Methods for host cell protein detection

Abstract

The present invention provides a method for measuring residual host cell protein (HCP) in a recombinant protein sample based on a combination of affinity chromatography and digestion under native conditions followed by analysis using reversed-phase liquid chromatography-tandem mass spectrometry (LC / MS-MS) wherein the liquid chromatography is performed at a multi-stage gradient of charge enhancer concentration.

Description

Field of the invention

[0001] The present invention relates to the field of recombinant antibody production and, in particular, to detecting residual host cell protein impurities in samples of proteins during purification or purified. BACKGROUND

[0003] Biotechnological products are in many cases recombinant proteins (but not limited to), which are obtained via complex production systems involving the use of genetically modified host cells (bacteria, yeast or mammalian cells). During the production of these biotechnological products, host systems (e.g. Chinese hamster ovary (CHO) cells) also express in different amounts endogenous proteins necessary for cell development and survival, which are commonly referred to as host cell proteins (HCPs) [1]. Regardless of the product and production system, residual HCPs must be routinely tested, since when biotechnological products are destined for pharmaceutical use and are ultimately administered to patients, it is necessary to ensure that these impurities are reduced to acceptable levels.

[0004] The removal of HCPs is usually performed with the chromatographic columns used during the purification of the desired biotechnological product and is often referred to as downstream processing (DSP).

[0005] However, it is known that in the field of recombinant antibody production, for example, a subset of endogenous HCPs has shown the possibility of co-purification via different DSP steps and are considered difficult to remove mainly due to interactions with antibodies or resins, and some of them are based on these effects are generally considered "high risk" [2]. In fact, these process-related impurities are considered critical quality attributes (CQAs) because they can pose a risk to clinical safety, product quality or efficacy [3], therefore, in order to support the risk assessment of HCP impurities in the production of multiple batches of therapeutic recombinant antibodies, it becomes essential to establish analytical procedures capable of identifying and monitoring HCP levels.

[0006] Traditionally, the quantification of HCPs has been performed by applying a summary method based on enzyme-linked immunosorbent assay (ELISA) using antibodies generated against various HCPs expressed in host cells [4]. However, ELISA can only quantify the total amount of HCPs and cannot indicate the identity or amount of any specific HCP. The application of mass spectrometry (MS)-based proteomics shows advantages in the quantitative analysis of individual HCPs. However, technical reproducibility, dynamic range and ensuring acceptable statistical significance of the scoring measurement are key obstacles that still need to be overcome.

[0007] Label free quantification (LFQ) methods are increasingly popular due to their direct quantification of the signal response of the relevant peptides to determine the amount of various HCPs in an antibody sample [5]. LFQ methods are generally divided into intensity-based measurements and spectral count measurements. Intensity-based measurements (e.g. Hi3) have become increasingly popular and show better accuracy when using high resolution MS compared to spectral counting.

[0008] Another aspect to consider is the type of data acquisition implemented on MS. Data independent acquisition (DIA) has been considered as an option, but has drawbacks in terms of loss of precursor selectivity. Data dependent acquisition (DDA) is widely used in discovery proteomics and has shown applicability in the field of HCP analysis [6]. However, DDA has a dynamic range problem that causes missing values that can affect the consistency of the results [7].

[0009] Several methods have been described herein to solve the dynamic range problem caused by the low abundance of HCPs in purified antibodies. Although limited in throughput, multidimensional LC-MS setups have successfully addressed sample complexity by providing higher sensitivity and lower detection limits. Other methods use affinity chromatography (protein A, G or L) to capture and deplete most of the antibodies from the sample. Huang et al. [8] describe a native digestion method, where an antibody sample is digested under non-denaturing conditions using very low enzyme-to-protein ratios.

[0010] Recently, high-field asymmetric waveform ion mobility spectrometry (FAIMS), affinity depletion and native digestion were combined and found to have a lower detection limit of 1 ppm, making it more efficient than established methods [9].

[0011] Therefore, there is still a need in the art for a method having sufficient sensitivity to detect HCPs present in a recombinant protein sample.

[0012] In particular, when considering recombinant proteins intended for use as a drug, in order to put the product on the market, specific requirements regarding purity must be met. Therefore, there is a particular need for a method that is suitable and / or compliant with quality guidelines (e.g. ICH quality guidelines, in particular ICH Q2 guideline). SUMMARY

[0014] In this context, the inventors provide a method for measuring residual host cell proteins (HCPs) in a recombinant protein sample, based on the combination of affinity chromatography and digestion under native conditions, followed by analysis using reversed-phase liquid chromatography-tandem mass spectrometry (LC / MS-MS), wherein the liquid chromatography is performed with a multi-step gradient of charge enhancer concentration. Furthermore, the inventors found that the use of a wash solution during the affinity chromatography step maximizes the recovery of difficult-to-remove HCPs and increases their identification. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 . Bar graph of total HCP proteome determined by normal digestion, native digestion, normal digestion with Prot A, normal digestion with Prot L, native digestion with Prot A and native digestion with Prot L.

[0017] Figure 2 . Bar graph of total HCP proteome determined by combination of Prot A and native digestion. No wash (resin rinsed with loading buffer only), wash with arginine buffer at basic pH, wash with citric acid + sodium citrate buffer at pH 5.5 and wash with arginine + sodium octanoate buffer at basic pH.

[0018] Figure 3 . Number of determined HCP proteome processed by global HCP database or internal HCP database using the method of the present invention. DETAILED DESCRIPTION

[0020] The present invention addresses the above-identified need by providing a novel method for analyzing (i.e. detecting and quantifying) residual host cell proteins in a recombinant antibody sample, which also allows to assess HCP removal during protein purification.

[0021] In a first aspect, the present invention refers to a method for detecting residual host cell proteins (HCPs) in a sample of a recombinant protein, comprising:

[0022] a) an affinity chromatography step, wherein a flow-through fraction is recovered,

[0023] b) trypsin digestion of the flow-through fraction recovered from a) under native conditions, followed by

[0024] c) reversed phase liquid chromatography-tandem mass spectrometry (LC-MS / MS), wherein the liquid chromatography is a reversed phase liquid chromatography with a multi-step gradient of concentration of a charge enhancer,

[0025] d) comparing the results of said LC-MS / MS with a HCP database.

[0026] In a second aspect, the present invention refers to a method for label-free quantification of residual HCPs in a sample of a recombinant protein, comprising:

[0027] a) an affinity chromatography step, wherein a flow-through fraction is recovered,

[0028] b) trypsin digestion of the flow-through fraction recovered from a) under native conditions, followed by

[0029] c) reversed phase liquid chromatography-tandem mass spectrometry (LC-MS / MS), wherein the liquid chromatography is a reversed phase liquid chromatography with a multi-step gradient of charge enhancer concentration,

[0030] d) comparing the results of the LC-MS / MS with an HCP database, and

[0031] e) quantification of HCPs.

[0032] Quantification of HCPs by label-free methods can be performed by available technologies, e.g. spectral counting or intensity-based measurements, e.g. via Hi3 label-free quantification. In a particular embodiment of the second aspect of the present application, the quantification of HCPs is performed via Hi3.

[0033] Affinity chromatography is a separation method based on specific binding interactions between an immobilized ligand and its binding partner. In the context of the present application, the affinity chromatography matrix is capable of binding the biotechnological product. Thus, the flow-through fraction recovered by affinity chromatography of the method of the present application is enriched in HCPs.

[0034] The person skilled in the art is aware of different affinity chromatography matrices suitable for recombinant proteins, e.g. immobilized metal affinity chromatography has been used for both recombinant protein and nucleic acid purification, or enzyme / substrate interactions and enzyme / inhibitor interactions have been used for recombinant protein isolation.

[0035] In a particular embodiment of the present application, the recombinant protein is a recombinant antibody.

[0036] The affinity chromatography matrix is capable of binding the antibody, preferably via the Fc region or the VH3 domain of the antibody.

[0037] In one embodiment, the affinity chromatography matrix is selected from the group consisting of: a protein A chromatography matrix, a protein G chromatography matrix and a protein L chromatography matrix.

[0038] There are many affinity chromatography materials containing protein A, protein G or protein L available to the person skilled in the art, e.g. (GE Healthcare), (Novasep), Captiv (Repligen), Praesto AP (Purolite) or (JSR) Nab TM Protein A (ThermoFisher) or Pierce TM Protein G (ThermoFisher).

[0039] Buffers suitable for use as wash and elution buffers in Protein A chromatography are readily available in the art and can be selected from, in a non-limiting manner, phosphate buffered saline (PBS), Tris, histidine, acetate, formate, citrate buffer or MES (2-(N-morpholino)ethanesulfonic acid imidazole), BES (N,N-(bis-2-hydroxyethyl)-2-aminoethanesulfonic acid), MOPS (3-(N-morpholino)-propane sulfonic acid) or HEPES (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid) buffer.

[0040] In a particular embodiment of the method of the application, the affinity chromatography step comprises:

[0041] i) contacting the sample with an affinity chromatography matrix,

[0042] ii) recovering the effluent from i),

[0043] iii) applying a wash buffer to the affinity chromatography matrix and recovering the effluent,

[0044] iv) combining the effluents from steps ii) and iii).

[0045] In a particular embodiment of the method of the application, the wash buffer used during affinity chromatography comprises benzoate, benzyl alcohol, arginine, sodium octanoate, citrate and / or a quaternary ammonium salt (e.g. tetramethylammonium chloride, tetrabutylammonium). In another preferred embodiment, the wash buffer comprises arginine, sodium octanoate or a combination of both.

[0046] In another particular embodiment of the application, the wash buffer comprises 0.2 M arginine to 1 M arginine, 0.2 M arginine to 0.8 M arginine, 0.2 M arginine to 0.6 M arginine, 0.2 M arginine to 0.5 M arginine or 0.2 M arginine to 0.4 M arginine, preferably 0.3 M arginine.

[0047] Arginine of different origin is available to the skilled person. In the context of the present application, arginine and L-arginine are used interchangeably. Typically, L-arginine can be used as a free base or as a salt, e.g. L-arginine monohydrochloride.

[0048] In a particular embodiment of the application, the wash buffer comprises 0.05 M sodium octanoate to 0.15 M sodium octanoate, 0.08 M sodium octanoate to 0.12 M sodium octanoate, 0.09 M sodium octanoate to 0.11 M sodium octanoate, preferably 0.1 M sodium octanoate.

[0049] In another specific embodiment of the application, the wash buffer has a pH value of 7 to 9, 7.2 to 9, 7.3 to 9, 7.4 to 9, 7.5 to 9, 7.6 to 9, 7.7 to 9, 7.8 to 9, 7.8 to 8.8, 7.8 to 8.6, 7.9 to 8.6, or 8.0 to 8.5.

[0050] Trypsin is a serine protease that cleaves proteins into peptides with an average size of 700-1500 Daltons, which is in the ideal range for MS detection. It cleaves with high specificity at the carboxy terminus of arginine and lysine residues, thereby rendering the resulting C-terminal arginine and lysine peptides charged and thus detectable via MS.

[0051] As will be appreciated by the skilled person, an alternative to trypsin is the use of a mixture of trypsin and Lys-C protease, which is sometimes considered advantageous because Lys-C protease can digest lysine cleavage sites that are missed by trypsin in the presence of residual trypsin inhibitor impurities.

[0052] In a specific embodiment of the method of the application, the trypsin digestion is performed using trypsin or a mixture of trypsin and Lys-C protease. Mixtures of trypsin and Lys-C protease are readily available from commercial sources, for example the Trypsin / Lys-C mix from Promega.

[0053] In a specific embodiment of the application, the trypsin digestion is performed using a ratio of enzyme to antibody of 1 / 250 to 1 / 2000, or 1 / 250 to 1 / 1500, or 1 / 250 to 1 / 1000, or 1 / 250 to 1 / 750, or 1 / 250 to 1 / 500.

[0054] The terms "digestion under native conditions" or "native digestion" are used interchangeably and in particular to distinguish from "conventional" or "normal" protein digestion, in which the protein has been denatured prior to digestion, i.e. prior to exposure to the digestion enzyme (e.g. trypsin).

[0055] In a specific embodiment of the method of the application, the trypsin digestion is followed by treatment with a mild reducing agent selected from dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP).

[0056] In a specific embodiment of the method of the application, the mild reducing agent is present at a concentration of 1 mM to 7 mM, preferably 1.5 mM to 6 mM, 1.5 mM to 5 mM, 1.5 mM to 4 mM, 2 mM to 4 mM, 2.5 mM to 3.5 mM, or 3 mM.

[0057] The complex mixture of resulting peptides is analyzed via reversed phase liquid chromatography (RP-LC) coupled with tandem mass spectrometry (MS / MS). Peptide and subsequent protein identification is accomplished by matching peptide fragment ion chromatograms to a database of host cell proteins.

[0058] Reversed phase liquid chromatography is an elution procedure used in liquid chromatography in which the mobile phase has substantially greater polarity than the stationary phase (e.g. a microporous silica-based material with chemically bonded alkyl chains).

[0059] In a particular embodiment of the method of the application, the reversed phase liquid chromatography is ultra-high performance liquid chromatography or high pressure liquid chromatography. In a preferred embodiment of the method of the application, the reversed phase liquid chromatography is ultra-high performance liquid chromatography.

[0060] In a particular embodiment of the method of the application, the reversed phase liquid chromatography is performed under heating, preferably at 45°C to 65°C, 55°C to 65°C or 60°C to 65°C. In a preferred embodiment of the method of the application, the reversed phase liquid chromatography is performed at 65°C.

[0061] Suitable columns for performing reversed phase liquid chromatography for peptide separation typically include C4, C8 or C18 columns. It is common knowledge in the art that the terms C4, C8 or C18 refer to the number of carbon atoms present in the alkyl chain of the stationary phase.

[0062] In a particular embodiment of the method of the application, the reversed phase liquid chromatography is performed using a C18 column.

[0063] The skilled person will be aware that mass spectrometry is one of the most popular methods for proteomic analysis. For the analysis of proteins and peptides by MS, it has a practical benefit of enhancing analyte charge as it can facilitate further structural analysis and increase resolution and accuracy by increasing the efficiency of peptide ionization and condensing ion currents into smaller charged states.

[0064] In a particular embodiment of the method of the application, the reversed phase liquid chromatography is performed in the presence of a charge-enhancing agent. In a preferred particular embodiment, the charge-enhancing agent comprises dimethyl sulfoxide (DMSO), m-nitrobenzyl alcohol (m-NBA), o-nitroanisole (o-NA), ethylene carbonate (EC), propylene carbonate (PC), or sulfolane.

[0065] In a particular embodiment of the method of the application, the multi-step gradient of charge-enhancing agent concentration is an increasing gradient or a decreasing gradient.

[0066] In a particular embodiment, the gradient of charge-enhancing agent concentration is 5% to 0.9%, 4% to 0.9%, 3.5% to 0.9%, 3% to 0.9%, 2.5% to 0.9%, 2% to 0.9%, or 1.5% to 0.9%.

[0067] In another embodiment, the gradient of charge-enhancing agent is 1.5% to 0.9% DMSO.

[0068] After the peptides resulting from the digestion of the protein are isolated, tandem mass spectrometry analysis is performed, which is commonly referred to as shotgun MS. There are currently two broad ways to generate this MS proteomic data: data-dependent acquisition and data-independent acquisition. In tandem mass spectrometry, the data-dependent acquisition method only sends certain peptides generated during the first cycle of MS into the second cycle for fragmentation, while in the case of data-independent methods, all peptides generated during the first MS cycle can be fragmented in the second round. During data-dependent acquisition, the measured spectra are compared to an established database, while in data-independent acquisition, the multiplex nature of the MS spectra requires deconvolution, so database-dependent search methods cannot be applied directly.

[0069] In one particular embodiment of the application, the mass spectrometry is performed using data-dependent acquisition.

[0070] In another particular embodiment of the application, the HCP database is an HCP database based on a specific HCP database originating from the same host cell line used to produce the recombinant protein. The HCP database can be created using a null cell line, i.e. a cell line that does not contain a gene expressing a recombinant protein, or a cell line expressing a recombinant protein. In one particular embodiment, the HCP database originates from a combination of a host cell line expressing a recombinant protein and the same host cell line without the gene for expression of the recombinant protein.

[0071] A commonly used database for the evaluation of the performance of the method, the performance of the instrument and the molecular characterization of the monoclonal antibody is the NIST Monoclonal Antibody (NIST mAb) Reference Material RM 8671 (https: / / www.nist.gov / programs-projects / nist-monoclonal-antibody-reference-material-8671) created based on the characterization of a single IgGlk. The NIST mAb is known to contain HCPs in the range of 100-300 ppm.

[0072] However, by generating the HCP database using the same host cell as for the production of the recombinant antibody, the comparison with the MS spectra obtained using the method of the application should be more reliable. Furthermore, when generating the HCP database, it is possible to improve the sensitivity by removing redundant entries, thus better defining the HCPs to be characterized.

[0073] In a specific method according to the application, the HCP database comprises between 3800 and 5000 protein sequences, between 4000 and 4800 protein sequences, between 4200 and 4600 protein sequences, and between 4300 and 4500 protein sequences.

[0074] Recombinant proteins produced for large scale commercial use can be produced by culturing prokaryotic (bacterial) or eukaryotic host cells transfected with one or more expression vectors encoding the recombinant protein.

[0075] In the context of the application as a whole, suitable eukaryotic host cells are mammalian host cells (also referred to as mammalian cells) and include Chinese hamster ovary (CHO cells), lymphocytic cell lines (e.g., NSO myeloma cells and SP2 cells, COS cells, myeloma or hybridoma cells). In a preferred embodiment, the mammalian cells are CHO cells. Suitable types of CHO cells can include CHO-K1, CHOK1-SV, dhfr-CHO (e.g., CHO-DG44, CHO-DXB11, CHO-DXB1, or CHO-S cells).

[0076] Mammalian cells can be cultured in any medium that supports their growth and antibody expression, preferably a chemically defined medium that is free of animal-derived products (e.g., animal serum and proteose peptone). Different cell culture media are available to the skilled person, comprising different combinations of vitamins, amino acids, hormones, growth factors, ions, buffers, nucleosides, glucose or equivalent energy sources, and present in appropriate concentrations to enable the cells to grow and produce proteins. Other cell culture medium ingredients known to the skilled person can be added to the cell culture medium at different times during the cell culture cycle.

[0077] Mammalian cell culture can be performed in any suitable vessel, such as a shake flask or a bioreactor, which can or can not be operated in fed-batch mode, depending on, for example, the production scale required. These bioreactors can be stirred tank reactors or airlift reactors. Various large-scale bioreactors can be used, which have a capacity of greater than 1,000 L to 50,000 L, preferably between 5,000 L and 20,000 L or to 10,000 L. Alternatively, smaller scale (e.g., between 2 L and 100 L) bioreactors can also be used to produce antibodies according to the methods of the application.

[0078] The antibody or antigen-binding fragment thereof produced according to the method of the present application is typically present in the supernatant of a mammalian host cell culture, typically a CHO cell culture. For CHO culture processes, in which the protein of interest, e.g. an antibody or antigen-binding fragment thereof, is secreted into the supernatant, the supernatant is collected by methods known in the art, typically by centrifugation. For the avoidance of doubt, supernatant refers to the liquid above the sedimented cells obtained by centrifugation of the cell culture.

[0079] Typically, the supernatant is filtered to remove charged particles, residual cell metabolites and cell debris, and the resulting liquid is typically referred to as clarified cell culture fluid, which is then further processed through a number of steps, typically including 2 or 3 chromatography steps and an ultrafiltration / diafiltration step, to obtain the purified antibody or antigen-binding fragment thereof.

[0080] During this further purification or downstream purification process, residual impurities, including host cell proteins, protein aggregates and degradation products, are also removed.

[0081] In this context, it can be of interest to measure the final concentration of HCPs in the purified antibody sample, typically referred to as drug substance, to ensure sufficient clearance, but it can also be of interest to perform this measurement at this purification step to improve control of the overall process.

[0082] Thus, in a particular embodiment of the method of the present application, the antibody sample is obtained from the final purified drug substance. And in an alternative embodiment of the method of the present application, the sample is obtained from a step in the purification flow of the recombinant antibody.

[0083] In a particular embodiment of the method of the present application, the antibody sample is a purified or partially purified antibody sample.

[0084] Definitions

[0085] The term "antibody" or "antibodies" as used herein refers to monoclonal antibodies or polyclonal antibodies. The term "antibody" or "antibodies" as used herein includes, but is not limited to, recombinant antibodies generated by known recombinant techniques in the art. "Antibody" or "antibodies" include antibodies of any species, in particular of a mammalian species; for example, antibodies of any isotype of a human, including IgD, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgE, and IgY. 2a 2b ​antibodies produced by IgG1, IgG2, IgG3, IgG4, IgE and dimers (including IgGA1, IgGA2) or pentamers (e.g. IgM and modified variants thereof) of this basic structure; non-human primate antibodies, e.g. from chimpanzee, baboon, rhesus or cynomolgus monkey; rodent antibodies, e.g. from mouse or rat; rabbit, goat or horse antibodies; and camelid antibodies (e.g. from camel or llama (e.g. nanobodies TM) and derivatives thereof; or an antibody of an avian species (e.g., chicken) or an antibody of a fish species (e.g., shark). The term "antibody" or "antibodies" also refers to "chimeric" antibodies in which at least a portion of a first heavy and / or light chain antibody sequence is altered to be non-human, and at least a portion of another heavy and / or light chain antibody sequence is altered to be non-human. Chimeric antibodies of interest herein include "primatized" antibodies wherein variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey (e.g., baboon, rhesus, or cynomolgus monkey) or Ape (e.g., chimpanzee)) and human constant region sequences are combined. "Humanized" antibodies are chimeric antibodies that contain sequences from a non-human antibody. In most instances, humanized antibodies are human antibodies (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, chicken, or non-human primate having the desired specificity, affinity, and activity. In most instances, the residues in the recipient antibody that are outside of the CDRs (i.e., in the framework regions (FRs)) are additionally replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. Humanization reduces immunogenicity of non-human antibodies in humans, thus facilitating the use of the antibodies in treating human disease. Humanized antibodies and several different techniques for making them are well known in the art. The term "antibody" or "antibodies" also refers to human antibodies, which can be generated as an alternative to humanization. For example, transgenic animals (e.g., mice) can be produced that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous mouse antibody production. For example, it has been described that the homozygous deletion of the mouse heavy chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice will result in the production of human antibodies upon antigen challenge.Other methods for obtaining human antibodies / antibody fragments in vitro are based on display technologies (e.g., phage display or ribosome display technologies) in which a recombinant DNA library is used that is generated at least in part artificially or from the immunoglobulin variable (V) domain gene repertoire of a donor. Phage and ribosome display technologies for generating human antibodies are well known in the art. Human antibodies can also be generated from isolated human B cells immunized ex vivo with the antigen of interest and then fused to generate hybridomas from which the best human antibodies can be selected. The term “antibody” or “antibodies” as used herein also refers to non-glycosylated antibodies.

[0086] The term “antibody” or “antibodies” as used herein refers not only to untruncated antibodies of any species (including from humans (e.g., IgG) and other mammalian species), but also to antibody fragments. Antibody fragments comprise at least one heavy or light chain immunoglobulin domain as known in the art and are capable of binding to one or more antigens. Examples of antibody fragments of the present invention include Fab, Fab', F(ab')2, and Fv and scFv fragments; as well as diabodies, triabodies, tetrabodies, minibodies, domain antibodies (dAbs) (e.g., single domain antibodies (sdAbs)), VHH fragments, and the like. Antibody fragments as defined above are all known in the art. H H and V NAR fragments, single chain antibodies formed from antibody fragments or antibodies, bispecific, trispecific, tetraspecific or multispecific antibodies (including but not limited to Fab-Fv or Fab-Fv-Fv constructs). Antibody fragments as defined above are all known in the art.

[0087] References:

[0088] 1. Wang, X., Hunter, A. K., and Mozier, N. M. Biotechnol. Bioeng. 2009, 103(3), 446-458.

[0089] 2. Singh, S. K., Mishra, A., Yadav, D., Budholiya, N., and Rathore, A. S. Biotechnol. Prog. 2020, 36(2), p. e2936.

[0090] 3. Jones, M., Palackal, N., Wang, F., Gaza-Bulseco, G., Hurkmans, K., Zhao, Y., Chitikila, C., Clavier, S., Liu, S., Menesale, E., Schonenbach, N. S., Sharma, S., Valax, P., Waerner, T., Zhang, L. and Connolly, T. Biotechnol. Bioeng. 2021, 118, 2870-2885.

[0091] 4. Bracewell, D. G., Francis, R., Smales, C. M. Biotechnol. Bioeng. 2015, 112, 1727-1737.

[0092] 5. Ahne, E., Molzahn, L., Glatter, T. and Schmidt, A. Proteomics 2013, 13, 2567-2578.

[0093] 6. Kreimer, S., Gao, Y., Ray, S., Jin, M., Tan, Z., Mussa, N. A., Tao, L., Li, Z., Ivanov, A. R. and Karger, B. L. Anal. Chem. 2017, 89, 5294-5302.

[0094] 7. Meier, F., Geyer, P. E., Virreira Winter, S., Cox, J. and Mann, M. Nat. Methods 2018, 15, 440-448.

[0095] 8. Huang, L., Wang, N., Mitchell, C. E., Brownlee, T., Maple, S. R. and De Felippis, M. R. Anal. Chem. 2017, 89, 5436-5444.

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[0097] 10. Silva, J.C., Gorenstein, M.V., Li, G.Z., Vissers, J.P., and Geromanos, S.J. Mol. Cell. Proteomics, 2006, vol. 5, no. 1, pp. 144-156. Examples

[0098] Materials

[0099] From readily available in-house UCB development product batches, empty (non-product expressing) harvest cell culture fluid (HCCF), upstream HCCF of a genetically engineered dihydrofolate reductase deficient (DG44) CHO cell line producing a recombinant full-length IgG antibody, and the corresponding purified mAb were obtained. 1M Tris hydrochloride (Tris-HCl, pH 8.0) buffer, sodium deoxycholate (SDC), Pierce High pH Reverse Phase Peptide Fractionation Kit, Trypsin / Lys-C protease mix, dithiothreitol (DTT), NAb Protein-A plus spin column, NAb Protein-L plus spin column, triethylammonium bicarbonate (TEAB), iodoacetamide (IAM), dimethyl sulfoxide (DMSO), 3K Da and 100KDa protein concentrator PES MWCO, Acclaim PepMap 100 C18 (3 pm, 1 mm x 150 mm) columns were all purchased from Thermo Scientific, Waltham, MA, USA. Quan-Recovery vials, MassPREP Bovine Serum Albumin (BSA, SwissProt P02769) digest standard, MassPREP Alcohol Dehydrogenase (ADH, SwissProt P00330) digest standard, MassPREP Phosphorylase b (PYGM, SwissProt P00489) digest standard, MassPREP Enolase 1 (ENOl, SwissProt P00924) digest standard, RapiGest SF surfactant, and ACQUITY UPLC CSH C18 chromatography columns (1.7 pm, 1 mm x 150 mm) were all purchased from Waters, Milford, MA, USA. Sodium octanoate, sodium citrate, citric acid, sodium hydroxide, sodium chloride, and arginine hydrochloride were purchased from Sigma-Aldrich, Overijse, Belgium. Formic acid (FA), trifluoroacetic acid (TFA), acetonitrile (ACN), and LC-MS grade water were from Biosolve, Valkenswaard, The Netherlands. 3 pm, 1 mm x 150 mm) columns were all purchased from Thermo Scientific, Waltham, MA, USA. Quan-Recovery vials, MassPREP Bovine Serum Albumin (BSA, SwissProt P02769) digest standard, MassPREP Alcohol Dehydrogenase (ADH, SwissProt P00330) digest standard, MassPREP Phosphorylase b (PYGM, SwissProt P00489) digest standard, MassPREP Enolase 1 (ENOl, SwissProt P00924) digest standard, RapiGest SF surfactant, and ACQUITY UPLC CSH C18 chromatography columns (1.7 pm, 1 mm x 150 mm) were all purchased from Waters, Milford, MA, USA. Sodium octanoate, sodium citrate, citric acid, sodium hydroxide, sodium chloride, and arginine hydrochloride were purchased from Sigma-Aldrich, Overijse, Belgium. Formic acid (FA), trifluoroacetic acid (TFA), acetonitrile (ACN), and LC-MS grade water were from Biosolve, Valkenswaard, The Netherlands. 1.7 pm, 1 mm x 150 mm) were all purchased from Waters, Milford, MA, USA. Sodium octanoate, sodium citrate, citric acid, sodium hydroxide, sodium chloride, and arginine hydrochloride were purchased from Sigma-Aldrich, Overijse, Belgium. Formic acid (FA), trifluoroacetic acid (TFA), acetonitrile (ACN), and LC-MS grade water were from Biosolve, Valkenswaard, The Netherlands.

[0100] Sample pretreatment

[0101] mAb normal digestion

[0102] 1 mg of mAb sample was diluted with 50 mM TEAB and denatured with 0.1% RapiGest. Then, the sample was reduced with 10 mM DTT for 60 min at 60 °C and carboxymethylated with 20 mM IAM for 30 min at room temperature in the dark. Next, the alkylated sample was digested with a trypsin / Lys-C mix at an enzyme to protein ratio of 1 :20 and incubated overnight at 37 °C. The reaction was terminated with 1% TFA for 20 min and centrifuged. The supernatant was acidified and dried using a speed-vac concentrator. The dried sample was dissolved in formic acid (FA), acetonitrile (ACN) and water (0.1 :2:98, v / v / v), four MassPREP proteins were added and transferred to a Quan-recovery vial before LC-MS / MS analysis.

[0103] mAb native digestion

[0104] A modified native digestion method by Huang et al. [8] was employed. 1 mg of mAb sample was diluted with 50 mM TEAB. Then, a trypsin / Lys-C protease mix (ratio 1 :500) was used for overnight digestion at 37 °C. Subsequently, disulfide bonds were reduced with 3 mM DTT and incubated at 90 °C for 10 min. The resulting digest was centrifuged at 15000 x g for 2 min. The supernatant was acidified and dried using a speed-vac concentrator. The dried sample was dissolved in FA, ACN and water (0.1 :2:98, v / v / v), four MassPREP proteins and an RTC mix were added and transferred to a Quan-recovery vial before LC-MS / MS analysis.

[0105] mAb affinity depletion - normal and native digestion

[0106] Protein-A and Protein-L bead agarose resins were used. Ten mg of mAb sample was taken for each, diluted with loading buffer (50 mM Tris.HCl, pH 7.0). The resins were equilibrated first and then the sample was applied. The flow-through was collected. To investigate the effect of wash solutions on HCP recovery during affinity depletion, different wash strategies were investigated. No wash (loading buffer), wash 1 (50 mM Tris-HCl, 0.5 M sodium chloride, 0.3 M arginine, pH 8.5), wash 2 (0.1% M citric acid, 0.5 M sodium chloride, pH 5.5), and wash 3 (50 mM Tris-HCl, 0.1 M sodium octanoate, 0.3 M arginine, pH 8.0) were used. The flow-through and wash solutions from each experiment were combined together and the buffer exchanged to 50 mM TEAB using a 3 K Da MWCO concentrator. The samples after buffer exchange were denatured with RapiGest or digested directly following the normal or native digestion protocol described previously.

[0107] HCCF and mAb off-line high pH fractionation

[0108] HCCF aliquots (empty and upstream) were filtered through 0.2 pm syringe filters and purified mAb samples were all diluted with 50 mM TEAB before denaturation, reduction, carboxymethylation, digestion, acidification, and centrifugation according to the normal digestion protocol described previously. The supernatants were fractionated using an off-line high pH fractionation kit. Eight fractions were collected from each sample using eluents containing 0.1% triethylamine and increasing percentages of ACN from 5% to 50%. The fractions were then vacuum dried, reconstituted in 0.1% FA, spiked with the four MassPREP proteins and RTC mix and transferred to Quan-recovery vials before LC-MS / MS analysis.

[0109] Reversed phase (RP)-LC-MS / MS for HCP analysis

[0110] Peptides were separated on an Acclaim PepMap 100 C18 column at 65 °C using a Thermo Fisher Scientific Ultimate 3000 UHPLC system equipped with a micro-LC setup. Samples were injected at a flow rate of 50 mΐ / min. Mobile phase A consisted of 1.5% DMSO and 0.1% FA in water, and mobile phase B consisted of 0.1% FA in ACN. DMSO was added to mobile phase A as an organic modifier to improve the efficiency of peptide ionization and to cause ion current to coalesce into fewer charge states. To improve peak capacity, a multi-step gradient was employed, ramping from 1% to 40% B over 160 min, followed by a 5 min column rinse at 80% B, and a 10 min re-equilibration at 1% B. The UHPLC system was coupled to an Exploris 480 mass spectrometer (Thermo Fisher Scientific). Tandem MS analysis was performed using DDA with the following settings: m / z range for MS scan was 360-1300 with a resolution of 120K full width at half maximum (FWHM), automatic gain control (AGC) target of 1E6, and maximum injection time (MIT) of 200 ms. The 40 most abundant ions were selected from each MS scan, and isotope was excluded. Ions with a charge state ranging from +2 to +4 and a count exceeding 5000 were subjected to MS / MS. High-energy collisional dissociation (HCD) was performed with a normalized energy of 28%, followed by ion trap analysis. A 2 m / z isolation window was used with a dynamic exclusion duration set to automatic. The resolution for MS / MS scan was 15K (FWHM), AGC target of 2E5, and MIT of 50 ms.

[0111] Construction of in-house database

[0112] The MS / MS data obtained from the high pH fractionation sample was searched against a custom database with 78,119 protein sequences, containing all Chinese hamster (Critecutulus griseus) entries from Uniprot.org (including Swiss-Prot and TrEMBL annotations), mAb heavy and light chains, and common contaminants. The search used the Byos v4.1 (Protein Metrics, San Carlos, CA) hybrid program for de novo sequencing assisted database searching. Search parameters included: precursor mass tolerance 10 ppm, fragment mass tolerance 20 ppm, semi-specific peptide end missing cleavage sites < 2, automatic peptide score cut and protein false discovery rate (FDR) cut of 2%. Cysteine carboxymethylation was considered a fixed modification, methionine or tryptophan oxidation was considered a common variable modification, and asparagine deamidation was considered a rare variable modification. A maximum of 1 common modification and 1 rare modification was allowed. Protein hits were adjusted to have a |Log Prob| (base 10 logarithm of the protein p-value) score at least 2.0 lower than the highest decoy protein score, and PSM filtering was postponed until after protein assembly. Common protein contaminants were excluded, and only double and triple charged peptides were considered. A total of 4380 proteins were identified and integrated to build an in-house database. The sequences of these proteins were used to generate a database in FASTA format.

[0113] Peptide and protein identification using in-house database

[0114] The resulting MS / MS data of the analyzed samples were searched by Byos against an in-house database (4380 protein sequences), immunoglobulin G binding protein A sequences, four standard protein digest of MassPREP (P02769, P00330, P00489, and P00924), mAb heavy and light chains, common contaminants. Search parameters included: precursor mass tolerance 10 ppm, fragment mass tolerance 20 ppm, complete specific peptide end missing cleavage sites < 2, automatic peptide score cut and protein FDR cut of 2%. Methionine or tryptophan oxidation was considered as a common variable modification. Asparagine deamidation, N-terminal glutamine forming pyroglutamic acid, N-terminal acetylation, and serine deamidation were considered as rare variable modifications. At most 1 common modification and 1 rare modification were allowed. Protein hits were adjusted to have a |Log Prob| score at least 2.0 lower than the highest decoy score. PSM filtering was delayed until protein assembly. Common protein contaminants were excluded and only double or triple charged peptides were considered. Further peptide / protein filtering was performed by eliminating peptides with less than six amino acids and all single-spectrum protein hits. Features missing in the replicate samples but correctly assigned in other replicate samples were inferred by Byos, reducing missing values and improving our subsequent differential abundance analysis.

[0115] Hi3 quantification

[0116] All filtered peptides by identification were exported together with their respective MS1 XIC peak areas. All intensities were normalized by dividing by the total intensity of each replicate and multiplying by the average of the total intensities of the three replicates. All modified peptides and peptides with a coefficient of variation greater than 25% in the three replicates were removed before Hi3 LFQ measurement. A universal signal response curve was generated by plotting the sum of the average MS1 intensities of the top three peptides of all four proteins of MassPREP against their concentrations (in femtomole (fmole)). A linear curve fit was shown and used to estimate the abundance (in mole) of individual HCPs based on the sum of the average intensities of the top three peptides of each identified HCP. The amount (in ng / mg) of each identified HCP was estimated using its molecular weight and the initial mAb amount.

[0117] Results

[0118] MS / MS data of purified mAb analyzed by the method of the present invention were searched against an in-house HCP database (4380 protein sequences) and a global HCP database containing all Chinese hamster entries (78119 protein sequences). When using the in-house database, a 28% increase in the number of identified HCP proteomes was observed Figure 3) and unique peptides had a 16% increase. The use of a low-redundancy database significantly improved the sensitivity of the scoring, enabling more comprehensive and higher quality HCP analysis.

Claims

1. A method for detecting residual host cell proteins (HCP) in a recombinant protein sample, comprising: a) an affinity chromatography step, wherein the effluent fraction is recovered; b) Trypsin digestion of the effluent fraction recovered from a) under native conditions, followed by c) liquid chromatography-tandem mass spectrometry (LC-MS / MS), wherein the liquid chromatography is a reversed-phase liquid chromatography performed in a multi-step gradient of charge enhancer concentration, and d) Comparing the LC-MS / MS results with the HCP database.

2. The method of claim 1, wherein the affinity chromatography step comprises: i) contacting the sample with an affinity chromatography matrix; ii) recovering the effluent from i); iii) applying a wash buffer to the affinity chromatography matrix and recovering the flow-through; and iv) combining the effluents from steps ii) and iii).

3. The method according to any one of the preceding claims, wherein the recombinant protein is a recombinant antibody.

4. The method according to any one of the preceding claims, wherein the affinity chromatography matrix is ​​selected from Protein A, Protein G or Protein L.

5. The method of claim 2, wherein the wash buffer comprises arginine, sodium caprylate, citrate, and / or a quaternary ammonium salt.

6. The method according to any one of the preceding claims, wherein the trypsin digestion is performed using a mixture of trypsin and lysine-C protease.

7. The method according to any one of the preceding claims, wherein the trypsin digestion is followed by treatment with a mild reducing agent selected from dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP).

8. The method according to any one of the preceding claims, wherein the reverse phase liquid chromatography is ultra performance liquid chromatography.

9. The method according to any one of the preceding claims, wherein the reverse phase liquid chromatography is performed under heating conditions, preferably between 45°C and 65°C.

10. The method according to any one of the preceding claims, wherein the reverse phase liquid chromatography is performed using a C18 column.

11. The method of any one of the preceding claims, wherein the charge enhancer comprises dimethyl sulfoxide (DMSO), m-nitrobenzyl alcohol (m-NBA), o-nitroanisole (o-NA), ethylene carbonate (EC), propylene carbonate (PC), or sulfolane.

12. The method according to any one of the preceding claims, wherein the gradient of the charge enhancer is 5% to 0.9%, preferably 1.5% to 0.9% DMSO.

13. The method of any preceding claim, wherein the mass spectrometry is performed using data-dependent acquisition.

14. The method according to any one of the preceding claims, wherein the HCP database is an HCP database based on an HCP database derived from a host cell line used to produce the antibody.

15. The method according to any one of the preceding claims, wherein the antibody sample is a purified or partially purified antibody sample.

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