EA046423B1 - WAYS TO REDUCE POLYSORBATE DEGRADATION IN PHARMACEUTICAL COMPOSITIONS OF DRUGS - Google Patents

Description

Cross reference to related applications

This application claims priority to U.S. Provisional Patent Application No. 62/982346, filed February 27, 2020, U.S. Provisional Patent Application No. 63/021181, filed May 7, 2020, and U.S. Provisional Patent Application No. 63/073125, filed September 1

2020, the contents of which are incorporated herein by reference in their entirety.

Technical field

This invention generally relates to compositions with reduced amounts of certain lipases, methods for preparing such compositions, and methods for reducing polysorbate degradation caused by the presence of such lipases. In particular, this invention generally relates to compositions and methods for preparing compositions with reduced presence of hepatic carboxylesterase B1-like protein and hepatic carboxylesterase 1-like protein.

State of the art

Among drugs, protein-based biotherapeutics are an important class of drugs that provide high levels of selectivity, potency, and efficacy, as evidenced by the significant increase in the number of clinical trials with monoclonal antibodies (mAbs) over the past few years. Bringing a protein-based biotherapeutic agent into the clinic can take many years and require coordinated efforts across multiple areas of research and development, including discovery, process and pharmaceutical formulation development, analytical characterization, and preclinical toxicology and pharmacology studies.

One of the most important aspects of a clinically and commercially viable biotherapeutic agent is the stability of the drug product in terms of the manufacturing process, as well as the length of shelf life. This often requires appropriate steps aimed at increasing the physical and chemical stability of protein-based biotherapeutic agents in the various solutions and media required for production and storage, with minimal impact on product quality, including the identification of molecules with greater primary stability, protein engineering and development pharmaceutical composition. Surfactants such as polysorbate are often used to enhance the physical stability of a protein-based biotherapeutic product. More than seventy percent of marketed monoclonal antibody therapeutics contain 0.001% to 0.1% polysorbate, a type of surfactant, to impart physical stability to protein-based biotherapeutics. Polysorbates are susceptible to auto-oxidation and hydrolysis, leading to the formation of free fatty acids and the subsequent formation of fatty acid particles. Degradation of polysorbate can have a negative effect on the quality of the drug product because polysorbate can protect against interfacial stresses such as aggregation and adsorption. The presence of certain lipases may be a likely cause of degradation of polysorbates in pharmaceutical formulations. Therefore, such lipases in drugs need to be identified, monitored, and their levels reduced.

Direct analysis of lipases may require isolation of the product in sufficiently large quantities for analysis, which is undesirable and only possible in selected cases. Therefore, determining the process technology and analytical tests required to characterize the lipases responsible for polysorbate degradation in a sample is challenging. In addition to identifying the lipases responsible for polysorbate degradation, the drug product must be prepared by purification methods that remove or reduce the level of such lipases.

It should be understood that there is a need for methods of depleting lipase from a pharmaceutical formulation of a drug product.

Brief description of the invention

Maintaining the stability of pharmaceutical drug formulations not only during storage, but also during production, transportation, processing and administration is a major challenge. Protein biotherapeutic agents are becoming increasingly popular among drug products due to their effectiveness and versatility. One of the major challenges in developing protein biotherapeutics is overcoming the limited stability of protein and excipients in products, which can be affected by the presence of lipases (present as host cell proteins). Assessing the effect of lipases on the pharmaceutical formulation of a drug and reducing the level of such lipases can be an important step in the development of a pharmaceutical formulation of a drug, followed by methods for producing a pharmaceutical formulation of a drug with reduced lipase content and increased stability due to the reduced level of lipases.

In one illustrative embodiment of the present invention, a method is provided for depleting lipase from a sample, comprising contacting said sample containing a lipase with a probe, wherein said probe has the ability to bind to said lipase to form a complex and separate said complex from said sample, thereby removing said lipase from said sample. In one aspect of the present invention, said sample may contain a protein of interest. In one aspect of the present invention, said sample may contain a polysorbate excipient. In a particular aspect of the present invention, said polysorbate excipient may be selected from polysorbate-20, polysorbate-60, polysorbate-80, or combinations thereof. In yet another specific aspect of the present invention, said polysorbate excipient is polysorbate-80.

In one aspect of the present invention, said lipase is a hepatic carboxylesterase B1-like protein. In another aspect of the present invention, said lipase is a hepatic carboxylesterase 1-like protein. In one aspect of the present invention, said lipase has the ability to cause degradation of polysorbate in said sample. Therefore, the method according to this embodiment of the present invention reduces the degradation of polysorbates by removing lipase from the specified sample.

In one aspect of the present invention, said probe may have the ability to bind to a solid support. In a particular aspect of the present invention, said solid support may be agarose beads or magnetic beads.

In one aspect of the present invention, said probe may be attached to a solid support using a ligand. In a particular aspect of the present invention, said ligand may be an indicator, a biotin molecule, a modified biotin molecule, a nucleic acid, a sequence, an epitope tag, an electron-poor molecule, or an electron-rich molecule.

In one aspect of the present invention, said method may further include extracting said lipase from said complex.

In one illustrative embodiment of the present invention, a method is provided for purifying a sample containing a protein of interest and a lipase, comprising contacting said sample with a probe, wherein said probe has the ability to bind to said lipase to form a complex and separate said complex from the specified sample. In one aspect of the present invention, said lipase is a hepatic carboxylesterase B1-like protein. In another aspect of the present invention, said lipase is a hepatic carboxylesterase 1-like protein.

In one aspect of the present invention, said sample contains a polysorbate excipient. In a particular aspect of the present invention, said polysorbate excipient may be selected from polysorbate-20, polysorbate-60, polysorbate-80, or combinations thereof. In yet another specific aspect of the present invention, said polysorbate excipient may be polysorbate-80.

In one aspect of the present invention, said probe may have the ability to bind to a solid support. In a particular aspect of the present invention, said solid support may be agarose beads or magnetic beads.

In one aspect of the present invention, said probe may be attached to a solid support using a ligand. In a particular aspect of the present invention, said ligand may be an indicator, a biotin molecule, a modified biotin molecule, a nucleic acid, a sequence, an epitope tag, an electron-poor molecule, or an electron-rich molecule.

In one illustrative embodiment of the present invention, a method of reducing the degradation of polysorbate in a sample is provided, comprising bringing said sample containing lipase and polysorbate into contact with a probe, wherein said probe has the ability to bind to said lipase to form a complex and separate said complex from of said sample, thereby reducing the degradation of polysorbate in said sample.

In one aspect of the present invention, said lipase is a hepatic carboxylesterase B1-like protein. In another aspect of the present invention, said lipase is a hepatic carboxylesterase 1-like protein.

In one aspect of the present invention, said sample may contain a protein of interest. In one aspect of the present invention, said sample may contain a polysorbate excipient. In a particular aspect of the present invention, said polysorbate excipient is selected from polysorbate-20, polysorbate-60, polysorbate-80, or combinations thereof. In another specific aspect of the present invention, said polysorbate excipient is polysorbate-80.

In one aspect of the present invention, said probe may have the ability to bind to a solid support. In a particular aspect of the present invention, said solid support may be agarose beads or magnetic beads.

In one aspect of the present invention, said probe may be attached to a solid support using a ligand. In a particular aspect of the present invention, said ligand may be an indicator, a biotin molecule, a modified biotin molecule, a nucleic acid, a sequence, an epitope tag, an electron-poor molecule, or an electron-rich molecule.

In one illustrative embodiment of the present invention, a composition is provided,

- 2 046423 containing a protein of interest purified from mammalian cells and a residual amount of hepatic carboxylesterase B1-like protein. In one aspect of the present invention, the residual amount of hepatic carboxylesterase B1-like protein is less than about 5 ppm. In another aspect of the present invention, said composition may further comprise a surfactant. In yet another aspect of the present invention, said surfactant may be a hydrophilic non-ionic surfactant. In another aspect of the present invention, said surfactant may be a sorbitan fatty acid ester. In a particular aspect of this invention, said surfactant may be a polysorbate. In another specific aspect of the present invention, the concentration of polysorbate in the composition may range from about 0.01% w/v. to about 0.2% w/v. In another specific aspect of the present invention, said surfactant may be polysorbate 80. In one aspect of the present invention, said mammalian cells may include a CHO cell.

In one aspect of the present invention, said hepatic carboxylesterase B1-like protein can cause degradation of polysorbate 80.

In one aspect of the present invention, said composition may be a pharmaceutical composition for parenteral administration.

In one aspect of the present invention, said protein of interest may be a monoclonal antibody, a polyclonal antibody, a bispecific antibody, an antibody fragment, a fusion protein, or an antibody-drug complex. In one aspect of the present invention, the concentration of said protein of interest may be from about 20 mg/ml to about 400 mg/ml.

In one aspect of the present invention, said composition may further comprise one or more pharmaceutically acceptable excipients. In another aspect of the present invention, the composition may further comprise a buffer selected from the group consisting of histidine buffer, citrate buffer, alginate buffer and arginine buffer. In one aspect of the present invention, said composition may further comprise a tonicity modifier. In yet another aspect of the present invention, the composition may further comprise sodium phosphate.

In one illustrative embodiment of the present invention, a composition is provided comprising a protein of interest purified from mammalian cells and a residual amount of hepatic carboxylesterase-1-like protein. In one aspect of the present invention, the residual amount of hepatic carboxylesterase 1-like protein is less than about 5 ppm. In another aspect of the present invention, said composition may further comprise a surfactant. In yet another aspect of the present invention, said surfactant may be a hydrophilic non-ionic surfactant. In another aspect of the present invention, said surfactant may be a sorbitan fatty acid ester. In a particular aspect of this invention, said surfactant may be a polysorbate. In another specific aspect of the present invention, the concentration of polysorbate in the composition may be from about 0.01% w/v to about 0.2% w/v. In another specific aspect of the present invention, said surfactant may be polysorbate 80. In one aspect of the present invention, said mammalian cells may include a CHO cell.

In one aspect of the present invention, said hepatic carboxylesterase 1-like protein can cause degradation of polysorbate 80.

In one aspect of the present invention, said composition may be a pharmaceutical composition for parenteral administration.

In one aspect of the present invention, said protein of interest may be a monoclonal antibody, a polyclonal antibody, a bispecific antibody, an antibody fragment, a fusion protein, or an antibody-drug complex. In one aspect of the present invention, the concentration of said protein of interest may be from about 20 mg/ml to about 400 mg/ml.

In one aspect of the present invention, said composition may further comprise one or more pharmaceutically acceptable excipients. In another aspect of the present invention, the composition may further comprise a buffer selected from the group consisting of histidine buffer, citrate buffer, alginate buffer and arginine buffer. In one aspect of the present invention, said composition may further comprise a tonicity modifier. In yet another aspect of the present invention, the composition may further comprise sodium phosphate.

In one illustrative embodiment of the present invention, a method for detecting lipase in a sample is provided. In one aspect of the present invention, said lipases may be hepatic carboxylesterase-1-like protein and hepatic carboxylesterase-like protein

- 3 046423 times B1. In one aspect of the present invention, said method of detecting lipase in a sample may include contacting said sample with a serine hydrolase probe. In one aspect of the present invention, said method of detecting lipase in a sample may include contacting said sample with a serine hydrolase probe and incubating them together to form a complex of the lipase with said serine hydrolase probe. In another aspect of the present invention, said method of detecting lipase in a sample may include filtering out a serine hydrolase probe that does not form said lipase complex with said serine hydrolase probe.

In one aspect of the present invention, said method of detecting lipase in a sample may further comprise contacting said sample with magnetic beads having the ability to bind to said serine hydrolase probe, such that said magnetic beads bind to said lipase complex with said serine hydrolase. hydrolase probe. Magnetic beads bound to said lipase complex with said serine hydrolase probe can then be removed from said sample and washed with buffer.

In another aspect of the present invention, said method may further comprise recovering said magnetic beads that are bound to said lipase complex with said serine hydrolase probe to form a lipase-rich solution.

In one aspect of the present invention, said method may further include adding a hydrolyzing agent to said solution to produce cleavage products. In a particular aspect of this invention, said hydrolyzing agent may be trypsin. In one aspect of the present invention, said method may further include analyzing said degradation products to identify said lipases. In one aspect of the present invention, said degradation products can be analyzed using a mass spectrometer. In a specific aspect of the present invention, said mass spectrometer may be a tandem mass spectrometer. In another specific aspect of the present invention, said mass spectrometer may be coupled to a liquid chromatography system. In yet another specific aspect of the present invention, said mass spectrometer can be coupled to a liquid chromatography and multiple reaction monitoring system.

In one aspect of the present invention, said method may further comprise adding a protein denaturing agent to said solution. In a particular aspect of the present invention, said protein denaturing agent may be urea. In one aspect of the present invention, said method may further comprise adding a protein reducing agent to said solution. In a specific aspect of the present invention, said protein reducing agent may be DTT (dithiothreitol; DTT). In one aspect of the present invention, said method may further comprise adding a protein alkylating agent to said solution. In a specific aspect of the present invention, said protein alkylating agent may be iodoacetamide.

These and other aspects of the present invention will be better appreciated and understood when taken in conjunction with the following description and accompanying drawings. The following description, although indicating various embodiments of the present invention and numerous specific details thereof, is presented by way of illustration and not limitation. Many substitutions, modifications, additions or permutations may be made within the scope of the present invention.

Brief description of graphic materials

In fig. Figure 1 shows the chemical structures of the main components of polysorbates. Polysorbates are primarily composed of fatty acid esters having a common head group of POE-sorbitan, POE-isosorbide, or POE, with oleic acid as the major fatty acid for PS80. Panel A on the right shows the total ion current (TIC) chromatogram of PS80 in the pharmaceutical mAb formulation obtained using online analysis by two-dimensional liquid chromatography with mass spectrometry (2DLC/MS). The designated peaks are identified as the following: (1) POE - POE-isosorbide - POEsorbitan, (2) POE-sorbitan-monolinoleate, (3) POE-sorbitan-monooleate, (4) POE-isosorbide-monooleate and POE-monooleate, (5 ) POE-sorbitan-linoleate/oleate diester, (6) POE-sorbitan-dioleate, (7) POE-isosorbide dioleate and POE-dioleate, (8) probably POE-isosorbide/POE-linoleate/oleate diester, since the mass spectra are too complex to interpret, (9) a mixture of POE-sorbitan trioleate and tetraoleate. Panel B on the right shows the charged aerosol detection (CAD) chromatogram of PS80 in the pharmaceutical mAb formulation obtained using online 2D-LC/CAD analysis.

In fig. 2 shows a chromatogram of 0.1% PS80 in 50 mg/ml mAb-1 after incubation at 5°C in 10 mM histidine, pH 6, for 0 hours and 36 hours according to an illustrative embodiment of the present invention. Peaks eluted between 11 and 17.5 min were POE, POEisosorbide, and POE-sorbitan.

In fig. Figure 3 shows a graph of the percentage of residual PS80 depending on the duration of incubation, where the original mAb-1, mAb-1 mixed with 0.125 µM, 0.5 µM and 2 µM probe

FFs are indicated by a filled circle with a black solid line, a filled diamond with a red dotted line, a filled square with an orange dotted line, and a filled triangle with a blue dotted line.

In fig. 4 is a diagram of a lipase(s) depletion experiment according to an exemplary embodiment of the present invention. Streptavidin magnetic Dynabeads were coupled to a desthiobiotin-FF probe and used to deplete lipase(s). The original mAb-1, eluted mAb-1, and process control mAb-1 were incubated with 0.1% PS80 at 5°C for 36 h and subjected to PS degradation measurements. The concentrated lipase(s) were digested and analyzed for HCP by mass spectrometry.

In fig. Figure 5 shows a graph of the percentage of residual PS80 in the original mAb-1, in the technological control mAb-1 and in mAb-1 after depletion of the lipase (lipases), where the original mAb-1, technological control mAb-1 and mAb-1 after depletion of the lipase ( lipases) are indicated by a filled diamond with a black solid line, a filled square with a blue dotted line, and a filled circle with an orange dotted line.

In fig. 6A shows a chromatogram of 0.1% PS80 in 20 μg/ml commercially purchased rabbit liver esterase after incubation at 5°C in 10 mM histidine, pH 6, for 0 h, 1.5 h, and 8 h according to the illustrative embodiment of this invention.

In fig. 6B shows a chromatogram of 0.1% PS80 in 100 μg/ml commercially purchased human liver carboxylesterase 1 after incubation at 5°C in 10 mM histidine, pH 6, for 0 hour, 5 hour and 18 hour according to the illustrative embodiment implementation of this invention.

In fig. 6C shows a chromatogram of 0.1% PS80 in 50 mg/ml mAb-1 after incubation at 5°C in 10 mM histidine, pH 6, for 0 hour, 18 hour and 36 hour according to an exemplary embodiment of the present invention.

In fig. 6D shows a sequence alignment of hepatic carboxylesterase-like protein B-1 (A0A061I7X9), hepatic carboxylesterase-like protein 1 (A0A061FE2) and human hepatic carboxylesterase (hCES-1).

Detailed description of the invention

Host cell proteins (HCPs) are a class of impurities that must be removed from all cell-derived protein therapeutics. The US Food and Drug Administration (FDA) has not established a maximum permissible level of HCP, but concentrations of HCP in the final drug product must be controlled and reproducible from batch to batch (FDA, 1999). The main safety concern relates to the possibility that CCPs may cause antigenic effects in human patients (Satish Kumar Singh, Impact of Product-Related Factors on Immunogenicity of Biotherapeutics, and 100 JOURNALS of Pharmaceutical Sciences 354-387 (2011)). In addition to adverse effects on patient health, CHOs containing enzymatic activity may potentially affect product quality during processing or long-term storage (Sharon X. Gao et al., Fragmentation of a highly purified monoclonal antibody attributed to residual CHO cell protease activity , 108 Biotechnology and Bioengineering 977-982 (2010) Flavie Robert et al., Degradation of an Fc-fusion recombinant protein by host cell proteases: Identification of a CHO cathepsin D protease, 104 Biotechnology and Bioengineering 1132-1141 (2009) . HCPs may pose the greatest risk due to their persistence after the manufacturing purification process in the final drug product. During long-term storage, the critical quality characteristics of the product molecule must be maintained, and the degradation of excipients in the final product, the pharmaceutical composition, must be minimized.

Some pharmaceutical drug formulations on the market contain polysorbate as one of the most commonly used nonionic surfactants in biopharmaceutical protein drugs, which can improve protein stability and protect drugs from aggregation and denaturation (Sylvia Kiese et al., Shaken, Not Stirred: Mechanical Stress Testing of an IgG1 Antibody, 97 Journal of Pharmaceutical Sciences 4347-4366 (2008); Ariadna Martos et al., Trends on Analytical Characterization of Polysorbates and Their Degradation Products in Biopharmaceutical Formulations, 106 Journal of Pharmaceutical Sciences 1722-1735 (2017)). Polysorbate 20 (PS20) and polysorbate 80 (PS80) are the most commonly used nonionic surfactants in biopharmaceutical protein drugs, which can improve protein stability and protect drugs from aggregation and denaturation. Typical polysorbate concentrations in a number of drug products may range from about 0.001% to about 0.1% (w/v) to provide sufficient protein stability.

Polysorbates, however, are susceptible to degradation, which can lead to unwanted particle formation in drug formulations. It is known that the degradation of polysorbates occurs in two main ways: autoxidation and hydrolysis. It was found that oxidation is more likely to occur in PS80 due to the high content of unsaturated fatty acid esters, while in PS20 oxidation is believed to occur at the ester bond in the polyoxyethylene chain, which is not often observed (Oleg V. Borisov , Junyan A. Ji & Y. John Wang, Oxidative Degradation of Polysorbate Surfactants Studied by Liquid Chromatography-Mass Spectrometry, 104 Journal of Pharmaceutical Sciences 1005-1018 (2015); Anthony Tomlinson et al., Polysorbate 20 Degradation in Biopharmaceutical Formulations: Quantification of Free Fatty Acids, Characterization of Particulates, and Insights into the Degradation Mechanism, 12 Molecular Pharmaceutics 3805-3815 (2015); Jia Yao et al., A Quantitative Kinetic Study of Polysorbate Autoxidation: The Role of Unsaturated Fatty Acid Ester Substituents, 26 Pharmaceutical Research 2303-2313 (2009)). In addition to this, polysorbates can also undergo hydrolysis by breaking the ester bond of the fatty acids. Particles generated by the degradation of polysorbates may be visible or even undetectable, which may increase the potential for immunogenicity in the patient and may have varying effects on the quality of the drug product. One such possible impurity may be fatty acid particles that are generated during the manufacture, transportation, storage, handling or administration of pharmaceutical drug formulations containing polysorbate. Fatty acid particles have the potential to cause adverse immunogenic effects and affect shelf life. In addition, the degradation of polysorbates can also lead to a decrease in the overall surfactant level in the pharmaceutical formulation, which affects the stability of the product during its manufacture, storage, handling and administration.

As a rule, degradation of polysorbate can be observed in medicinal products only after quite long storage. However, degradation of PS80 was observed in one monoclonal antibody (mAb) preparation for 24 hours at 4°C, although no clearly highly concentrated lipase was detected, suggesting that unknown lipase(s) were present in this drug product ). It is critical to identify and reduce the concentration(s) of such lipase(s) to maintain the stability of the pharmaceutical formulation of the drug product.

Hypothetical phospholipase B domain-containing protein 2 (PLBD2) is the first host cell protein that has been proposed to cause enzymatic hydrolysis of PS20 (Nitin Dixit et al., Residual Host Cell Protein Promotes Polysorbate 20 Degradation in a Sulfatase Drug Product Leading to Free Fatty Acid Particles, 105 Journal of Pharmaceutical Sciences 1657-1666 (2016)). Porcine liver esterase has been reported to be able to specifically hydrolyze polysorbate 80 (not PS20) and lead to the formation of PS85 over time in a mAb drug product (Steven R. Labrenz, Ester Hydrolysis of Polysorbate 80 in mAb Drug Product: Evidence in Support of the Hypothesized Risk After the Observation of Visible Particulates in mAb Formulations, 103 Journal of Pharmaceutical Sciences 2268-2277 (2014)). Group XV lysosomal phospholipase A ₂ isomer X1 (LPLA2) has demonstrated the ability to degrade PS20 and PS80 at concentrations less than 1 ppm (Troii Hall et al., Polysorbates 20 and 80 Degradation by Group XV Lysosomal Phospholipase A 2 Isomer X1 in Monoclonal Antibody Formulations, 105 Journal of Pharmaceutical Sciences 1633-1642 (2016), and Ying Cheng et al., A Rapid High-Sensitivity Reversed-Phase Ultra High Performance Liquid Chromatography Mass Spectrometry Method for Assessing Polysorbate 20 Degradation in Protein Therapeutics, 108 Journal of Pharmaceutical Sciences 2880-2886 (2019)).

Recently, a number of carboxyesters, including pseudomonas cepacia lipase on immobead 150 (PCL), candida antarctica lipase B on immobead 150 (CALB), thermomyces lanuginosus lipase on immobead 150 (LTL), rabbit liver esterase (PEC, English RLE), lipase B from Candida antarctica (LPCA, English CALB) and porcine pancreatic lipase type II (LPZHS, English PPL), was chosen to study the hydrolysis of two unique PS20 and PS80, which contained 99% laurate and 98% oleate esters, respectively. Different carboxylesters exhibit unique degradation patterns, indicating that the degradation pattern can be used to differentiate polysorbate hydrolyzing enzymes (A.C. Mcshan et al., Hydrolysis of Polysorbate 20 and 80 by a Range of Carboxylester Hydrolases, 70 PDA Journal of Pharmaceutical Science and Technology 332-345 (2016)). It may be necessary to evaluate the effect of host cell protein copurified with the drug on polysorbates to ensure stability of the pharmaceutical formulation of the drug. This may require identification of the host cell protein and its ability to cause polysorbate degradation. Identification of host cell proteins can be particularly challenging because the presence of HCP is typically in the ppm range, making isolation and identification of HCP difficult.

The present invention provides improved compositions containing polysorbate with reduced levels of host cell proteins that may cause degradation of the polysorbate(s), methods for detecting such host cell proteins, and methods for depleting such host cell proteins.

Unless otherwise specified, all technical and scientific terms used in the context of this document have the same meaning as commonly understood by one skilled in the art to which this invention relates. Although any methods or materials similar or equivalent to those described herein may be used in practice or testing,

- 6 046423 document, preferred methods and materials are presented in this document. All publications referenced herein are incorporated herein by reference.

The singular term should be understood to mean at least one; the terms about and roughly should be understood to be subject to standard variations as understood by those of ordinary skill in the art; For all ranges specified in this document, their scope includes the extreme points of the specified ranges.

In some illustrative embodiments of the present invention, a composition is provided comprising a protein of interest, a polysorbate, and a residual amount of lipase.

As used herein, the term composition refers to an active pharmaceutical agent that is formulated in admixture together with one or more pharmaceutically acceptable carriers.

As used herein, the term active pharmaceutical agent may include the biologically active component of a drug product. An active pharmaceutical agent can refer to any compound or combination of compounds used in a drug product intended to provide pharmacological activity or other direct effect in the diagnosis, treatment, mitigation, therapy or prevention of a disease, or for a direct effect in restoring, correcting or modifying physiological functions in animals. Non-limiting methods for preparing the active pharmaceutical agent may include the use of a fermentation process, the use of recombinant DNA technology, isolation and extraction from natural resources, chemical synthesis, or combinations thereof.

In some illustrative embodiments of the present invention, the content of the active pharmaceutical agent in the specified pharmaceutical composition can vary from about 0.01 mg/ml to about 600 mg/ml. In some specific embodiments of this invention, the content of the active pharmaceutical agent in the specified pharmaceutical composition may be about 0.01 mg/ml, about 0.02 mg/ml, about 0.03 mg/ml, about 0.04 mg/ml, about 0.05 mg/ml, about 0.06 mg/ml, about 0.07 mg/ml, about 0.08 mg/ml, about 0.09 mg/ml, about 0.1 mg/ml, about 0. 2 mg/ml, about 0.3 mg/ml, about 0.4 mg/ml, about 0.5 mg/ml, about 0.6 mg/ml, about 0.7 mg/ml, about 0.8 mg /ml, about 0.9 mg/ml, about 1 mg/ml, about 2 mg/ml, about 3 mg/ml, about 4 mg/ml, about 5 mg/ml, about 6 mg/ml, about 7 mg /ml, about 8 mg/ml, about 9 mg/ml, about 10 mg/ml, about 15 mg/ml, about 20 mg/ml, about 25 mg/ml, about 30 mg/ml, about 35 mg/ml , about 40 mg/ml, about 45 mg/ml, about 50 mg/ml, about 55 mg/ml, about 60 mg/ml, about 65 mg/ml, about 70 mg/ml, about 5 mg/ml, about 80 mg/ml, about 85 mg/ml, about 90 mg/ml, about 100 mg/ml, about 110 mg/ml, about 120 mg/ml, about 130 mg/ml, about 140 mg/ml, about 150 mg /ml, about 160 mg/ml, about 170 mg/ml, about 180 mg/ml, about 190 mg/ml, about 200 mg/ml, about 225 mg/ml, about 250 mg/ml, about 275 mg/ml , about 300 mg/ml, about 325 mg/ml, about 350 mg/ml, about 375 mg/ml, about 400 mg/ml, about 425 mg/ml, about 450 mg/ml, about 475 mg/ml, about 500 mg/ml, about 525 mg/ml, about 550 mg/ml, about 575 mg/ml or about 600 mg/ml.

In some illustrative embodiments of this invention, the pH of the composition may be greater than about 5.0. In one illustrative embodiment of the present invention, said pH may be greater than about 5.0, greater than about 5.5, greater than about 6, greater than about 6.5, greater than about 7, greater than about 7.5, greater than than about 8 or greater than about 8.5.

In some illustrative embodiments of the present invention, said active pharmaceutical agent may be a protein of interest.

As used herein, the term protein or protein of interest may include any polymer of amino acids containing covalent amide bonds. Proteins include one or more polymer chains of amino acids, generally known in the art as polypeptides. A polypeptide refers to a polymer consisting of amino acid residues, their related naturally occurring structural variants and their non-naturally occurring synthetic analogues linked by peptide bonds, their related naturally occurring structural variants and their non-naturally occurring synthetic analogues. Synthetic peptides or polypeptides refers to a non-naturally occurring peptide or polypeptide. Synthetic peptides or polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. Various methods for solid-phase synthesis of peptides are known to those skilled in the art. A protein may contain one or more polypeptides to form a single functioning biomolecule. The protein may include any biotherapeutic protein, recombinant protein used in research or therapy, decoy proteins and other chimeric receptor Fc fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, polyclonal antibodies, human antibodies and bispecific antibodies. In another illustrative aspect of the present invention, the protein may include antibody fragments, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, and the like. Proteins can be produced using recombinant cell production systems such as the insect baculovirus system, yeast systems (e.g. Pichia sp.), mammalian systems (e.g. CHO cells and CHO cell derivatives such as CHO-K1 cells) . For a recent review discussing biotherapeutic proteins and their production, see Ghaderi et al., Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation, (Darius Ghaderi et al., Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation, 28 Biotechnology and Genetic Engineering Reviews 147-176 (2012)) . In some embodiments of the present invention, the proteins contain modifications, adducts, and other covalently linked moieties. These modifications, adducts and fragments include, for example, avidin, streptavidin, biotin molecule, modified biotin molecule, glycans (for example, N-acetylgalactosamine, galactose, neuraminic acid, N-acetylglucosamine, fucose, mannose and other monosaccharides), polyethylene glycol ( PEG), polyhistidine, FLAGtag, maltose binding protein (MBP), chitin binding protein (CBP), glutathione B-transferase (GST), myc epitope, fluorescent tags and other dyes, and the like. Proteins can be classified based on composition and solubility and thus may include simple proteins such as globular proteins and fibril proteins; conjugated proteins such as nucleoproteins, glycoproteins, mucoproteins, chromoproteins, phosphoproteins, metalloproteins and lipoproteins; and derived proteins, such as primary derived proteins and secondary derived proteins.

In some illustrative embodiments of the present invention, the protein of interest may be an antibody, a bispecific antibody, a multispecific antibody, an antibody fragment, a monoclonal antibody, a fusion protein, or combinations thereof.

In a particular aspect of the present invention, the protein of interest may be aflibercept (see US Pat. No. 7,279,159, the entire contents of which are incorporated herein by reference).

As used herein, the term antibody includes immunoglobulin molecules containing four polypeptide chains, two heavy (H) chains and two light (L) chains, linked by disulfide bonds, as well as their multimers (eg, IgM). Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or _VH ) and a heavy chain constant region. The heavy chain constant region contains three domains: _CH1 , CH2 and CH3. Each light chain contains a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain (CL1). The VH and _VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), with insertions of more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. In various embodiments of the present invention, the FR regions of an anti-big-ET-1 antibody (or an antigen-binding fragment thereof) may be identical to human germline sequences or may be naturally or artificially modified. A consensus amino acid sequence can be determined based on parallel analysis of two or more CDRs.

As used herein, the term antibody also includes antigen-binding portions of complete antibody molecules. As used herein, the terms antigen binding portion of an antibody, antigen binding fragment of an antibody, and the like include any naturally occurring, enzymatically produced, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding antibody fragments can be prepared, for example, from whole antibody molecules using any standard techniques, such as proteolytic digestion or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding variable and, optionally, constant domains. Such DNA is known and/or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration or to introduce codons, create cysteine residues, modify, add or remove amino acids, etc. .d.

As used herein, an antibody fragment includes a portion of an intact antibody, such as, for example, an antigen binding or antibody variable region. Examples of antibody fragments include, but are not limited to, Fab fragment, Fab' fragment, F(ab')2 fragment, scFv fragment, Fv fragment, dsFv diabody, dAb fragment, Fd' fragment, Fd fragment and selected region defining complementarity (CDR), as well as tribodies, tetrabodies, linear antibodies, single-chain antibody molecules and multispecific antibodies formed from antibody fragments. Fv fragments are a combination of immunoglobulin heavy and light chain variable regions, and ScFv proteins are recombinant single chain polypeptide molecules in which the immunoglobulin light and heavy chain variable regions are combined

- 8 046423 connected by a peptide linker. In some illustrative embodiments of the present invention, the antibody fragment contains sufficient amino acid sequence of the parent antibody of which it is a fragment so that it binds to the same antigen as the parent antibody; in some illustrative embodiments of the present invention, the fragment binds to an antigen with an affinity similar to that of the specified parent antibody and/or competes with the specified parent antibody for binding to the corresponding antigen. The antibody fragment can be obtained by any means. For example, an antibody fragment can be produced enzymatically or chemically by fragmentation of an intact antibody and/or it can be produced recombinantly from a gene encoding a partial sequence of the antibody. Alternatively or in addition, the antibody fragment can be obtained in whole or in part synthetically. The antibody fragment may optionally include a single chain antibody fragment. Alternatively or in addition, an antibody fragment may include multiple chains that are linked together, for example, by disulfide bonds. The antibody fragment may optionally include a multimolecular complex. A functional antibody fragment typically contains at least about 50 amino acids and, more often, contains at least about 200 amino acids.

The phrase bispecific antibody includes an antibody capable of selectively binding two or more epitopes. Bispecific antibodies typically contain two different heavy chains, with each heavy chain specifically binding to a different epitope - either on two different molecules (such as antigens) or on the same molecule (such as the same antigen) . If a bispecific antibody is capable of selectively binding two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for said first epitope will generally be at least one to two or three to four orders of magnitude lower than the affinity of said first heavy chain. chain to the specified second epitope, and vice versa. The epitopes recognized by a bispecific antibody may be on the same target or on different targets (eg, on the same protein or on different proteins). Bispecific antibodies can be produced, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of the same antigen can be fused to nucleic acid sequences encoding different heavy chain constant regions, and such sequences can be expressed in a cell that expresses an immunoglobulin light chain .

An exemplary bispecific antibody has two heavy chains, each containing three heavy chain CDRs followed by a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain, and an immunoglobulin light chain that either does not confer antigen binding specificity but that can be associated with each heavy chain, or which can associate with each heavy chain and which can bind one or more epitopes linked by the antigen-binding regions of the heavy chain, or which can bind with each heavy chain and cause one or both heavy chains to bind to one or both epitopes. Bispecific antibodies (bsAb) can be divided into two main classes: those that contain an Fc region (IgG-like) and those that lack the Fc region, the latter usually being smaller in size than IgG and IgG-like bispecifics. molecules containing Fc. IgG-like bsAbs may come in a variety of formats, such as, but not limited to, triomab, IgG-Kih IgG, and crossMab. , ortho-Fab IgG (English orth-Fab IgG), Ig with two variable domains (Ig-DVD, English DVD-Ig), double Fab or double-action Fab (DFD, English DAF), IgG with single-chain Fv ( IgG-otsFv, English IgG-scFv) or κλ-bodies. Non-IgG-like various formats include tandem scFv, diabody format, single chain diabody, tandem diabody (TandAb), dual affinity retargeting molecule (DART), DART-Fc Fc), nanobodies or antibodies obtained by dock-and-lock (DNL) method (Gaowei Fan, Zujian Wang & minutes gju Hao, Bispecific antibodies and their applications, 8 Journal of Hematology & Oncology 130; Dafne Muller & Roland E. Kontermann, Bispecific Antibodies, Handbook of Therapeutic Antibodies 265-310 (2014)).

Methods for producing bsAbs are not limited to quadroma technology, which is based on the somatic fusion of two different hybridoma cell lines, chemical conjugation, which includes chemical cross-linkers, and genetic approaches using recombinant DNA technology. Examples of bsAts include those disclosed in the following patent applications, which are incorporated herein by reference: US Patent Application No. 12/823,838, filed June 25, 2010; US Patent Application No. 13/488628, filed June 5, 2012; US Patent Application No. 14/031075, filed September 19, 2013; US Patent Application No. 14/808171, filed July 24, 2015; US Patent Application No. 15/713574, filed September 22, 2017; US Patent Application No. 15/713569, filed September 22, 2017; US Patent Application No. 15/386453, filed December 21, 2016; US Patent Application No. 15/386,443, filed December 21, 2016; application for pa

- 9 046423 US tent No. 15/22343 filed July 29, 2016; and US Patent Application No. 15814095, filed November 15, 2017. Low levels of homodimer impurities may be present at several stages of bispecific antibody production. Detection of such homodimer impurities can be challenging if it involves the use of intact mass analysis, due to the low content of homodimer impurities and the co-eluting of these impurities with major fragments when using conventional liquid chromatography techniques.

As used herein, the term multispecific antibody, or mAb, refers to an antibody having binding specificity for at least two different antigens. Although such molecules typically bind only two antigens (ie, bispecific antibodies, bsAbs), antibodies with additional specificity, such as trispecific antibody and trispecific BB antibody, can also be used in the system and method presented herein.

When used in the context of this document, the term monoclonal antibody is not limited to antibodies produced using hybridoma technology. A monoclonal antibody can be obtained from a single clone, including any eukaryotic, prokaryotic or phage clone, by any method available or known in the art. Monoclonal antibodies useful for this invention can be obtained using a wide range of methods known in the art, including the use of hybridoma technologies, recombinant technologies and phage display technologies, or combinations thereof.

In some illustrative embodiments of the present invention, the protein of interest may have a pI ranging from about 4.5 to about 9.0. In one illustrative specific embodiment of the present invention, said pI may be about 4.5, about 5.0, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6, 0, about 6.1 about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1 about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8 ,1 about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9 or about 9.0.

In some illustrative embodiments of the present invention, the compositions contain at least two types of proteins of interest. In some specific embodiments of the present invention, one of the at least two proteins of interest may be a monoclonal antibody, a polyclonal antibody, a bispecific antibody, an antibody fragment, a fusion protein, or an antibody-drug complex. In some other specific embodiments of the present invention, the concentration of one of the at least two proteins of interest may be from about 20 mg/ml to about 400 mg/ml. In some illustrative embodiments of the present invention, the compositions contain two types of proteins of interest. In some illustrative embodiments of the present invention, the compositions contain three types of proteins of interest. In some illustrative embodiments of the present invention, the compositions contain five types of proteins of interest.

In some illustrative embodiments of the present invention, said two or more proteins of interest contained in said composition may be selected from decoy proteins, chimeric receptor Fc fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, polyclonal antibodies, human antibodies , bispecific antibodies, multispecific antibodies, antibody fragments, nanobodies, recombinant antibody chimeras, cytokines, chemokines or peptide hormones.

In some illustrative embodiments of the present invention, the composition may be a combination pharmaceutical composition.

In some illustrative embodiments of the present invention, the specified protein of interest can be purified from mammalian cells. These mammalian cells may or may not be of human origin and may include primary epithelial cells (eg, keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, renal epithelial cells and retinal epithelial cells), established cell lines and their strains (e.g., fetal kidney 293 cells, BHK cells, HeLa cervical epithelial cells and PER-C6 retinal cells, MDBK (NBL1) cells, 911 cells, CRFK cells, MDCK cells, CHO cells, BeWo cells, Chang cells, Detroit 562 cells, HeLa 229 cells, HeLa S3 cells, Hep-2 cells, KB cells, LSI80 cells, LS174T cells, NCI-H-548 cells, RPMI2650 cells, SW-13 cells, T24 cells, WI-28 VA13 cells, 2RA cells, WISH cells, BS-C-I cells, LLC-MK2 cells, M-3 clone cells, 1-10 cells, RAG cells, TCMK-1 cells, Yl cells, LLC-PKi cells, PK(15) cells GHi cells, GH3 cells, L2 cells, LLC-RC 256 cells, MHiCi cells, XC cells, MDOK cells, VSW cells and TH-I cells, B1 cells, BSC-1 cells, RAf cells, RK cells, PK-15 cells or their derivatives), fibroblast cells from any tissue or organ (including, but not limited to, heart, liver, kidney, colon, intestines, esophagus, stomach,

- 10 046423 nervous tissue (brain, spinal cord), lung, vascular tissue (artery, vein, capillary), lymphoid tissue (lymph gland, adenoid, tonsil, bone marrow and blood), spleen, and fibroblasts and fibroblast-like cell lines (eg, CHO cells, TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells, Citrullinemia cells, Dempsey cells, Detroit 551 cells, Detroit 510 cells, Detroit 525 cells, Detroit 529 cells, Detroit 532 cells, Detroit 539 cells, Detroit 548 cells, Detroit 573 cells, HEL 299 cells, IMR-90 cells, MRC-5 cells, WI-38 cells, WI-26 cells, Midi cells, CHO cells, CV-1 cells, COS- cells 1, COS-3 cells, COS-7 cells, Vero cells, DBSFrhL-2 cells, BALB/3T3 cells, F9 cells, SV-T2 cells, M-MSV-BALB/3T3 cells, K-BALB cells, BLO- cells 11, NOR-10 cells, C3H/IOTI/2 cells, HSDMiC3 cells, KLN205 cells, McCoy cells, mouse L-cells, strain 2071 cells (mouse L-cells), strain L-M cells (mouse L-cells), L- MTK' (Mouse L cells), NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss/3T3 cells, Indian muntjac cells, SIRC cells, Cn cells and Jensen cells, Sp2/0, NS0, NS1 cells or their derivatives) .

In some illustrative embodiments of this invention, the composition may be stable. The stability of the composition may include assessing the chemical stability, physical stability, or functional stability of the active pharmaceutical agent. The pharmaceutical compositions of this invention generally exhibit high levels of stability of the active pharmaceutical agent.

With respect to protein pharmaceutical formulations, the term stable as used herein refers to a protein of interest in a pharmaceutical formulation that is capable of retaining an acceptable degree of chemical structure or biological function after storage under the exemplary conditions defined herein. A pharmaceutical formulation may be stable even if the protein of interest it contains does not retain 100% of its chemical structure or biological function after storage for a certain period of time. Under certain circumstances, retention of about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of a protein's structure or function after storage for a certain period of time may be considered stable.

Stability can be measured, among other things, by determining the percentage of native protein(s) that remains in the pharmaceutical composition after storage for a certain period of time at a certain temperature. The percentage of native protein can be determined, among other things, by size exclusion chromatography (eg, size exclusion liquid chromatography [SE-HPLC]), where native means without aggregation and without degradation. When used in the context of this document, the phrase acceptable degree of stability means that at least 90% of the native form of the protein can be recovered in the pharmaceutical composition after storage for a specified period of time at a specified temperature. In certain embodiments of the present invention, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the native form of the protein can be detected in the pharmaceutical composition after storage for a certain period of time at a certain temperature. The specified period of time after which stability is measured may be at least 14 days, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months or more.

Stability can be measured, among other things, by determining the percentage of protein that forms in the aggregate in the pharmaceutical composition after storage for a certain period of time at a certain temperature, with stability being inversely proportional to the percentage of aggregate formed. This form of stability is also referred to herein as colloidal stability. The percentage of aggregated protein can be determined, among other things, by size exclusion chromatography (eg, high performance liquid size exclusion chromatography [HPLC-SEC]). When used in the context of this document, the phrase acceptable degree of stability means that no more than 6% of the protein in an aggregated form can be detected in a pharmaceutical formulation after storage for a specified period of time at a specified temperature. In certain embodiments of the present invention, an acceptable degree of stability means that no more than 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the protein in the aggregate can be detected in the pharmaceutical composition. form after storage for a certain period of time at a given temperature. The specified period of time after which stability is measured may be about at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months or more. Temperature at which pharmaceuticals can be stored

- 11 046423 composition when assessing stability, can be at any temperature from about -80°C to about 45°C, for example, storage at about -80°C, about -30°C, about -20°C, about 0 °C, about 4°C, about 5°C, about 25°C, about 35°C, about 37°C or about 45°C. For example, a pharmaceutical formulation may be considered stable if, after six months of storage at 5°C, less than about 3%, 2%, 1%, 0.5%, or 0.1% of the protein is detected in aggregated form. The pharmaceutical composition may also be considered stable if, after six months of storage at about 25°C, less than about 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the protein is detected in aggregated form. The pharmaceutical composition can also be considered stable if, after 28 days of storage at 45°C, less than about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% protein is detected in aggregated form. A pharmaceutical composition may also be considered stable if, after three months of storage at -20°C, -30°C or -80°C, it is less than about 3%, 2%, 1%, 0.5%, or 0.1% protein is detected in aggregated form.

Stability can also be measured, among other things, by determining the percentage of protein that forms in the aggregate in the pharmaceutical composition after storage for a certain period of time at a certain temperature, with stability being inversely proportional to the percentage of aggregate formed. This form of stability is also referred to herein as colloidal stability. The percentage of aggregated protein can be determined, among other things, by size exclusion chromatography (eg, high performance liquid size exclusion chromatography [HPLC-SEC]). When used in the context of this document, the phrase acceptable degree of stability means that no more than about 6% of the protein in the pharmaceutical composition can be detected in aggregated form after storage for a specified period of time at a given temperature. In certain embodiments of the present invention, an acceptable degree of stability means that no more than 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the protein in the aggregate can be detected in the pharmaceutical composition. form after storage for a certain period of time at a given temperature. The specified period of time after which stability is measured may be about at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months or more. The temperature at which the pharmaceutical composition may be stored when assessing stability may be any temperature from about -80°C to about 45°C, for example, storage at about -80°C, about -30°C, about -20°C , about 0°C, about 4°8°C, about 5°C, about 25°C, about 35°C, about 37°C or about 45°C. For example, a pharmaceutical formulation may be considered stable if, after six months of storage at about 5°C, less than about 3%, 2%, 1%, 0.5%, or 0.1% of the protein is detected in aggregated form. The pharmaceutical composition may also be considered stable if, after six months of storage at about 25°C, less than about 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the protein is detected in aggregated form. The pharmaceutical composition may also be considered stable if, after about 28 days of storage at 45°C, there is less than about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% protein identified in aggregate form. The pharmaceutical composition can also be considered stable if, after three months of storage at a temperature of about -20°C, -30°C or -80°C, there is less than about 3%, 2%, 1%, 0.5%, or 0.1 % protein is detected in aggregated form.

Stability can also be measured, among other things, by determining the percentage of protein that migrates in the more acidic fraction during ion exchange (acidic form) than in the bulk protein fraction (major contaminated form), with stability being inversely proportional to the content of the protein fraction in the acidic form. Without being limited by theory, deamidation of a protein can cause the protein to become more negatively charged and therefore more acidic than a non-deamidated protein (see, for example, Robinson, N. (2002) “Protein Deamidation” PNAS, 99 (8) ): 5283-5288). The percentage of acidic protein can be determined, among other things, by ion exchange chromatography (eg, cation exchange high performance liquid chromatography [CEX-HPLC]). When used in the context of this document, the phrase acceptable degree of stability means that no more than 49% of the protein in a pharmaceutical formulation can be recovered in a more acidic form after storage for a specified period of time at a specified temperature. In certain illustrative embodiments of the present invention, an acceptable degree of stability means that no more than 49%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% can be detected in the pharmaceutical composition , 4%, 3%, 2%, 1%, 0.5% or 0.1% protein in acidic form after storage for a specified period of time at a given temperature. The specified period of time after which stability is measured may be about at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12

- 12 046423 months, at least 18 months, at least 24 months or more.

The temperature at which the pharmaceutical composition may be stored when assessing stability may be any temperature from about -80°C to about 45°C, for example, storage at about -80°C, about -30°C, about -20°C , about 0°C, about 4°-8°C, about 5°C, about 25°C or about 45°C. For example, a pharmaceutical composition can be considered stable if, after three months of storage at -80°C, -30°C or -20°C, it is less than about 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7% , 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the protein is detected in a more acidic form. The pharmaceutical composition can also be considered stable if, after six months of storage at 5°C, it is less than about 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5% 4%, 3%, 2%, 1%, 0.5% or 0.1% of the protein is in the more acidic form. The pharmaceutical composition can also be considered stable if, after six months of storage at 25°C, it is less than about 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17% , 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the protein is in a more acidic form. The pharmaceutical composition can also be considered stable if after 28 days of storage at a temperature of 45°C there is less than about 9%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23% , 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5 %, 4%, 3%, 2%, 1%, 0.5% or 0.1% protein can be detected in a more acidic form.

Other methods may be used to evaluate the stability of the pharmaceutical compositions of this invention, such as, for example, differential scanning calorimetry (DSC) to determine thermal stability, controlled stirring to determine mechanical stability, and absorbance at about 350 nm or about 405 nm for determination of solution turbidity. For example, a pharmaceutical composition of the present invention may be considered stable if, after 6 or more months of storage at a temperature of from about 5°C to about 25°C, the change in optical density OD405 of the pharmaceutical composition is less than about 0.05 (e.g., 0. 04, 0.03, 0.02, 0.01 or less) from OD405 of the specified pharmaceutical composition at time zero. Measuring the biological activity or binding affinity of a protein to its target can also be used to assess stability. For example, a pharmaceutical composition can be considered stable if, after storage at a temperature of, for example, 5°C, 25°C, 45°C, etc. over a period of time (eg, 1 to 12 months), the protein contained in said pharmaceutical composition binds to its target with an affinity that is at least 90%, 95% or greater of the binding affinity of said protein prior to said storage. Binding affinity can be determined, for example, using enzyme-linked immunosorbent assay (ELISA) or plasmon resonance. Biological activity can be determined using a protein activity assay, such as, for example, bringing a cell that expresses the specified protein into contact with a pharmaceutical composition containing the specified protein. The binding of the protein to such a cell can be measured directly, for example, using fluorescence-activated cell sorting (FACS) analysis. Alternatively, the subsequent activity of the protein system can be measured in the presence of protein and compared with the activity of the protein system in the absence of protein.

In some illustrative embodiments of the present invention, the composition may be used to treat, prevent, and/or alleviate a disease or disorder. Non-limiting examples of diseases and disorders that can be treated and/or prevented by the administration of pharmaceutical compositions according to this invention include: infections; respiratory diseases; pain resulting from any pathological condition associated with neurogenic, neuropathic or nociceptive pain; genetic disorder; congenital disorder; cancer; herpetiform disease; chronic idiopathic urticaria; scleroderma, hypertrophic scarring; Whipple's disease; benign prostatic hyperplasia; lung diseases such as mild, moderate or severe asthma, allergic reactions; Kawasaki disease, sickle cell anemia; Churg-Strauss syndrome; Graves' disease; preeclampsia; Sjögren's syndrome; autoimmune lymphoproliferative syndrome; autoimmune hemolytic anemia; Barrett's esophagus; autoimmune uveitis; tuberculosis; nephrosis; arthritis, including chronic rheumatoid arthritis; inflammatory bowel diseases, including Crohn's disease and ulcerative colitis; systemic lupus erythematosus; inflammatory diseases; HIV infection; AIDS; LDL apheresis; disorders caused by mutations activating proprotein convertase subtilisin-kexin type (PCSK9) (gain of functional mutations, UFM, GOF), disorders caused by heterozygous familial hypercholesterolemia (heFH); primary hypercholesterolemia; dyslipidemia; cholestatic liver diseases; nephrotic syndrome; hypothyroidism; obesity; atherosclerosis; cardiovascular diseases; neurodegenerative diseases; multisystem inflammatory disease in newborns (English NOM ID/CINCA);

- 13 046423

Muckle-Wells syndrome (MWS); familial cold autoinflammatory syndrome (FCAS); familial Mediterranean fever (FMF); tumor necrosis factor receptor-associated periodic fever syndrome (TRAPS); juvenile idiopathic arthritis with systemic onset (Still's disease); diabetes mellitus type 1 and 2; autoimmune diseases; motor neuron diseases; eye diseases; sexually transmitted diseases; tuberculosis; a disease or pathological condition that is attenuated, inhibited or alleviated by a vascular endothelial growth factor antagonist (VEGF antagonist); a disease or pathological condition that is attenuated, inhibited or ameliorated by a programmed cell death protein inhibitor PD-1; a disease or pathological condition that is attenuated, inhibited or ameliorated by an anti-interleukin antibody; a disease or condition that is attenuated, inhibited, or ameliorated by an anti-nerve growth factor antibody (NGF antibody); a disease or condition that is attenuated, inhibited, or ameliorated by an anti-PCSK9 antibody; a disease or pathological condition that is attenuated, inhibited or ameliorated by an antibody against angiopoietin-like proteins (anti-ALP antibody, ANGPTL); a disease or pathological condition that is attenuated, inhibited or ameliorated by an anti-activin antibody; a disease or pathological condition that is attenuated, inhibited or ameliorated by an anti-growth and differentiation factor antibody (anti-GDF antibody); a disease or pathological condition that is attenuated, inhibited or ameliorated by an antibody against the Fel d1 protein; a disease or pathological condition that is attenuated, inhibited or ameliorated by an anti-cluster of differentiation antibody (anti-CD antibody); a disease or pathological condition that is attenuated, inhibited or ameliorated by an antibody against complement component C5, or a combination thereof.

In some illustrative embodiments of the present invention, the composition may be administered to a patient. Administration can be accomplished by any route acceptable to those skilled in the art. Non-limiting routes of administration include oral, topical or parenteral. Administration by certain parenteral routes may involve administering the pharmaceutical compositions of this invention to a patient through a needle or catheter and actuating said compositions with a sterile syringe or some other mechanical device, such as a continuous infusion system. The composition presented in this invention can be administered using a syringe, injector, pump or any other device known in the art for parenteral administration. The composition of this invention may also be administered as an aerosol for absorption into the lungs or nasal cavity. These compositions can also be administered for absorption through mucous membranes, for example, by buccal administration.

As used herein, the term polysorbate refers to a commonly used excipient used in the development of pharmaceutical formulations to protect antibodies from various physical stresses such as agitation, freeze-thaw processes, and air/water interface interactions (Emily Ha, Wei Wang & Y. John Wang, Peroxide formation in polysorbate 80 and protein stability, 91 Journal of Pharmaceutical Sciences 2252-2264 (2002); Bruce A. Kerwin, Polysorbates 20 and 80 Used in the Formulation of Protein Biotherapeutics: Structure and Degradation Pathways, 97 Journal of Pharmaceutical Sciences 2924-2935 (2008); Hanns-Christian Mahler et al., Adsorption Behavior of a Surfactant and a Monoclonal Antibody to Sterilizing-Grade Filters, 99 Journal of Pharmaceutical Sciences 2620-2627 (2010)), and may include itself is a nonionic amphipathic surfactant consisting of polyoxyethylene sorbitan fatty acid esters. These esters may include a polyoxyethylene sorbitan head group and either a saturated monolaurate side chain (polysorbate 20; PS20) or an unsaturated monooleate side chain (polysorbate 80; PS80). In some illustrative embodiments of the present invention, the polysorbate may be present in the specified pharmaceutical composition in the range of 0.001% to 2% (w/v). Polysorbate may also contain a mixture of different fatty acid chains; for example, polysorbate 80 contains oleic, palmitic, myristic and stearic fatty acids, with the monooleate fraction making up about 58% of the polydisperse mixture (Nitin Dixit et al., Residual Host Cell Protein Promotes Polysorbate 20 Degradation in a Sulfatase Drug Product Leading to Free Fatty Acid Particles, 105 Journal of Pharmaceutical Sciences 1657-1666 (2016)). Non-limiting examples of polysorbates include polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65 and polysorbate-80.

Polysorbate may be susceptible to auto-oxidation depending on pH and temperature, and in addition, exposure to ultraviolet light can also lead to instability (Ravuri S.k. Kishore et al., Degradation of Polysorbates 20 and 80: Studies on Thermal Autoxidation and Hydrolysis, 100 Journal of Pharmaceutical Sciences 721-731 (2011)), resulting in the formation of free fatty acids in solution along with the sorbitan head group. Free fatty acids derived from polysorbate may include any aliphatic fatty acid with six to twenty carbon atoms. Non-limiting examples of free fatty acids include oleic acid, palmitic acid, stearic acid, myristic acid, lauric acid, or combinations thereof.

In some illustrative embodiments of the present invention, the polysorbate may form free fatty acid species. The free fatty acid particle size may be at least 5 microns. In addition to this, these fatty acid particles can be classified according to their size as visible (> 100 µm), sub-visible (< 100 µm, which can be divided into micron (1-100 µm) and sub-micron (100-1000 nm )) and nanometer particles (< 100 nm) (Linda Narhi, Jeremy Schmit & Deepak Sharma, Classification of protein aggregates, 101 Journal of Pharmaceutical Sciences 493-498). In some illustrative embodiments of the present invention, said fatty acid particles may be visible particles. Visible particles can be identified by visual inspection. In some illustrative embodiments of the present invention, said fatty acid particles may be sub-visible particles. Sub-visible particles can be monitored using the USP light blocking method.

In some illustrative embodiments of the present invention, the concentration of polysorbate in the composition may be about 0.001% w/v, about 0.002% w/v, about 0.003% w/v, about 0.004% w/v, about 0.005% w/v, about 0.006% w/v, about 0.007% w/v, about 0.008% w/v, about 0.009% w/v, about 0.01% w/v, about 0.011% w/v, about 0.015% w/v, about 0.02% w/v, 0.025% w/v, about 0.03% w/v. /vol., about 0.035% w/v, about 0.04% w/v, about 0.045% w/v, about 0.05% w/v, about 0.055% w/v vol., about 0.06% w/v, about 0.065% w/v, about 0.07% w/v, about 0.075% w/v, about 0.08% wt. /vol., about 0.085% w/v, about 0.09% w/v, about 0.095% w/v, about 0.1% w/v, about 0.11% wt. ./vol., about 0.115% w/v, about 0.12% w/v, about 0.125% w/v, about 0.13% w/v, about 0.135% wt. /vol., about 0.14% w/v, about 0.145% w/v, about 0.15% w/v, about 0.155% w/v, about 0.16% wt. ./vol., about 0.165% w/v, about 0.17% w/v, about 0.175% w/v, about 0.18% w/v, about 0.185% wt. /vol., about 0.19% w/v, about 0.195% w/v. or about 0.2% w/v.

In some illustrative embodiments of the present invention, degradation of the polysorbate may be accomplished by lipase(s) present in the composition. The lipase(s) may be industrial impurities that may arise during the manufacturing process and may include three general categories: cell substrate-derived, cell culture-derived, and post-exposure-derived. Cell substrate-derived impurities include, but are not limited to, host-derived proteins and nucleic acid (host cell genomic, vector, or total DNA). Impurities originating from cell culture include, but are not limited to, inducers, antibiotics, serum and other components of the culture medium. Impurities resulting from subsequent exposure include, but are not limited to, enzymes, chemical and biochemical reactants (e.g., cyanogen bromide, guanidine, oxidizing and reducing agents), inorganic salts (e.g., heavy metals, arsenic, nonmetallic ion) , solvents, carriers, ligands (eg monoclonal antibodies) and other leachables.

In one aspect of the present invention, said lipase may be a serine hydrolase. In a specific aspect of the present invention, said lipase may be a carboxylesterase B-1-like protein (A0A061I7X9). In another specific aspect of the present invention, said lipase may be a hepatic carboxylesterase 1-like protein (A0A061IFE2). In yet another specific aspect of the present invention, said lipase may be either a carboxylesterase B-1-like protein or a hepatic carboxylesterase 1-like protein.

The effect of lipases on polysorbate degradation was identified using detection methods in accordance with certain exemplary embodiments of the present invention.

Having identified lipases that can degrade polysorbates in certain protein formulations, it would be extremely advantageous and desirable to have reagents, methods and kits for specific, sensitive and quantitative determination and/or depletion of the level of such lipases, as well as to develop methods for preparing compositions with low lipase levels.

Some illustrative embodiments of the present invention provide compositions that contain less than about 5 ppm of carboxylesterase B1-like protein and/or hepatic carboxylesterase 1-like protein.

In some illustrative embodiments of the present invention, the residual amount of carboxylesterase B-1-like protein and/or hepatic carboxylesterase 1-like protein in the composition may be less than about 5 ppm. In some illustrative embodiments of the present invention, the residual amount of carboxylesterase B-1-like protein and/or hepatic carboxylesterase 1-like protein is less than about 0.01 ppm, about 0.02 ppm, about 0.03 ppm, about 0.04 ppm, about 0.05 ppm, about 0.06 ppm, 0.07 ppm, 0.08 ppm,

- 15 046423

0.09 ppm, about 0.1 ppm, about 0.2 ppm, about 0.3 ppm, about 0.4 ppm, about 0.5 ppm, about 0.6 ppm, 0.7 ppm, 0.8 ppm , 0.9 ppm, about 1 ppm, about 2 ppm, about 3 ppm, about 4 ppm or about 5 ppm.

Some illustrative embodiments of the present invention provide various methods for preparing a composition containing a protein of interest with less than about 5 ppm of carboxylesterase B-1-like protein and/or hepatic carboxylesterase 1-like protein.

The present invention also provides a method of preparing a composition containing a protein of interest containing less than about 5 ppm of carboxylesterase B-1-like protein and/or hepatic carboxylesterase 1-like protein, the method comprising forming a sample with said a protein of interest and a lipase, contacting said sample with a probe, wherein said probe has the ability to bind to said lipase to form a complex, and separating said complex from said sample.

In some exemplary embodiments of the present invention, the sample may be obtained at any stage of the bioprocess, e.g., cell culture fluid (CCL), collected cell culture fluid (CCL), process performance qualification sample (PQS), any downstream processing stage, for example, a drug solution (DDS) or drug product (DP) containing the final product formulated as a pharmaceutical composition. In some illustrative embodiments of the present invention, the sample can be obtained at any stage of the subsequent clarification, chromatographic purification, viral inactivation, or filtration process. In some specific illustrative embodiments of the present invention, said drug product may be selected from a manufactured drug product in a clinical setting, during transportation, storage, or handling. In some other specific illustrative embodiments of this invention, the specified drug may contain polysorbate(s).

In some illustrative embodiments of the present invention, said method of producing a composition comprising a protein of interest containing less than about 5 ppm of carboxylesterase B-1-like protein and/or hepatic carboxylesterase 1-like protein may also include additional chromatographic stages.

In some illustrative embodiments of the present invention, a method of preparing a composition comprising a protein of interest and less than about 5 ppm of carboxylesterase B-1-like protein and/or hepatic carboxylesterase 1-like protein may further comprise filtering one or all of the following: a sample, an eluate from one or more chromatographic steps, and/or an eluent from one or more chromatographic steps.

As used herein, the term viral filtration may include filtration using suitable filters including, but not limited to, Planova 20N™, 50 N or BioEx from Asahi Kasei Pharma, Viresolve™ filters from EMD Millipore, ViroSart CPV from Sartorius or Ultipor DV20 or DV50™ filters from Pall Corporation. It will be apparent to one of ordinary skill in the art how to select an appropriate filter to obtain the desired filtration performance.

In some illustrative embodiments of the present invention, a method of producing a composition comprising a protein of interest and less than about 5 ppm of carboxylesterase B-1-like protein and/or hepatic carboxylesterase 1-like protein may further include performing UV/ DF of one or all of the following: sample, eluate from one or more chromatographic steps, and/or eluent from one or more chromatographic steps.

As used herein, the term ultrafiltration, or UF, may include a membrane filtration process similar to reverse osmosis, using hydrostatic pressure to force water through a semi-permeable membrane. Ultrafiltration is described in detail in Leos J. Zeman & Andrew L. Zydney, Microfiltration and ultrafiltration: principles and applications (1996). For ultrafiltration, filters with a pore size of less than 0.1 microns can be used. When filters having such small pore sizes are used, sample volume can be reduced by passing the sample buffer through the filter while the antibodies are retained behind the filter.

As used herein, the term diafiltration, or DF, may include the method of using ultrafilters to remove and exchange salts, sugars and non-aqueous solvents, to separate free particles from bound particles, to remove low molecular weight material, and/or to rapidly change ionic environment and (or) pH conditions of the environment. Dissolved microsolutes are most effectively removed by adding solvent to the ultrafiltration solution at a rate approximately equal to the ultrafiltration rate. This flushes microfragments out of solution in a constant volume, effectively producing retained antibody. In certain

- 16 046423 embodiments of the present invention, the diafiltration step can be used to replace various buffers used in connection with the present invention, optionally before further chromatography or other purification steps, as well as to remove impurities from the antibody preparation.

In some illustrative embodiments of the present invention, said probe may have the ability to bind to a solid support. Said solid support may be any of the well-known supports or matrices that are currently widely used or proposed for immobilization, separation, etc. They may take the form of particles, sheets, gels, filters, membranes, fibers, capillaries or microtiter strips, tubes, plates or wells, etc. A convenient substrate can be made of glass, silicon, latex or polymer material. Particulate materials, such as granules, are generally preferred due to their greater binding capacity, especially for polymer granules. The particulate solid support used in this invention contains spherical granules. Non-magnetic polymer beads suitable for use in the method of this invention are available from Dyno Particles AS (Lillestrøm, Norway), as well as from Qiagen, Pharmacia and Serotec.

However, for ease of handling and separation, magnetic beads are preferred. As used herein, the term magnetic means that the substrate is capable of having a magnetic moment imparted to it when placed in a magnetic field, and thus can move under the influence of that field. In other words, the support containing the magnetic particles can be easily removed by magnetic aggregation, which provides a fast, simple and efficient way to separate the particles after the nucleic acid binding step and is much less harsh than traditional techniques such as centrifugation that generate shear forces , which can destroy nucleic acids. Therefore, using the method according to the present invention, the complex formed between the probe and the lipase can be removed by applying a magnetic field, for example using a permanent magnet. Typically, it is sufficient to apply a magnet to the wall of a vessel containing a sample mixture so that the particles adhere to said vessel wall, and pour out the remainder of the sample. In certain specific aspects of the present invention, superparamagnetic particles, such as those described by Sintef in patent document EP-A-106873, can be used because magnetic aggregation and sticking together of the particles during the reaction can be avoided, thereby ensuring uniform extraction of nucleic acids. The well known magnetic particles sold by Dynal AS (Oslo, Norway) as DYNABEADS are particularly suitable for use in the present invention. In addition, it is possible to obtain beads or other supports having various types of functionalized surface, for example, positively charged or hydrophobic. Weakly and strongly positively charged surfaces, weakly negatively charged neutral surfaces, and hydrophobic surfaces such as polyurethane coatings have been shown to work well.

In some illustrative embodiments of the present invention, the specified probe may have the ability to bind to a solid support using a ligand. Non-limiting examples may include an indicator, a biotin molecule, a modified biotin molecule, a modified biotin molecule, a modified biotin molecule, a nucleic acid, a protein sequence, an epitope tag, an electron-poor molecule, or an electron-rich molecule. Specific examples of ligands may include, but are not limited to, a biotin molecule or a modified one such as a desiminobiotin molecule, a desthiobiotin molecule, vicinal diols such as 1,2-dihydroxyethane, 1,2-dihydroxycyclohexane, etc., digoxigenin, maltose, oligohistidine, glutathione, 2,4-dinitrobenzene, phenylarsenate, single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), peptide or polypeptide, metal chelate, saccharide, rhodamine or fluorescein, or any hapten , to which an antibody can be obtained. Examples of ligands and reagents for their capture include, but are not limited to: desthiobiotin or structurally modified biotin-based reagents, including desiminobiotin molecule, modified biotin molecule, which bind to proteins of the avidin/streptavidin family, which can, for example, be used in forms streptavidin - agarose, oligomeric avidin - agarose or monomeric avidin - agarose; any 1,2-diol, such as 1,2-dihydroxyethane (HO-CH ₂ -CH ₂ -OH), and other 1,2-dihydroxyalkanes, including cyclic alkanes, such as 1,2-dihydroxycyclohexane, which bind to an alkyl or arylboronic acid an acid or boronic acid esters such as phenyl-B(OH) ₂ or hexyl-B(O-ethyl) ₂ which can be attached via an alkyl or aryl group to a solid support material such as agarose; maltose, which binds to maltose-binding protein (as well as to any other sugar/sugar-binding protein pair or, more generally, to any ligand/ligand-binding protein pair that has the properties discussed above); a hapten, such as a dinitrophenyl group, for any antibody, wherein said hapten binds to an anti-hapten antibody that recognizes said hapten, for example, a dinitrophenyl group will bind to antidinitrophenyl-lgG; a ligand that binds to a transition metal, for example an oligomeric histidine will bind to

- 17 046423

The Ni(II) transition metal trapping reagent may be used in the form of a resin-bound chelated transition metal such as nitrilotriacetic acid chelated Ni(II) or imine diacetic acid chelated Ni(II); glutathione, which binds to glutathione Stransferase.

The present invention also provides a method for detecting hepatic carboxylesterase 1-like protein or hepatic carboxylesterase B-1 like protein in a sample by contacting said sample with a serine hydrolase probe. In one aspect of the present invention, said method of detecting lipase in a sample may include contacting said sample with a serine hydrolase probe and incubating them together to form a complex of the lipase with said serine hydrolase probe. In another aspect of the present invention, said method of detecting lipase in a sample may include filtering out a serine hydrolase probe that does not form said lipase complex with said serine hydrolase probe.

In some illustrative embodiments of the present invention, said method of detecting lipase in a sample may further comprise contacting said sample with magnetic beads having the ability to bind to said serine hydrolase probe, such that said magnetic beads bind to said lipase complex with said serine hydrolase. hydrolase probe.

In some specific illustrative embodiments of the present invention, said magnetic beads associated with said lipase complex with said serine hydrolase probe can be further removed from said sample and washed with buffer.

In some illustrative embodiments of the present invention, said method of detecting lipase in a sample may further include retrieving said magnetic beads that are bound to said lipase complex with said serine hydrolase probe to form a lipase-enriched solution.

In some exemplary embodiments of the present invention, said method of detecting lipase in a sample may further include adding a hydrolyzing agent to said solution to produce degradation products.

As used herein, the term hydrolyzing agent refers to any one or combination of a large number of different agents that can cause protein degradation. Non-limiting examples of hydrolyzing agents that can perform enzymatic degradation include protease from Aspergillus Saitoi, elastase, subtilisin, protease XIII, pepsin, trypsin, Tryp-N, chymotrypsin, aspergillopepsin I, LysN protease (Lys-N), endoproteinase LysC ( Lys-C), endoproteinase Asp-N (Asp-N), endoproteinase Arg-C (Arg-C), endoproteinase Glu-C (Glu-C) or outer membrane protein T (OMPT), an enzyme that carries out degradation of immunoglobulins from Streptococcus pyogenes (FDIS, English IdeS), thermolysin, papain, pronase, V8 protease or their biologically active fragments or homologues, or combinations thereof. Non-limiting examples of hydrolyzing agents that can perform non-enzymatic digestion include the use of high temperature, microwave, ultrasound, high pressure, infrared, solvents (non-limiting examples include ethanol and acetonitrile), immobilized enzyme digestion (IMER), enzymes immobilized on magnetic particles and enzymes immobilized on a chip. For a recent review discussing available protein digestion methods, see Switzar et al., Protein Digestion: An Overview of the Available Techniques and Recent Developments (Linda Switzar, Martin Giera & Wilfried M. A. Niessen, Protein Digestion: An Overview of the Available Techniques and Recent Developments, 12 Journal of Proteome Research 1067-1077 (2013)). A single hydrolyzing agent or a combination of them can cleave peptide bonds in a protein or polypeptide in a sequence-specific manner, creating a predictable set of shorter peptides.

The ratio of hydrolyzing agent to lipase and the time required for cleavage can be selected appropriately to obtain lipase cleavage. When the enzyme to substrate ratio is unsuitably high, the corresponding high digestion rate will not provide sufficient time for analysis of the peptides by the mass spectrometer, and sequence coverage will be compromised. On the other hand, a low enzyme-to-substrate (F/S) ratio requires longer digestion times and hence longer acquisition times. The enzyme to substrate ratio can vary from about 1:0.5 to about 1:200. As used herein, the term cleavage refers to the hydrolysis of one or more peptide bonds of a protein. There are several approaches to achieving protein digestion in a sample using an appropriate hydrolyzing agent, such as enzymatic digestion or non-enzymatic digestion.

In some illustrative embodiments of the present invention, said method of detecting lipase in a sample may further comprise adding a protein denaturing agent to said solution.

When used in the context of this document, protein denaturation may refer to

- 18 046423 a process in which the three-dimensional shape of a molecule changes compared to its native state without breaking the peptide bonds. Protein denaturation can be accomplished using a protein denaturing agent. Non-limiting examples of protein denaturing agents include heat, high or low pH, or exposure to chaotropic agents. A number of chaotropic agents can be used as protein denaturing agents. Chaotropic solutes increase the entropy of a system by interfering with intramolecular interactions mediated by noncovalent forces such as hydrogen bonds, van der Waals forces, and hydrophobic effects. Non-limiting examples of chaotropic agents include butanol, ethanol, guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea, Nlauroyl sarcosine, urea and their salts. In a specific aspect of the present invention, said protein denaturing agent may be urea.

In some illustrative embodiments of the present invention, said method of detecting lipase in a sample may further comprise adding a protein denaturing or protein reducing agent to said solution.

As used herein, the term protein reducing agent refers to an agent used to reduce disulfide bridges in a protein. Non-limiting examples of protein reducing agents used for protein reduction include dithiothreitol (DTT), β-mercaptoethanol, Ellman's reagent, hydroxylamine hydrochloride, sodium cyanoborohydride, tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl). . TCEPHCl) or combinations thereof. In one aspect of the present invention, said protein reducing agent may be DTT.

In some illustrative embodiments of the present invention, said method of detecting lipase in a sample may further comprise adding a protein alkylating agent to said solution.

As used herein, the term protein alkylating agent refers to an agent used to alkylate certain free amino acid residues in a protein. Non-limiting examples of protein alkylating agents include iodoacetamide (IOA), chloroacetamide (CAA), acrylamide (AA), N-ethylmaleimide (NEM), methyl methanethiosulfonate (MMTS) and 4-vinylpyridine or combinations thereof.

In some illustrative embodiments of the present invention, said method of detecting lipase in a sample may further include analyzing said degradation products to detect said lipases. In one aspect of the present invention, said degradation products can be analyzed using a mass spectrometer. In a specific aspect of the present invention, said mass spectrometer may be a tandem mass spectrometer. In another specific aspect of the present invention, said mass spectrometer may be coupled to a liquid chromatography system. In another specific aspect of the present invention, said mass spectrometer can be coupled to a liquid chromatography and multiple reaction monitoring system.

As used herein, the term mass spectrometer includes a device capable of identifying specific molecular moieties and measuring their exact masses. The term is intended to include any molecular detector into which a polypeptide or peptide can be eluted for detection and/or characterization. A mass spectrometer may include three main parts: an ion source, a mass analyzer, and a detector. The role of the ion source is to create ions in the gas phase. The atoms, molecules or clusters to be analyzed can be transferred into the gas phase and ionized simultaneously (as in electrospray ionization) or through separate processes. The choice of ion source largely depends on the application.

As used herein, the term tandem mass spectrometry includes a technique that obtains structural information about the molecules of a sample using multiple mass selection and mass separation steps. A necessary condition is the ability to transfer the molecules into the gas phase and ionize them without damage, and the ability to induce their decay in some predictable and controllable manner after the first mass selection step. Multi-step MS/MS, or MC ⁿ , can be performed by initially selecting and isolating a precursor ion (MS ² ), fragmenting it, isolating the primary fragment ion (MS ³ ), fragmenting it, isolating a secondary fragment (MS ⁴ ), and so on until as long as meaningful information can be obtained or as long as a signal from a fragment ion is detected. Tandem MS has been successfully performed using a wide range of analyzer combinations. The choice of analyzers for a particular application can be determined by many different factors, such as sensitivity, selectivity and speed, as well as size, cost and availability. The two main categories of tandem MS methods are spatially resolved tandem MS methods and time-resolved tandem MS methods, but there are also hybrid methods in which time-resolved tandem MS analyzers are coupled spatially or to analyzers

- 19 046423 tandem MS with spatial resolution. A spatially resolved tandem mass spectrometer contains an ion source, a precursor ion activation device, and at least two non-capture mass analyzers. Specific m/z separation functions can be configured so that ions are sampled in one part of the device and dissociated in an intermediate part of the device, and the resulting ions are then transferred to another analyzer for m/z separation and data collection. In a time-resolved mass spectrometer, ions produced in the ion source can be captured, released, fragmented, and m/z separated in the same physical device.

Peptides identified by mass spectrometry can be used as surrogate representatives of the intact protein and their post-translational modifications. They can be used to characterize proteins by correlating experimental and theoretical data from MS/MS, the latter being generated from candidate peptides in a protein sequence database. Said characterization includes, but is not limited to, amino acid sequencing of protein fragments, protein sequencing, de novo protein sequencing, location of post-translational modifications or identification of post-translational modifications, or comparability analysis, or combinations thereof.

When used in the context of this document, the term database refers to bioinformatics tools that provide the ability to search uninterpreted MS/MS spectra against all possible sequences in the database(s). Non-limiting examples of such tools include Mascot (http://www.matrixscience.com), Spectrum Mill (http://www.chem.agilent.com), PLGS (http://www.waters.com), PEAKS (http://www.bioinformaticssolutions.com), Proteinpilot (http://download.appliedbiosystems.com//proteinpilot), Phenyx (http://www.phenyxms.com), Sorcerer (http://www.sagenresearch .com), OMSSA (http://www.pubchem.ncbi.nlm.nih.gov/omssa/), X!Tandem (http://www.thegpm.org/TANDEM/), Protein Prospector (http:/ /www.http://prospector.ucsf.edu/prospector/mshome.htm), Byonic (https://www.proteinmetrics.com/products/byonic) or Sequest (http://fields.scripps.edu/sequest ).

In some illustrative embodiments of the present invention, said mass spectrometer may be coupled to a liquid chromatography system.

As used herein, the term chromatography refers to a process in which a chemical mixture carried by a liquid or gas can be separated into components as a result of the differential distribution of chemical compounds as they flow around or over stationary liquid or solid phases. Non-limiting examples of chromatography include classical reversed phase (RP), ion exchange (IE), and normal phase (NP) chromatography. Unlike RP, NP, and IO chromatography, in which hydrophobic interaction, hydrophilic interaction, and ionic interaction, respectively, are the dominant interaction modes, mixed chromatography can employ a combination of two or more of these interaction modes. Several types of liquid chromatography can be used with a mass spectrometer, such as rapid resolution liquid chromatography (RRLC), ultra-performance liquid chromatography (UPLC), ultrafast liquid chromatography (UFLC), and nano- liquid chromatography (nLC). For more detailed information on the method and principles of chromatography, see the work of Colin et al. (Colin F. Poole et al., Liquid chromatography fundamentals and instrumentation (2017)).

In some illustrative embodiments of the present invention, said mass spectrometer may be coupled to a nano-liquid chromatography system. In some illustrative embodiments of the present invention, the mobile phase used to elute the protein in liquid chromatography may be a mobile phase that may be compatible with the mass spectrometer. In some specific illustrative embodiments of the present invention, said mobile phase may be ammonium acetate, ammonium bicarbonate or ammonium formate, or combinations thereof.

In some illustrative embodiments of the present invention, the mass spectrometer may be coupled to a liquid chromatography and multiple reaction monitoring system.

As used in the context of this document, the term multiple reaction monitoring, or MRM, refers to a mass spectrometry-based technique that allows the precise quantification of small molecules, peptides, and proteins in complex matrices with high sensitivity, specificity, and wide dynamic range (Paola Picotti & Ruedi Aebersold, Selected reaction monitoring-based proteomics: workflows, potential, pitfalls and future directions, 9 Nature Methods 555-566 (2012). Typically, MMR can be performed using triple quadrupole mass spectrometers, in which the precursor ion corresponding to selected small molecules/peptides is selected in the first quadrupole, and the fragment ion of the precursor ion is selected for monitoring in the third quadrupole (Yong Seok Choi et al ., Targeted human cerebrospinal fluid proteomics for the validation of multiple Alzheimers disease biomarker candidates, 930 Journal of Chromatography B 129-20 046423

135 (2013)).

In some illustrative embodiments of the present invention, said mass spectrometer may be coupled to a liquid chromatography system and monitor selected reactions.

It should be understood that this invention is not limited to any of the foregoing chromatography resins, excipients, filtration methods, hydrolyzing agents, protein denaturing agents, protein alkylating agents, instruments used for identification, and any/any chromatographic resin(s), excipient(s), filtration method(s), hydrolyzing agent(s), protein denaturing agent(s), protein alkylating agent(s), instrument(s) used ) for identification, can be selected in any suitable way.

A variety of publications are cited throughout this document, including patents, patent applications, published patent applications, accession numbers, technical articles, and scientific articles. Each of these cited references is incorporated herein by reference in its entirety for all purposes.

The following examples are presented to provide a more complete understanding of the present invention. However, they should not be construed as limiting the scope of the present invention in any way.

Examples

Materials

Dynabeads MyOne Streptavidin T1 beads were purchased from Invitrogen from Thermo Fisher Scientific (Waltham, MA, USA). Serine hydrolase probe desthiobiotin-FF ActivX, formic acid, acetonitrile, dithiothreitol (DTT) and tetramethylbenzidine (TMB) solution for 1-step blotting were purchased from Thermo Fisher Scientific (Waltham, MA, USA). acid, 10× Tris-buffered saline (TBS), iodoacetamide (IAA), bovine serum albumin (BSA), and urea were purchased from Sigma-Aldrich (St. Louis, MO, USA). EDTA solution and 0.005% v/v surfactant P-20 (HBSEP) were purchased from GE (Boston, MA, USA) Monoclonal antibody drug substance was from Regeneron Pharmaceutical Inc. Polysorbate 80 was purchased from Croda (East Yorkshire, UK) Rabbit liver esterase (PEC, rLE) was purchased from Sigma Aldrich (St Louis, MO, USA) Human IES-1 was purchased from Abcam (Cambridge, UK Modified trypsin of sequencing purity was purchased from Promega (Russia). Madison, Wisconsin, USA). Oasis Max column (2.1 x 20 mm, 30 µm) and Acquity UPLC BEH C4 column (2.1 x 50 mm, 1.7 µm) were purchased from Waters (Milford, MA, USA). Acclaim PepMap 100 C18 Analytical Column (0.075 x 250 mm, 3 µm) and Acclaim PepMap 100 C18 Trap Column (0.075 x 20 mm, 3 µm) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). 10× Dulbecco's phosphate-buffered saline (DPBS) was purchased from Gibco (Thermo Fisher Scientific, Waltham, MA, USA), and Tween-20 was purchased from J.T. Baker (Phillipsburg, New Jersey, USA). Q-Exactive Plus with electrospray ionization (ESI) source was purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Analysis of polysorbate degradation by two-dimensional liquid chromatography with charged aerosol detection (DZA)/mass spectrometry (MS)

Degradation of PS20 and PS80 in cell-free CHO medium or pharmaceutically formulated antibodies was analyzed by two-dimensional HPLC-DNA/MS as previously described by Genentech (Yi Li et al., Characterization and Stability Study of Polysorbate 20 in Therapeutic Monoclonal Antibody Formulation by Multidimensional Ultrahigh-Performance Liquid Chromatography-Charged Aerosol Detection-Mass Spectrometry, 86 Analytical Chemistry 5150-5157 (2014)). First, polysorbates were separated from the mAb pharmaceutical formulation using an Oasis MAX column (2.1 x 20 mm, 30 μm) pre-equilibrated with 99% solvent A (0.1% formic acid in water) and 1% solvent B (0.1% formic acid in acetonitrile). After sample injection, the equilibrium gradient was maintained for 1 min, followed by a linear increase in solvent B to 15% over 4 min to separate the polysorbate from the mAb. The eluted polysorbates were then applied to an Acquity BEH C4 column (2.1 x 50 mm, 1.7 μm) using a switching valve for reverse phase chromatography separation. At the beginning of the separation, solvent B was rapidly increased to 20% in 1.5 min, then gradually increased to 99% by 45 min and held for 5 min, followed by an equilibration step with 1% solvent B for 5 min. The flow rate was maintained at 0.1 mL/min and the column temperature at 40 °C.

The 2D LC system was powered by a Thermo UltiMate 3000 and coupled to a Corona Ultra CAD detector operating at 75 psi nitrogen pressure for quantitation. Chromeleon 7 was used to control this system and analyze the data. The Q-Exactive Plus with ESI source was interfaced with the 2D LC system only for

- 21 046423 characterization. This device was operated in positive mode with a capillary voltage of 3.8 kV, a capillary temperature of 350°C, a crimp flow rate of 40, and an auxiliary flow rate of 10. Full scan spectra were collected over an m/z range of

150-2000. Thermo Xcalibur software was used for MS data acquisition and analysis.

The peak area of each ester was determined from the DZA chromatogram and summed to account for intact PS80. The percentage of PS80 remaining after degradation was calculated by comparing the sum of the peak areas of the monoester eluted between 25 and 30 min at each time point with the sum of the peak areas at time zero. The relative percentage of esters of different orders or total esters can be calculated in a similar manner.

Analysis of PS80 degradation with human CES-1, rabbit PES and pharmaceutical composition of the antibody

The effect of human carboxylesterase 1 (CES-1) and rabbit liver esterase (LES) on PS80 was studied by mixing 2 μl of 0.1 mg/ml human CES-1 or 0.02 mg/ml rabbit PES with 2 μl 1 % PS80 in 16 μl of 10 mM histidine buffer, pH 6.0, followed by incubation at 4°C for 1.5, 8 and 18 h, respectively. One aliquot (3 μl) of each solution was diluted 25-fold by adding 72 μl 10 mM histidine, pH 6.0, before analysis by LC-DNA.

Hydrolysis of PS80 in a pharmaceutical mAb formulation was studied by mixing 18 μl of 50 mg/ml mAb (buffer exchanged with 10 mM histidine, pH 6.0) with 2 μl of 1% PS80 and subsequent incubation at 5°C for 18, 24 and 36 h. One aliquot (3 μl) of each solution was diluted 25 times in 10 mM histidine, pH 6.0, before analysis by LC-DZA.

Inhibition of lipases by CHO-derived antibodies

The serine hydrolase probe desthiobiotin-FF ActivX was diluted in DMSO to a concentration of 0.1 mM to obtain a stock solution. Aliquots of 1.25 µl, 5 µl and 20 µl of the probe stock solution were mixed with 5 mg mAb in 1× phosphate-buffered saline (PBS) in a final volume of 1 ml, then gently rotated at room temperature for 1 hour. Each mixture was then buffer exchanged with 10 mM histidine, pH 6.0, to remove free probes, and the mAb concentration was adjusted to 50 mg/ml. Each sample, after buffer exchange, was incubated with 0.1% PS80 at 5°C, followed by analysis of PS80 degradation by LC-DSA.

Depletion of lipases from CHO-derived antibodies

The lipase depletion experiment was performed using the immobilized serine hydrolase probe desthiobiotin-FF ActivX. To immobilize the indicated probe, 35 μl of the ActivX serine hydrolase probe desthiobiotin-FF (0.1 mM stock solution in DMSO) was first coupled to 2 mg of streptavidin Dynabeads to a final volume of 1 ml in 1× PBS by gentle rotation at room temperature for 2 h A process control sample was prepared by mixing 35 µl of DMSO with 2 mg of streptavidin Dynabeads to a final volume of 1 ml in 1x PBS and gentle rotation at room temperature for 2 hours. These beads were washed 3 times in 1x PBS and then resuspended in 800 µl of 1x PBS. Then, 5 mg of mAb sample was added to the indicated streptavidin Dynabeads connected to the FF probe and incubated at room temperature with gentle rotation for 1 hour. The supernatant was buffer exchanged with 10 mM histidine, pH 6.0, and the mAb concentration was adjusted up to 50 mg/ml. Subsequently, the supernatant samples, after buffer exchange, were incubated with 0.1% PS80 at 5°C, followed by analysis of PS80 degradation by LC-DNA.

Detection of host cell proteins (HCPs) in CHO-derived antibodies using activity-based protein profiling (ABPP)

The serine hydrolase probe desthiobiotin-FF ActivX was diluted in DMSO to a concentration of 0.1 mM to obtain a stock solution. A 20 μL aliquot of the probe stock solution was first mixed with 5 mg mAb in 1× PBS in a final volume of 1 mL and then gently rotated at room temperature for 1 h. Free probes were removed by filtration and the protein was extracted with 5 M urea in FSB. 2 mg of streptavidin Dynabeads were added to the solution and incubated with gentle rotation at room temperature for 2 hours. After removal of the supernatant, Dynabeads were collected using a magnet and washed with 5 M urea in PBS, and then resuspended in 5 M urea/ 50 mM Tris solution with 5 mM TCEP. Proteins were denatured and reduced at 55°C for 30 min and then incubated with 10 mM iodoacetamide for 30 min in the dark. Alkylated proteins were diluted 5-fold and digested with 1 μg of trypsin at 37°C overnight. Dynabeads were removed with a magnet, and the peptide mixture supernatant was acidified with 5 μL of 10% MK, desalted using a GL-Tip™ SDB desalting tip (GL science, Japan), and resuspended in 40 μL of 0.1% MK. 15 μl was transferred into Eppendorf tubes for analysis by nano-LC-MS/MS, and the remaining volume was stored at -80°C. Negative control samples were prepared by initially heating the mAb sample at 80°C for 5 min to denature all proteins to prevent host cell proteins from binding to the serine hydrolase probe desthiobiotin-FF ActivX.

Analysis by LC-MS/MS

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The peptide mixture was dissolved in 40 μl of 0.1% formic acid (FA) and 10 μl was first loaded onto a 20 cm x 0.075 mm Acclaim PepMap 100 C18 trap column (Thermo Fisher Scientific) for desalting and then separated on an Acclaim PepMap analytical column 100 C18 measuring 250 mm x 0.075 mm in an UltiMate 3000 nanoLC system (Thermo Fisher Scientific). Mobile phase A was prepared from 0.1% LA in ultrapure water, and mobile phase B was prepared from 0.1% LA in 80% acetonitrile (ACN). Peptides were separated in a 150-minute linear gradient of 2–32% buffer B at a flow rate of 300 nL/min. The UltiMate 3000 nanoLC was coupled to a Q-Exactive HFX mass spectrometer (Thermo Fisher Scientific). The mass spectrometer was operated in a data-dependent mode in which the 10 most intense ions were fragmented by higher energy collision dissociation (HCD) with a normalized collision energy (NCE) of 27%, AGC equal to 3e6, maximum injection time equal to 60 ms, for each full MS scan (from m/z equal to 375-1500, with resolution equal to 120000) and with AGC equal to 1e5, maximum injection time equal to 60 ms, for MS/MS events (from m/z equal to 200-2000, with resolution equal to 30000).

Direct cleavage of mAb-1

100 μg of mAb-1 was dried in a high-speed vacuum, then redissolved in 20 μl of 8 M urea containing 10 mM DTT. The protein was denatured and reduced at 55°C for 30 min and then incubated with 6 μl of 50 mg/ml iodoacetamide for 30 min in the dark. The alkylated protein was digested with 100 μl of 0.1 μg/μl trypsin at 37°C overnight. The peptide mixture was acidified with 5 μl of 10% trifluoroacetic acid (TFA). The sample was diluted to 0.4 μg/μl and 2 μl was injected onto the column for LCMS/MS analysis.

MPR analysis of KES-BIL and KES-Il in mAb-1

Digest mixture samples (0.8 μg) were directly loaded onto a 20 cm x 0.075 mm Acclaim PepMap 100 C18 trap column (Thermo Fisher Scientific) for desalting and then separated on a 250 mm x 0.075 mm Acclaim PepMap 100 C18 analytical column. UltiMate 3000 nanoLC system (Thermo Fisher Scientific). The column was pre-equilibrated with 98% mobile phase A (prepared from 0.1% formic acid in water) and 2% mobile phase B (prepared from 0.1% formic acid in 80% ACN) at a flow rate of 300 nL/min. After sample injection, a linear gradient from 2% to 37% mobile phase B was applied over 100 min to separate the peptides. Mass spectrometry data were obtained using parallel reaction monitoring (PRM) targeting 3 peptides - LNVQGDTK [m/z 437.73512+], AISESGVILVPGLFTK [m/z 815.97442+] and ENHAFVPTVLDGVLLPK [m/z 925.01452+] - from KES-1b, 3 peptides - APEEILAEK [m/z 500.27152+], DGASEEETNLSK [m/z 640.28612+] and IRDGVLDILGDLTFGIPSVIVSR [m/z 819.13553+] - from KES- BIL, and 3 peptides - GPSVFPLAPCSR [644.32932+], LLIYDASNRPTGIPAR [586.32833+] and STSESTAALGCLVK [712.35852+] - from mAb-1. In all experiments, a full mass spectrum with a resolution of 120,000 relative to m/z 200 (AGC target: 1e6, maximum injection time: 60 ms, m/z: 350-2000) was accompanied by a scheduled MPR scan with a resolution of 30,000 (AGC target: 1e5 , maximum injection time: 100 ms). Higher energy collision dissociation (HCD) was used with a normalized collision energy of 27 eV and an isolation window of 2 m/z for MS/MS analysis.

Example 1. Detection of polysorbate in a pharmaceutical mAb composition using two-dimensional LCDS/MS

Polysorbates from pharmaceutical mAb formulations were separated, identified and quantified by 2D LC-DNA/MS following a slightly modified method according to Yi Li et al., supra, and Oleg V. Borisov et al., Toward Understanding Molecular Heterogeneity of Polysorbates by Application of Liquid Chromatography-Mass Spectrometry with Computer-Aided Data Analysis, 83 Analytical Chemistry 3934-3942 (2011). An Oasis Max column in the first dimension of LC was used to remove mAbs, and reverse phase chromatography in the second dimension was used to separate the remaining POEs and POE esters based on their content and fatty acid type. PS80 fragments were eluted in the following order: POE, POE-isosorbide, POE-sorbitan, monoesters, diesters, triesters, and tetraesters (Fig. 1, right panel). The structure of each ester was elucidated by mass spectrometry based on the chemical formula of the polymer and the dioxolanilium ion formed by fragmentation at the source. In fig. 1 panel A on the right is a representative total ion current (TIC) chromatogram of PS80 with the main peaks labeled in order of elution: POE - POE-isosorbide - POE-sorbitan, POE-sorbitan monolinoleate, POE-sorbitan monooleate, POE-isosorbide monooleate and POE monooleate, POE sorbitan linoleate/oleate diester, POE sorbitan dioleate, POE isosorbide dioleate and POE dioleate, probably POE isosorbide/POE linoleate/oleate diester, and a mixture of POE sorbitan trioleate and tetraoleate. It should be noted that peak 8 in Fig. 1 was designated as a possible POEisosorbide/POE-linoleate/oleate diester mixture because its mass spectra were too complex to interpret. Quantitative determination of polysorbates was carried out using chromatographic analysis with charged aerosol detection (CAD) (Fig. 1, panel B right).

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Example 2. Rapid degradation of PS80 in the pharmaceutical composition of mAb-1

Rapid degradation of PS80 in mAb-1 was observed when stored at 5°C for 36 h. A significant decrease was observed in peaks eluting between 25 and 30 min, representing POE monoesters, i.e. POE-sorbitan monolinoleate, POE-sorbitan monooleate, POE-isosorbide monooleate and POE-monooleate, while POE elution between 10 and 18 min revealed a significant increase (Figure 2). There were no changes in POE di-, tri-, and tetra-esters eluting between 32-45 minutes. This unique degradation pattern suggests that a single family of lipases/esterases is most likely responsible for PS80 degradation. This family of hydrolases can only degrade the monoester portion of PS80, leaving higher order esters intact. If more than one type of hydrolase were involved in degradation, the degradation pattern would be more complex.

Example 3. Inhibition of lipases with a desthiobiotin-fluorophosphonate (FF) probe eliminates PS80 degradation

Since most of the lipases that have been reported to degrade polysorbates belong to the serine hydrolase family, we performed inhibition experiments using the FF probe. This experiment allows the identification and discrimination of enzymatically active hydrolases from other inactive hydrolases or in their zymogenic form , or with endogenous inhibitors. The rationale for this experiment is that if any active serine hydrolase is present, adding its inhibitor will stop the enzyme from functioning, in this case the degradation of PS80. The desthiobiotin-FF probe is one of the commercially available serine hydrolase probes that contains a reactive fluorophosphonate group that forms a covalent bond with Ser at the catalytic site of the serine-type hydrolase and blocks its enzymatic activity. This inhibition experiment clearly demonstrated that with the addition of as little as 0.125 μM FF probe, enzymatic activity was completely abolished (Figure 3). This experiment also demonstrated that only serine-type lipase was present in the pharmaceutical composition of the drug studied, since the desthiobiotin-FF probe is specific for serine hydrolase.

Example 4. Depletion of lipases with a desthiobiotin-fluorophosphonate (FF) probe leads to a decrease in the degradation of PS80 in the pharmaceutical composition of mAbs

We then performed lipase depletion using the immobilized serine hydrolase probe FF ActivX. The biotin moiety of the probe can be captured and immobilized on the streptavidin surface, allowing the trapped serine hydrolases to be concentrated and purified. The depletion experimental design serves two purposes: 1) if PS80 degradation is caused by lipase(s) belonging to the serine hydrolase family, depletion will result in decreased PS80 degradation; 2) the lipase(s) captured by the desthiobiotin-FF probe can be further identified by mass spectrometric analysis.

This depletion experiment was performed as described in Materials and Methods, with the mAb depletion diagram shown in FIG. 4. The desthiobiotin-FF probe was coupled to streptavidin Dynabeads to deplete lipases. As shown in FIG. 5, before lipase depletion, about 44.7% degradation of PS80 monoesters in mAb-1 was observed after 18 h incubation at 5°C. An additional 18-hour incubation resulted in complete loss of PS80 monoesters. Following lipase depletion, less than 8% PS80 degradation was observed after both 18 h and 36 h of incubation. The results of this depletion experiment indicate that the lipase(s) that caused PS80 degradation in mAb-1 were removed by the desthiobiotin-FF probe. To ensure that it was the probe, and not the magnetic streptavidin beads, that interacted with lipases, a control technological sample was included in these experiments. The specified technological control sample was obtained by mixing mAb-1 only with magnetic streptavidin beads, without adding the desthiobiotin-FF probe. About 29% and 86% of PS80 degradation in mAb-1 was observed after 18 h and 36 h of incubation at 5°C, respectively, indicating the presence of certain nonspecific interactions between lipases and magnetic beads. Compared with the FF probe, there was significantly less lipase removed through nonspecific interactions, so most of the lipase was removed by specific binding between the immobilized FF probe and the lipase.

Example 5. Hepatic carboxylesterases were identified in the mAb-1 fraction concentrated using the FF probe

Host cell proteins captured by the desthiobiotin-FF probe were subjected to trypsin digestion and mass spectrometric analysis as described in Materials and Methods. Among the 15 identified host cell proteins (Table 1), carboxylesterases CES-BIL (CES-B1L) and CES-LL (CES-1L) were identified for the first time. By comparison with a denatured control sample (Table 2), it was concluded that both proteins were captured by specific binding to the FF probe. KES-fL was identified only in the active form, but not in the denatured form, suggesting that this carboxylesterase was biologically active in

- 24 046423 mAb-1. IES-BIL was identified in the active form with 13 unique peptides, while only 2 unique peptides were in denatured forms, suggesting that a small amount of IES-BIL protein was able to bind nonspecifically to the magnetic beads, and these results are consistent with the control results. technological sample in the depletion experiment (Fig. 5). However, the much larger number of unique peptides identified in the active forms suggests that CES-BIL is also responsible for PS80 degradation. Regarding the other host cell proteins identified, most of them could be easily identified as inactive enzymes as they were detected in both conditions with the same number of unique peptides, for example, cullin-like protein 9 and ceruloplasmin. Anionic trypsin-2 was identified in active form in mAb-1 because it is also a serine protease, but it can be excluded due to its protease function unrelated to PS80 degradation. Two other co-entrapped proteins, actin and annexin, can also be excluded from the PS80 degradation process due to their lack of enzymatic functions.

To determine the content of newly identified lipases, KES-BIL and KES-Il, analysis was carried out using the MPR method. The concentrations of KES-BIL and KES-Il were determined to be 9.6 and 9.0 ppm, respectively. The relatively low content of these two lipases suggests their strong enzymatic activity towards PS80 degradation.

Table 1 Host cell protein concentrated using the FF probe identified in native mAb-1

Protein in mAb-1 (native) Account number Number of unique peptides Hepatic carboxylesterase-like protein B-1 А0А06117X9 13 Hepatic carboxylesterase-like protein 1 A0A061IFE2 7 Peroxiredoxin-1 Q9JKY1 7 Cullin-like protein 9 A0A061IMU 7 7 Connective plakoglobin G3HLU9 6 Transthyretin G3I4M9 6 Glyceraldehyde-3-phosphate dehydrogenase A0A061IDP2 3 Ceruloplasmin A0A061INJ7 3 Annexations G3IG05 3 Anionic trypsin-2 G3HL18 3 Vitamin D binding protein G3IHJ6 3 Ubiquitin 40 S ribosomal protein S27a-like protein A0A061IQ58 3 Desmocollin 2-like protein A0A061IEG0 2 Receptor tyrosine protein kinase G3HG67 2 Actin, aortic smooth muscle G3HQY2 2

table 2

Host cell protein concentrated with FF probe identified in denatured mAb-1

Protein in mAb-1 (denatured) Account number Number of unique peptides Cullin-like protein 9 A0A061IMU7 7 Transthyretin G3I4M9 4 Ubiquitin 40 S ribosomal protein S27a-like protein A0A061IQ58 3 Glyceraldehyde-3-phosphate dehydrogenase A0A061IDP2 3 Peroxiredoxin-1 Q9JKY1 3 Vitamin D binding protein G3IHJ6 3 Ceruloplasmin A0A061INJ7 2 Receptor tyrosine protein kinase G3HG67 2 Desmocollin 2-like protein A0A061TEG0 2 Hepatic carboxylesterase-like protein B-1 А0А06117X9 2 Connective plakoglobin G3HLU9 1

Example 6: PS80 Degradation Pattern with Human Liver Carboxylesterase 1 and Rabbit Liver Esterase

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One common and important practice in HCP analysis is to test the function of lipase activity. These inhibition and depletion experiments provided strong evidence that IES-NGE and IES-GE are most likely the lipases responsible for PS80 degradation. However, given that the FF probe used is specific not for one protein but for a family of proteins, it is possible that other lipase(s) present in the drug tested may also play a role in degradation paths. A spiking experiment may provide the necessary validation of whether the putative lipases are the primary cause of PS80 degradation. If the specifically added lipase can cause exactly the same degradation pattern as the endogenous lipase, then the identified lipase can be confirmed as a key element for PS degradation. Such confirmation is usually difficult due to the lack of available active lipases. To further confirm the role of these two newly identified lipases, a BLAST search was performed. The search results showed that commercially available rabbit liver esterase and human liver carboxylesterase 1 are functionally similar, with each having 56.0% and 69.7% sequence homology with the first segment of IES-BIL and IES-IL, respectively (Fig. .6D). Both human liver carboxylesterase 1 and rabbit liver carboxylesterase were selected to compare the degradation pattern of PS80 in mAb-1. In fig. Figure 6 shows that human and rabbit liver esterases show a degradation pattern equivalent to that shown for mAb-1 (Figure 6, A-C). In all three samples, the rapidly degrading components of PS80 were the monoesters, which eluted between 25 and 30 min, while the di-, tri-, and tetraesters, which eluted after 32 min, remained unchanged. These experiments characterizing degradation patterns using known lipases confirmed that KES-BIL and KES-IL are responsible for the degradation of PS80 in mAb-1.

Claims (19)

1. A method for depleting lipase from a sample, including: bringing a sample containing at least one lipase into contact with a probe, wherein said probe has the ability to bind to said lipase to form a complex; and separating said complex from said sample, thereby depleting said lipase from said sample, wherein said probe preferentially binds to the enzymatically active lipase. 2. The method according to claim 1, characterized in that said sample contains a protein of interest. 3. The method according to claim 1, characterized in that said sample contains a polysorbate excipient. 4. The method of claim 3, wherein said polysorbate excipient is selected from polysorbate-20, polysorbate-60, polysorbate-80, or combinations thereof. 5. The method according to claim 1, characterized in that said lipase is a protein similar to hepatic carboxylesterase B1. 6. The method according to claim 1, characterized in that said lipase is a protein similar to hepatic carboxylesterase 1. 7. The method according to claim 1, characterized in that said probe has the ability to bind to a solid substrate. 8. The method according to claim 1, characterized in that said probe is attached to a solid support using a ligand. 9. The method of claim 8, wherein said ligand may be an indicator, a biotin molecule, a modified biotin molecule, a nucleic acid, a sequence, an epitope tag, an electron-poor molecule, or an electron-rich molecule. 10. The method of claim 1, further comprising extracting at least one fragment of said lipase from said complex. 11. A method for purifying a sample containing a protein of interest and a lipase, comprising: bringing a sample containing a protein of interest and at least one lipase into contact with a probe, wherein said probe has the ability to bind to said lipase to form a complex; and separating said complex from said sample, thereby purifying said protein of interest in said sample, wherein said probe preferentially binds to an enzymatically active lipase. 12. The method according to claim 11, characterized in that said sample contains a polysorbate excipient. 13. The method of claim 12, wherein said polysorbate excipient is selected from polysorbate-20, polysorbate-60, polysorbate-80, or combinations thereof. - 26 046423 14. The method according to claim 11, characterized in that said lipase is a protein similar to hepatic carboxylesterase B1. 15. The method according to claim 11, characterized in that said lipase is a protein similar to hepatic carboxylesterase 1. 16. The method according to claim 11, characterized in that said probe has the ability to bind to a solid substrate. 17. The method according to claim 11, characterized in that said probe is attached to a solid support using a ligand. 18. The method of claim 17, wherein said ligand may be an indicator, a biotin molecule, a modified biotin molecule, a nucleic acid, a sequence, an epitope tag, an electron-poor molecule, or an electron-rich molecule. 19. The method of claim 11, further comprising extracting at least one fragment of said lipase from said complex.