acid sequence of pembrolizumab as shown in SEQ ID NO:2, the method comprising: The composition or pharmaceutical preparation is digested with trypsin to obtain the digested product; The enzyme digestion product is subjected to peptide mass fingerprint analysis, for example, using LC-MS / MS, to determine the presence or absence of the pembrolizumab containing glucuronidation modification based on the primary and secondary mass spectra.

[0035] In some embodiments of the third aspect, the pembrolizumab-containing composition or pharmaceutical formulation contains pembrolizumab or contains glucuronidated modified pembrolizumab in the amount described in the first aspect.

[0036] In some embodiments of the third aspect, the method further includes obtaining extracted ion chromatograms (XICs) of the glucuronidated and unmodified peptides after peptide mass fingerprinting analysis, and calculating the content of the glucuronidated pembrolizumab based on the corresponding peak areas.

[0037] In a fourth aspect, this disclosure provides a method for quality inspection or quality control of products containing pembrolizumab, wherein a portion of the pembrolizumab contains a glucuronidation modification at the 27th lysine residue of the light chain as shown in SEQ ID NO:2, and the method includes detecting the content of the glucuronidated pembrolizumab in the product containing the pembrolizumab.

[0038] In some embodiments, the composition containing the glucuronidated pembrolizumab light chain content as a percentage of the total light chain content of pembrolizumab in the composition is less than or equal to 3.0%, less than or equal to 2.9%, less than or equal to 2.8%, less than or equal to 2.7%, less than or equal to 2.6%, less than or equal to 2.5%, less than or equal to 2.4%, less than or equal to 2.3%, less than or equal to 2.2%, less than or equal to 2.1%, less than or equal to 2.0%, less than or equal to 1.9%, and less than or equal to 3.0%, 2.9%, 2.0%, 1.9%, and so on. A concentration of 1.8%, less than or equal to 1.7%, less than or equal to 1.6%, less than or equal to 1.5%, less than or equal to 1.4%, less than or equal to 1.3%, less than or equal to 1.2%, less than or equal to 1.1%, less than or equal to 1.0%, less than or equal to 0.9%, less than or equal to 0.8%, less than or equal to 0.7%, less than or equal to 0.6%, less than or equal to 0.5%, less than or equal to 0.4%, less than or equal to 0.3%, less than or equal to 0.2%, less than or equal to 0.1%, less than or equal to 0.05%, or less than or equal to 0.01% indicates that the product meets pharmaceutical requirements.

[0039] In a fifth aspect, this disclosure provides the use of pembrolizumab containing glucuronidation modification in the quality inspection or quality control of products containing pembrolizumab, wherein the glucuronidation modification occurs at the 27th lysine residue of the light chain of the amino acid sequence of pembrolizumab as shown in SEQ ID NO:2.

[0040] In some embodiments of the fourth and / or fifth aspects, the content of pembrolizumab or pembrolizumab containing glucuronidation modification in the product is as described in the first aspect.

[0041] In some implementations, this disclosure achieves at least one or more of the following beneficial technical effects: 1. The glucuronidation modification of a portion of the pembrolizumab in the compositions disclosed herein is located at the CDR position of the antibody light chain (at lysine residue position 27 of the light chain as shown in SEQ ID NO:2). Therefore, the biological activity and function (including, for example, antigen-binding activity) of the glucuronidated pembrolizumab relative to the unmodified pembrolizumab may be affected. However, this disclosure demonstrates that when the content of the glucuronidated pembrolizumab in the composition is less than or equal to 3% of the total pembrolizumab in the composition, the overall biological activity and function of the composition are not significantly changed.

[0042] 2. This disclosure demonstrates that if the content of glucuronidated pembrolizumab in the preparation of pembrolizumab is evaluated to be no more than 3% of the total pembrolizumab content in the sample (calculated as the percentage of the content of the light chain containing the modified light chain to the total content of the light chain containing the modified light chain and the unmodified light chain), it is generally not necessary to perform operations to remove variants or impurities. This simplifies the production process to a certain extent and saves production costs.

[0043] It should be understood that the features, characteristics, components, or steps described in a particular aspect, implementation, or embodiment of this disclosure may be applied to any other aspect, implementation, or embodiment described herein, unless there is any conflict therewith.

[0044] The foregoing disclosure generally describes the present invention, and the following embodiments further illustrate the invention. These embodiments are described merely to illustrate the invention and not to limit its scope. Although specific terms and values ​​are used herein, they are also understood to be exemplary and do not limit the scope of the invention. Unless specifically indicated, the experimental methods and techniques described herein are methods and techniques well known to those skilled in the art. Attached Figure Description

[0045] Figure 1 The glucuronidated modified peptide ASK of pembrolizumab was shown. 27 Extraction ion chromatogram (XIC) of GVSTSGYSYLHWYQQKPGQAPR (SEQ ID NO:3).

[0046] Figure 2 The glucuronidated modified peptide ASK of pembrolizumab was shown. 27 The first-order mass spectrum of GVSTSGYSYLHWYQQKPGQAPR (SEQ ID NO:3).

[0047] Figure 3 The glucuronidated modified peptide ASK of pembrolizumab was shown. 27The secondary mass spectrum of GVSTSGYSYLHWYQQKPGQAPR (SEQ ID NO:3). Glucuronidated peptides are prone to neutral loss during HCD fragmentation. Figure 3 "in "This is a +121.99 Da fragment ion generated by the neutral loss of lysine peptides after glucuronidation modification." Detailed Implementation

[0048] Example 1: Preparation process of pembrolizumab Upstream cell culture process The expression system used in this embodiment is the Merck CHOZN® GS expression system, which contains CHO-K1 host cells constructed with cGMP. The expression vector is the Merck pCGS3.2 plasmid, enabling efficient expression of exogenous recombinant proteins in mammalian cells. The culture medium system includes EX-CELL® CD CHO Fusion medium, QL001 medium, Cell Boost 5 medium, and OPMA XF04 high-efficiency fed medium.

[0049] Referring to the typical cell culture process for antibody drugs, pembrolizumab antigen solution was produced following the process of cell resuscitation, stepwise expansion in shake flasks and bioreactors, and production in a 500L disposable bioreactor. The production stage adopted a fed-batch culture process.

[0050] Take a cryopreserved tube from the working cell bank, thaw it in a 37°C water bath, and culture it in a shake flask expansion medium. After continuous expansion culture in shake flasks and a bioreactor, follow the formula (4.5±1.0)×10⁻⁶. 6 Cells were seeded at the target density of 500L single-use bioreactor.

[0051] The growth stage temperature was set at 36.5℃, and the expression stage temperature was lowered to 30℃. The pH was set at 7.0±0.3, and dissolved oxygen was set at 40%. The stirring speed was 50rpm~70rpm. Aeration was provided at the top (0~15) L / min and at the bottom (0~10) L / min. pH control was coupled with bottom aeration (carbon dioxide) and sodium carbonate solution, while dissolved oxygen control was coupled with bottom aeration (oxygen). During culture, 28%~48% of the initial culture medium volume was added, along with glucose solution and antifoaming agent solution as needed. The culture time was 16 days. The cell culture medium was purified by deep filtration and 0.2μm sterile filtration, and the supernatant was collected.

[0052] Downstream purification process This embodiment employs a typical purification process for antibody drugs, including: affinity chromatography, low-pH virus inactivation, anion exchange chromatography, cation exchange chromatography, virus removal filtration, ultrafiltration concentration, and finally, the addition of excipients followed by sterile filtration to obtain the stock solution.

[0053] Protein A affinity chromatography: Protein A affinity chromatography was used as the primary capture step, employing ATProtein A Diamond plus packing material from BorgLyn. The target antibody bound to the packing material, while media components and host cell protein impurities did not bind. After equilibrating the column with Tris solution, the loading phase began. After loading, the column was washed, and the eluted protein was collected.

[0054] Low-pH virus inactivation: The eluent from protein A affinity chromatography was adjusted to a low pH using acetic acid to inactivate any remaining virus. The inactivated product was neutralized with Tris solution and then filtered through a depth filter and a sterile filter to obtain the virus-inactivated and neutralized filtered sample. This sample was then further processed in a subsequent anion exchange chromatography step.

[0055] Anion exchange chromatography: Anion exchange chromatography was performed using Toyopearl® NH2-750F packing material from TOSOH. The column was equilibrated with Tris buffer, and the virus-inactivated and neutralized filtered product was loaded. During loading, the target protein flowed through the chromatography column. The target protein was collected according to the collection interval during loading and post-loading equilibration to obtain the anion exchange chromatography collect solution.

[0056] Cation exchange chromatography: Cation chromatography was performed using Capto SP ImpRes packing material from Cytiva, and the column was equilibrated with sodium acetate-acetic acid buffer. The protein solution collected from anion exchange chromatography was loaded onto the packing material to adsorb the protein. After loading, a gradient elution method was used to elute the target protein from the cation exchange chromatography column, and the eluent was collected.

[0057] Virus removal filtration: The cation exchange chromatography collection solution was filtered for virus removal using a pre-filter (A1HC, cellulose diatomaceous earth material) and a virus removal filter (Viresolve® Pro, PES material). After filtration, the tubing and filter were rinsed with buffer solution, and the filtered sample was collected for sterile filtration.

[0058] Ultrafiltration Concentration: The antibody concentration is adjusted to the desired level using ultrafiltration concentration. After ultrafiltration concentration, the excipients to be added are added to the antibody solution, and the protein concentration is finally adjusted to approximately 165 mg / ml. The final product also contains 10 mM histidine, 2% sucrose, 2% L-arginine, 15 mM methionine, 0.4 mg / ml polysorbate 80, pH 5.5, and hyaluronidase 2000 IU / ml. The sample is sterilely filtered through a 0.22 μm filter and filled into sterile 2 ml vials under controlled conditions, sealed with a membrane-coated rubber stopper and an aluminum-plastic composite cap.

[0059] Example 2: Discovery of glucuronidation modification in pembrolizumab The sample prepared in Example 1 was analyzed by liquid chromatography-tandem high-resolution mass spectrometry, and the steps are as follows: Urea and TCEP were added to the sample to a final concentration of 6M, and incubated at 37ºC for 20 min for denaturation and reduction. After incubation, iodoacetamide (IAM) was added to a final concentration of 20mM, and the sample was incubated at 37ºC in the dark for 15 min for alkylation. Then, urea was diluted to 1M with 50mM ammonium bicarbonate solution, and sequencing-grade trypsin (Rhinogen, catalog number: QIP-003A) was added at a protein:enzyme ratio of 10:1 (mass ratio), and digestion was performed at 37ºC for 25 min. After digestion, formic acid was added to a final concentration of 1% to terminate the digestion. The sample was centrifuged at 12000 rpm for 3 min, and the supernatant was transferred to a sample vial.

[0060] The sample was analyzed using liquid chromatography-tandem high-resolution mass spectrometry (LC-MS / MS) with an injection volume of 10 μg. The glucuronidated peptide (peptide sequence: ASK) in the light chain CDR1 was targeted for acquisition. 27 The primary and secondary mass spectrometry information for the major ion form (mass-to-charge ratio of 743.86) of GVSTSGYSYLHWYQQKPGQAPR (SEQ ID NO:3) is as follows, with the chromatographic and mass spectrometry conditions used: Chromatographic conditions:

[0061] Mass spectrometry conditions:

[0062] Data analysis was performed using Xcalibur software.

[0063] Figures 1 to 3 The extracted ion current chromatograms (XIC) of the modified peptide are shown below. Figure 1 ), first-order mass spectrum ( Figure 2 ) and secondary mass spectra ( Figure 3 ).

[0064] The above analysis results indicate that the lysine residue at position 27 of the light chain of pembrolizumab (LC:K) 27 It exhibits glucuronidation modification.

[0065] Example 3: Preparation and content determination of pembrolizumab-forced glucuronidated samples In this embodiment, pembrolizumab was artificially glucuronidated with glucuronic acid to obtain a glucuronidated sample of pembrolizumab (referred to herein as a “forced glucuronidated sample” or “forced sample”).

[0066] Using commercially available glucuronic acid reagent, pembrolizumab was placed in a 128 mM glucuronic acid solution and incubated at 37°C for 6 h, 12 h, and 24 h, respectively. After incubation, the forced-incubation samples were treated with formulation excipients to change the incubation solution.

[0067] Then, the LC:K content in the forced sample was determined using peptide mass fingerprinting analysis. 27 The proportion of glucuronidation modification at each site. The analytical steps are as follows: Approximately 150 μg of sample was added to a final concentration of 6 M urea and 10 mM TCEP, and incubated at 37 °C for 20 min for denaturation and reduction. Then, 20 mM IAM was added, and the mixture was incubated at 37 °C for 15 min for alkylation. The urea concentration was diluted to 1 M with 50 mM ammonium bicarbonate buffer, and sequencing-grade trypsin (manufacturer: Rhinogen, catalog number: QIP-003A) was added at a protein:enzyme ratio of 10:1 (mass ratio), and digestion was performed at 37 °C for 25 min. After digestion, 1% formic acid was added to terminate the digestion. The sample was centrifuged at 12000 rpm for 3 min, and the supernatant was transferred to a sample vial.

[0068] Peptide mass fingerprinting was performed using liquid chromatography-tandem high-resolution mass spectrometry (LC-MS / MS) with an injection volume of 3 μg. The chromatographic and mass spectrometric conditions were as follows: Chromatographic conditions:

[0069] Mass spectrometry conditions:

[0070] Data analysis was performed using Byos software from Protein Metrics to extract modified peptides (ASK). 27GVSTSGYSYLHWYQQKPGQAPR (SEQ ID NO:3), with mass-to-charge ratios of the major ions of 743.86 and 595.29, and its corresponding unmodified peptide (GVSTSGYSYLHWYQQKPGQAPR (SEQ ID NO:4), with mass-to-charge ratios of the major ions of 628.31 and 837.41), showed the strongest XIC response in the two charge forms. The peak areas of each target component were obtained by integration, and the modification ratio was calculated according to the following formula:

[0071] The modification ratios for each sample are shown in Table 1 below. The analytical results showed that pembrolizumab, after incubation in glucuronic acid solution at 37°C for 24 hours, [LC:K] 27 The site glucuronidation modification rate reached 2.59%.

[0072] Table 1. LC:K in forced samples 27 The proportion of glucuronidated modified components exists.

[0073] Example 4: Activity assay of pembrolizumab in forced glucuronidation samples In this embodiment, the binding activity and biological activity of the samples prepared in Example 1 and Example 3 are studied.

[0074] The ability of the above test samples to competitively bind PD-1 with PD-L1 was determined by time-resolved fluorescence resonance energy transfer (TR-FRET) method, as follows: First, add serially diluted control and test samples (5 μl / well) to a 96-well shallow plate. Then, add diluted PD1 (His-tagged) protein and biotinylated PD-L1 protein (5 μl / well) to the plate. Next, mix equal volumes of 100-fold diluted streptavidin-d2 and MAb Anti-6HIS-Tb cryptate Gold and add 10 μl / well to the 96-well shallow plate. Seal the plate with sealing film and incubate at room temperature for 2 hours. Briefly centrifuge the incubated 96-well shallow plate at 1000 rpm and read the plate using a time-resolved fluorescence microplate reader (TECAN SPARK multi-functional microplate reader) with excitation wavelength: 320 nm; emission wavelengths: 620 nm and 665 nm.

[0075] Using sample concentration as the X-axis and TR-FRET ratio (665nm signal value ÷ 620nm signal value × 10000, automatically calculated after software settings) as the Y-axis, a four-parameter fitting was performed, and the calculation results were obtained.

[0076] Using the sample prepared in Example 1 as a control (the PD-1 binding activity of the control sample was recorded as 100%), the binding activity of the forced glucuronidation sample prepared in Example 3 was determined. The activity determination results are shown in Table 2 below. The analysis results show that the PD-1 binding activity of each sample is between 80% and 120% of that of the control sample.

[0077] Table 2. Results of binding activity assay for forced glucuronidation samples

[0078] The biological activities of various samples were detected using a reporter gene assay. CHO cells transfected with PD-L1 and an anti-CD3 single-chain antibody fragment (scFv) were used as target cells, and Jurkat cells transfected with PD-1 and the NFAT-regulated luciferase gene were used as effector cells. After the anti-CD3-scFv on the CHO cell membrane binds to CD3 on the surface of Jurkat cells, it presents an activation signal to the Jurkat cells, thereby expressing luciferase. PD-L1 on the CHO cell surface binds to PD-1 on the surface of Jurkat cells, delivering an inhibitory signal to the Jurkat cells, inhibiting luciferase expression. Anti-PD-1 / anti-PD-L1 antibodies can block the binding of PD-1 to PD-L1, thereby releasing the inhibitory signal, restoring luciferase expression, and generating a fluorescent signal. Therefore, this reporter gene assay was used to determine the biological activities of different types of anti-PD-1 / anti-PD-L1 antibodies.

[0079] First, adjust the CHO-PD-L1-CD3L cell density to 5 × 10⁶ cells using complete culture medium. 5 Cells / ml were added at 80 µl / well to 96-well cell culture plates and incubated at 37°C in a 5% CO2 incubator for 14–18 hours. The control sample and test sample were serially diluted with assay medium. The cell density of the Jurkat-PD1-NFAT cell suspension was adjusted to 2 × 10⁶ cells / ml with assay medium. 6 Cells / ml. Remove the culture plate, discard the supernatant, and add 40 µl / well of each diluted control sample and test sample at various concentration gradients. Then add 40 µl / well of Jurkat-PD1-NFAT cell suspension and incubate at 37°C, 5% CO2 for 6 hours. After incubation, remove the 96-well culture plate, add 80 µl of BioLite staining solution to each well, and mix thoroughly by shaking at room temperature in the dark. Read the plate using a chemiluminescence microplate reader. Perform a four-parameter fitting with sample concentration as the X-axis and the average chemiluminescence signal value of the control sample and test sample as the Y-axis to calculate the results.

[0080] Using the sample prepared in Example 1 as a control (the biological activity of the control sample PD-1 was recorded as 100%), the biological activity of the forced glucuronidation sample prepared in Example 3 was determined. The results of the biological activity analysis are shown in Table 3 below. The analysis results show that the biological activity of each sample is between 80% and 120% of that of the control sample.

[0081] Table 3. Results of biological activity analysis of forced glucuronidation samples

[0082] The results above show that the binding activity and biological activity of the sample prepared in Example 3 are basically the same as those of the sample in Example 1.

[0083] Various changes and equivalent substitutions may be made to the embodiments disclosed herein without departing from the spirit and scope of this disclosure. Unless otherwise stated in the context, any feature, step, or implementation of the embodiments of this disclosure may be combined with any other feature or implementation.

Claims

1. A composition comprising at least 100 mg / ml of pembrolizumab, wherein a portion of the pembrolizumab contains a glucuronidation modification at lysine residue 27 of its light chain as shown in SEQ ID NO:2, and wherein the light chain content of the glucuronidated pembrolizumab in the composition is less than or equal to 3%, calculated as a percentage of the light chain content of the pembrolizumab containing the glucuronidation modification relative to the total light chain content of pembrolizumab in the composition, based on trypsin peptide mass fingerprinting analysis.

2. The composition of claim 1, wherein the light chain content of the glucuronidated pembrolizumab in the composition is less than or equal to 2.7% based on the percentage of the light chain content of the glucuronidated pembrolizumab in the composition relative to the total light chain content of pembrolizumab in the composition.

3. A pharmaceutical preparation comprising the composition of claim 1 or 2, and one or more pharmaceutically acceptable carriers.

4. A method for detecting glucuronidated pembrolizumab in a composition or pharmaceutical formulation containing pembrolizumab, wherein the method is used only to detect glucuronidation modification occurring at lysine residue 27 of the light chain of pembrolizumab as shown in SEQ ID NO:2, the method comprising: The composition or pharmaceutical preparation is digested with trypsin to obtain the digested product; The presence or absence of the pembrolizumab containing glucuronidation modification was determined by peptide mass fingerprinting analysis of the enzyme digestion product. The composition or pharmaceutical preparation described herein contains at least 100 mg / ml of pembrolizumab.

5. The method as described in claim 4, characterized in that... Peptide mass fingerprinting was performed using LC-MS / MS to determine the presence or absence of the glucuronidated pembrolizumab based on primary and secondary mass spectra.

6. The method of claim 4 or 5, further comprising obtaining an extracted ion current chromatogram (XIC) of the peptide containing the glucuronidated modified peptide and the unmodified peptide after peptide mass fingerprint analysis, and calculating the content of the glucuronidated pembrolizumab based on the corresponding peak area.

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