Methods for the detection of host cell proteins

Abstract

The present invention provides a method for measuring residual host cell proteins (HCPs) in recombinant protein samples based on a combination of affinity chromatography and digestion under native conditions prior to analysis using reversed-phase liquid chromatography coupled with tandem mass spectrometry (LC / MS-MS), where the liquid chromatography is performed with a multi-step gradient of charge enhancing agent concentrations.

Description

[Technical Field]

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

[0002] Biotechnology products, often recombinant proteins, are obtained through complex production systems that include, but are not limited to, the use of genetically engineered host cells (bacteria, yeast, or mammalian cells). During the manufacture of such biotechnology products, host systems, such as Chinese hamster ovary (CHO) cells, also express, to varying degrees, a significant number of endogenous proteins essential for cellular development and survival, commonly referred to as host cell proteins (HCPs). [1] When biotechnology products are intended for pharmaceutical use and ultimately for administration to patients, residual HCPs must be routinely tested, regardless of the product and manufacturing system, to ensure that such impurities are reduced to acceptable levels.

[0003] Removal of HCPs typically utilizes chromatography columns used during the purification of the desired biotechnology product, often referred to as downstream processing (DSP).

[0004] However, in the field of recombinant antibody production, for example, some endogenous HCPs have been shown to potentially co-purify through different DSP steps and are considered difficult to remove, primarily due to their interactions with the antibody or resin. Some endogenous HCPs are generally considered "high risk" based on their impact. [2] Indeed, such process-related impurities are considered critical quality attributes (CQAs) because they may pose risks to clinical safety, product quality, or efficacy. [3] Therefore, it is becoming essential to establish analytical procedures that can identify and monitor HCP levels to support risk assessment of HCP impurities across production batches of therapeutic recombinant antibodies.

[0005] Traditionally, HCP quantification has been performed using a global approach based on enzyme-linked immunosorbent assays (ELISAs), which utilize antibodies raised against various HCPs expressed in host cells. [4] However, ELISAs only quantify the total amount of HCPs and do not indicate the identity or quantity of any specific HCP. The application of mass spectrometry (MS)-based proteomics has demonstrated the benefits of quantitatively profiling individual HCPs. However, ensuring technical reproducibility, dynamic range, and acceptable statistical significance of scoring measurements remain key challenges that need to be overcome.

[0006] Label-free quantitation (LFQ) methodologies have become widely used to directly quantify the signal response of related peptides to determine the amount of various HCPs in antibody samples [5]. Label-free quantitation methods are typically divided into intensity-based and spectral counting measurements. Intensity-based measurements, such as Hi3, have become widely used and have demonstrated better accuracy compared to spectral counting when using high-resolution MS.

[0007] Another aspect to consider is the type of data acquisition performed in MS. Data-independent acquisition (DIA) has been explored as an option, but it suffers from the drawback of loss of precursor selectivity. Data-dependent acquisition (DDA) has been widely used in exploratory proteomics and has demonstrated applicability in the field of HCP analysis [6]. However, DDA suffers from dynamic range issues that result in missing values ​​that can affect the consistency of results [7].

[0008] Several approaches have been proposed to address the dynamic range problem caused by the low abundance of HCPs in purified antibodies. Multidimensional LC-MS setups effectively address sample complexity by offering higher sensitivity and lower detection limits, despite limited throughput. Other methods use affinity chromatography (Protein A, G, or L) to capture and remove most of the antibody from the sample. Huang et al. [8] describe a native digestion approach in which antibody samples are digested under non-denaturing conditions with a very low enzyme-to-protein ratio.

[0009] Recently, high-field asymmetric waveform ion mobility spectrometry (FAIMS), affinity depletion, and native digestion have been combined and found to be more effective than existing methods, with a detection limit of 1 ppm [9].

[0010] Thus, there remains a need in the art for methods that are sensitive enough to detect HCPs present in samples of recombinant proteins.

[0011] Particularly when considering recombinant proteins destined for pharmaceutical use, special requirements regarding purity must be met in order for the product to be brought to market, and as such there is a particular need for methods that meet and / or comply with quality guidelines such as ICH quality guidelines, especially the ICH Q2 guidelines. Summary of the Invention

[0012] Against this background, the present inventors provide a method for measuring residual host cell proteins (HCPs) in recombinant protein samples based on a combination of affinity chromatography and digestion under native conditions prior to analysis using reversed-phase liquid chromatography coupled with tandem mass spectrometry (LC / MS-MS), where the liquid chromatography is performed with a multi-step gradient of charge enhancer concentrations. Furthermore, the present inventors have found that utilizing a wash solution during the affinity chromatography step maximizes recovery and increases identification of difficult-to-remove HCPs. [Brief explanation of the drawings]

[0013] [Figure 1] 1 is a bar graph of the total HCP protein groups identified by normal digestion, native digestion, Prot A and normal digestion, Prot L and normal digestion, Pro A and native digestion, and Pro L and native digestion. [Figure 2] Bar graph of total HCP proteins identified by a combination of Prot A and native digestion: no wash (resin washed only with loading buffer), wash with arginine buffer at alkaline pH, wash with citric acid + sodium citrate buffer at pH 5.5, and wash with arginine + sodium caprylate buffer at alkaline pH. [Figure 3] FIG. 1 shows the number of HCP protein groups identified using the methods of the present invention processed in either a global HCP database or an in-house HCP database. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention solves the above-stated need by providing a new method for the analysis, i.e., detection and quantification, of host cell proteins in recombinant antibody samples that also allows for the assessment of HCP removal during protein purification.

[0015] In a first aspect, the present invention provides a method for detecting residual host cell proteins (HCPs) in a recombinant protein sample, comprising: a) an affinity chromatography step in which the flow-through fraction is collected; b) Trypsin digestion under native conditions of the flow-through fraction collected from a), followed by c) liquid chromatography coupled with tandem mass spectrometry (LC-MS / MS), wherein the liquid chromatography is reversed-phase liquid chromatography performed with a multi-step gradient of charge enhancer concentration; and d) Comparing the results from the LC-MS / MS with the HCP database The present invention relates to a method, including:

[0016] In a second aspect, the present invention provides a label-free method for quantitation of residual HCPs in a recombinant protein sample, comprising: a) an affinity chromatography step in which the flow-through fraction is collected; b) Trypsin digestion under native conditions of the flow-through fraction collected from a), followed by c) liquid chromatography coupled with tandem mass spectrometry (LC-MS / MS), wherein the liquid chromatography is reversed-phase liquid chromatography performed with a multi-step gradient of charge enhancer concentrations; d) comparing the results from the LC-MS / MS with the HCP database; and e) Quantification of HCP The present invention relates to a label-free quantitative method, comprising:

[0017] Quantification of HCPs by label-free methods can be performed by available techniques such as spectral counting or intensity-based measurements such as Hi3 label-free quantification. In certain embodiments of the second aspect of the invention, quantification of HCPs is performed via Hi3.

[0018] Affinity chromatography is a separation method based on the specific binding interaction between an immobilized ligand and its binding partner. In the context of the present invention, the affinity chromatography matrix can bind to biotechnology products. Therefore, the flow-through fraction collected from the affinity chromatography method of the present invention is enriched in HCPs.

[0019] Those skilled in the art are aware of a variety of affinity chromatography matrices suitable for recombinant proteins, for example, immobilized metal affinity chromatography has been used for both recombinant protein and nucleic acid purification, or enzyme / substrate and enzyme / inhibitor interactions have been used for recombinant protein isolation.

[0020] In a particular embodiment of the invention, the recombinant protein is a recombinant antibody.

[0021] The affinity chromatography matrix is ​​preferably capable of binding to the antibody via the Fc region or the VH3 domain of the antibody.

[0022] In one embodiment, the affinity chromatography matrix is ​​selected from a Protein A chromatography matrix, a Protein G chromatography matrix, and a Protein L chromatography matrix.

[0023] There are many affinity chromatography materials containing Protein A, Protein G or Protein L available to those skilled in the art, such as MabSelect® (GE Healthcare), Absolute® (Novasep), Captiv A® (Repligen), Praesto AP (Purolite) or Amsphere® (JSR), Nab™ Proteins (ThermoFisher), or Pierce™ Proteins (ThermoFisher).

[0024] Buffers suitable for use as wash and elution buffers for Protein A chromatography are readily available in the art and may be selected from, for example, but not limited to, phosphate buffered saline (PBS), Tris, histidine, acetate, formate, citrate buffers, or MES (2-(N-morpholino)ethanesulfonic acid imidazole), BES (N,N-(bis-2-hydroxyethyl)-2-aminoethanesulfonic acid), MOPS (3-(N-morpholino)-propanesulfonic acid), or HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffers.

[0025] In certain embodiments of the method of the present invention, the affinity chromatography step comprises: i) contacting the sample with an affinity chromatography matrix; ii) collecting the flow-through from i); iii) applying a wash buffer to the affinity chromatography matrix and collecting the flow-through; iv) combining the flow-through from steps ii) and iii); Includes.

[0026] In certain embodiments of the methods of the invention, the wash buffer used during affinity chromatography comprises benzoate, benzyl alcohol, arginine, sodium caprylate, citrate, and / or a quaternary ammonium salt such as tetramethylammonium chloride, tetrabutylammonium, etc. In a further preferred embodiment, the wash buffer comprises arginine, sodium caprylate, or a combination of both.

[0027] In further particular embodiments of the invention, the wash buffer comprises 0.2M arginine to 1M arginine, 0.2M arginine to 0.8M arginine, 0.2M arginine to 0.6M arginine, 0.2M arginine to 0.5M arginine, or 0.2M arginine to 0.4M arginine, preferably 0.3M arginine.

[0028] A variety of arginine sources are available to those skilled in the art. In the context of the present invention, arginine and L-arginine are used interchangeably. Typically, L-arginine is available as a free base or as a salt, such as L-arginine monohydrochloride.

[0029] In certain embodiments of the invention, the wash buffer comprises 0.05 M sodium caprylate to 0.15 M sodium caprylate, 0.08 M sodium caprylate to 0.12 M sodium caprylate, 0.09 M sodium caprylate to 0.11 M sodium caprylate, preferably 0.1 M sodium caprylate.

[0030] In further particular embodiments of the invention, the wash buffer has a pH 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.

[0031] Trypsin is a serine protease that cleaves proteins into peptides with an average size of 700-1500 daltons, an ideal range for MS detection. Trypsin is highly specific, cleaving at the carboxyl side of arginine and lysine residues, making the resulting C-terminal arginine and lysine peptides charged and therefore detectable by MS.

[0032] As those skilled in the art will recognize, an alternative to trypsin is the use of a mixture of trypsin and lysine-C protease, which may be considered advantageous because the lysine-C protease may digest lysine cleavage sites that were not cleaved by trypsin in the presence of residual trypsin inhibitor impurities.

[0033] In certain embodiments of the methods of the present invention, tryptic digestion is carried out using trypsin or a mixture of trypsin and lysine-C protease, which is readily available from commercial sources, such as the Trypsin / Lysine-C mix from Promega.

[0034] In certain embodiments of the invention, trypsin digestion is performed at an enzyme-to-antibody ratio 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.

[0035] The terms "digestion under native conditions" or "native digestion" are used interchangeably, particularly to distinguish from "conventional" or "normal" protein digestion in which proteins are denatured prior to digestion, i.e., exposure to digestive enzymes such as trypsin.

[0036] In a particular embodiment of the method of the invention, trypsin digestion is followed by treatment with a mild reducing agent selected from dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP).

[0037] In certain embodiments of the methods of the present invention, 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.

[0038] The resulting complex mixture of peptides is analyzed by reversed-phase liquid chromatography (RP-LC) coupled with tandem mass spectrometry (MS / MS). Identification of peptides and subsequent proteins is completed by matching peptide fragment ion spectra against a host cell protein database.

[0039] Reversed-phase liquid chromatography is an elution procedure used in liquid chromatography in which the mobile phase is significantly more polar than the stationary phase, for example a microporous silica-based material to which alkyl chains have been chemically bonded.

[0040] In certain embodiments of the method of the present invention, 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 present invention, the reversed phase liquid chromatography is ultra high performance liquid chromatography.

[0041] In certain embodiments of the method of the present invention, reversed-phase liquid chromatography is preferably carried out under heating conditions of 45° C. to 65° C., 55° C. to 65° C., or 60° C. to 65° C. In particularly preferred embodiments of the method of the present invention, reversed-phase liquid chromatography is carried out at 65° C.

[0042] Columns suitable 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.

[0043] In a particular embodiment of the method of the present invention, reverse phase liquid chromatography is performed using a C18 column.

[0044] As those skilled in the art will appreciate, mass spectrometry is one of the most common methods used in proteomics analysis. In protein and peptide analysis by MS, there are practical benefits to increasing the charge of the analyte, as it can increase the efficiency of peptide ionization and the convergence of ion current to less charged states, thereby facilitating further structural analysis and increasing resolution and accuracy.

[0045] In certain embodiments of the methods of the present invention, the reversed-phase liquid chromatography is carried out in the presence of a charge enhancing agent, which in certain preferred embodiments comprises dimethyl sulfoxide (DMSO), meta-nitrobenzyl alcohol (m-NBA), ortho-nitroanisole (o-NA), ethylene carbonate (EC), propylene carbonate (PC), or sulfolane.

[0046] In certain embodiments of the method of the present invention, the multi-step gradient of charge enhancing agent concentration is either an increasing gradient or a decreasing gradient.

[0047] In certain embodiments, the gradient of the charge enhancer concentration is between 5% and 0.9%, between 4% and 0.9%, between 3.5% and 0.9%, between 3% and 0.9%, between 2.5% and 0.9%, between 2% and 0.9%, or between 1.5% and 0.9%.

[0048] In a further embodiment, the gradient of charge enhancer is 1.5% to 0.9% DMSO.

[0049] The separation of peptides resulting from protein digestion followed by tandem mass spectrometry is often referred to as a shotgun MS approach. Currently, there are two broad approaches to generating such MS proteomics data: data-dependent acquisition and data-independent acquisition. In tandem mass spectrometry, data-dependent acquisition methods present only specific peptides generated during the first cycle of MS for fragmentation during the second cycle, whereas data-independent methods allow all peptides generated during the first MS cycle to be fragmented in the second round. In data-dependent acquisition, the measured spectrum is compared to spectra in an established database, but in data-independent acquisition, the multiplexed nature of MS spectra requires deconvolution, and therefore database-dependent search methods cannot be directly applied.

[0050] In certain embodiments of the invention, mass spectrometry is performed using data-dependent acquisition.

[0051] In a further specific embodiment of the present invention, the HCP database is based on a specific HCP database derived from the same host cell line used to produce the recombinant protein. The HCP database can be generated using a null cell line, i.e., a cell line that does not contain a gene that expresses the recombinant protein, or a cell line that expresses the recombinant protein. In a specific embodiment, the HCP database is derived from a combination of a recombinant protein-expressing host cell line and the same host cell line that does not contain a recombinant protein-expressing gene.

[0052] A frequently used database for evaluating method performance, instrument performance, and molecular characterization of monoclonal antibodies is the NIST Monoclonal Antibody (NISTmAb) Reference Material RM8671 (https: / / www.nist.gov / programs-projects / nist-monoclonal-antibody-reference-material-8671), which was created based on the characterization of a single IgG1k. NISTmAb is known to contain HCPs in the range of 100-300 ppm.

[0053] However, by generating an HCP database using the same host cells as those used to produce the recombinant antibody, comparison with MS spectra obtained using the methods of the present invention will be more reliable. Furthermore, when generating an HCP database, sensitivity can be improved by removing redundant entries and therefore more clearly defining the HCPs being characterized.

[0054] In particular methods according to the invention, the HCP database comprises between 3800 and 5000 protein sequences, between 4000 and 4800 protein sequences, between 4200 and 4600 protein sequences, between 4300 and 4500 protein sequences.

[0055] Recombinant proteins produced for large-scale commercial purposes can be produced by culturing prokaryotic (bacterial) or eukaryotic host cells that have been transfected with one or more expression vectors encoding the recombinant protein.

[0056] In the context of the present invention, suitable eukaryotic host cells are generally mammalian host cells (also called mammalian cells), including Chinese hamster ovary (CHO cells), lymphoid cell lines such as NSO myeloma cells and SP2 cells, COS cells, myeloma cells, or hybridoma cells. In a preferred embodiment, the mammalian cells are CHO cells. Suitable types of CHO cells may include CHO-K1, CHOK1-SV, dhfr-CHO, such as CHO-DG44, CHO-DXB11, CHO-DXB1, or CHO-S cells.

[0057] Mammalian cells can be cultured in any medium that promotes their growth and antibody expression, preferably a chemically defined medium that is free of animal serum and animal-derived products such as peptone. A variety of cell culture media are available to those skilled in the art, containing various combinations of vitamins, amino acids, hormones, growth factors, ions, buffers, nucleosides, glucose or equivalent energy sources present at appropriate concentrations to enable cell growth and protein production. Additional cell culture medium components can be included in the cell culture medium at appropriate concentrations at different times during the cell culture cycle, as would be known to those skilled in the art.

[0058] Mammalian cell culture can be carried out in any suitable vessel, such as a shake flask or a bioreactor, which may or may not be operated in fed-batch mode, depending, for example, on the scale of production required. These bioreactors may be either stirred tank or airlift reactors. A variety of large-scale bioreactors are available, with volumes of more than 1,000 L up to 50,000 L, preferably 5,000 L to 20,000 L or up to 10,000 L. Alternatively, small-scale bioreactors, such as 2 L to 100 L, can also be used to produce antibodies according to the methods of the invention.

[0059] The antibodies or antigen-binding fragments thereof that can be produced according to the methods of the present invention are typically found in the supernatant of mammalian host cell cultures, typically CHO cell cultures. In the case of CHO culture processes in which the protein of interest, such as 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 resulting from centrifugation of the cell culture.

[0060] The supernatant is typically filtered to remove charged particles, residual cellular metabolic products, and cellular debris, and the resulting fluid is typically called clarified cell culture fluid. The clarified cell culture fluid is then further processed in several steps, typically including two or three chromatography steps and an ultrafiltration / diafiltration step, to obtain the purified antibody or antigen-binding fragment thereof.

[0061] During this further or downstream purification process, residual impurities including host cell proteins, protein aggregates and degradation products are also removed.

[0062] In this regard, it may be important to measure the final concentration of HCPs in purified antibody samples (typically referred to as drug substances) to ensure adequate clearance, but also to perform this measurement during the purification step to improve control over the process.

[0063] Thus, in certain embodiments of the method of the invention, the antibody sample is obtained from the final purified drug substance. In alternative embodiments of the method of the invention, the sample is obtained from a step in the purification stream of said recombinant antibody.

[0064] In certain embodiments of the methods of the present invention, the antibody sample is a purified antibody sample or a partially purified antibody sample.

[0065] definition As used herein, the term "antibody" or "antibodies" refers to monoclonal or polyclonal antibodies. As used herein, the term "antibody" or "antibodies" includes, but is not limited to, recombinant antibodies produced by recombinant techniques known in the art. "Antibody" or "antibodies" includes antibodies of any species, particularly antibodies of mammalian species, e.g., IgD, IgG1, IgG 2a , IgG 2b, IgG3, IgG4, IgE, and antibodies produced as dimers of this basic structure, including IgGA1, IgGA2, or pentamers such as IgM and engineered variants thereof, non-human primate antibodies, for example, from chimpanzee, baboon, rhesus monkey, or cynomolgus monkey; rodent antibodies, for example, from mouse or rat; rabbit, goat, or horse antibodies; and camelid antibodies (e.g., from camel or llama, such as Nanobodies™) and derivatives thereof; or antibodies from avian species, such as chicken antibodies, or antibodies from fish species, such as shark antibodies. The term "antibody" or "antibodies" may also refer to "chimeric" antibodies, in which a first portion of at least one heavy and / or light chain antibody sequence is derived from a first species and a second portion of the heavy and / or light chain antibody sequence is derived from a second species. Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g., an Old World monkey such as a baboon, rhesus monkey, or cynomolgus monkey) and human constant region sequences. "Humanized" antibodies are chimeric antibodies comprising sequences derived from a non-human antibody. In most cases, humanized antibodies are human antibodies (recipient antibodies) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region [or complementarity-determining region (CDR)] 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 cases, residues outside the CDRs of the human (recipient) antibody, i.e., residues in the framework regions (FR), are additionally replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues not found in either the recipient antibody or the donor antibody. These modifications are made to further refine antibody performance. Humanization reduces the immunogenicity of non-human antibodies in humans, thus facilitating the application of antibodies to the treatment of human diseases. Humanized antibodies and several different techniques for producing them are well known in the art. The term "antibody" or "antibodies" can also refer to human antibodies, which can be produced as an alternative to humanization.For example, it is possible to generate transgenic animals (e.g., mice) that, upon immunization, are capable of producing a full repertoire of human antibodies in the absence of endogenous mouse antibody production. For example, homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germline mutant mice has been described, resulting in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene array into such germline mutant mice will result in the production of human antibodies with specificity for a particular antigen when the transgenic mice carrying the human germline immunoglobulin genes are immunized with the antigen. Techniques for generating such transgenic animals, as well as techniques for isolating and producing human antibodies from such transgenic animals, are known in the art. Alternatively, in transgenic animals, e.g., mice, only the immunoglobulin genes encoding the variable regions of mouse antibodies are replaced with the corresponding human variable immunoglobulin gene sequences. The mouse germline immunoglobulin genes encoding the antibody constant regions remain unchanged. In this way, antibody effectors function in the immune system of the transgenic mouse, resulting in essentially unchanged B cell development, which can lead to improved antibody responses upon antigen challenge in vivo. Once genes encoding a specific antibody of interest are isolated from such a transgenic animal, the genes encoding the constant regions can be replaced with human constant region genes to obtain fully human antibodies. Another method for obtaining human antibodies / antibody fragments in vitro is based on display technologies such as phage display or ribosome display. These display technologies use recombinant DNA libraries, at least in part, artificially generated or generated from a donor's immunoglobulin variable (V) domain gene repertoire. Phage and ribosome display technologies for generating human antibodies are well known in the art. Human antibodies may also be produced by ex vivo immunization of isolated human B cells with an antigen of interest, followed by fusion to generate hybridomas that can be screened for optimal human antibodies.As used herein, the term "antibody" or "antibodies" may also refer to aglycosylated antibodies.

[0066] As used herein, the term "antibody" or "antibodies" refers not only to uncleaved antibodies of any species, including humans (e.g., IgG) and other mammalian species, but also to antibody fragments. Antibody fragments contain at least one heavy or light chain immunoglobulin domain, as known in the art, and bind to one or more antigens. Antibody fragments according to the present invention include Fab, Fab', F(ab')2, and Fv and scFv fragments, as well as diabodies, triabodies, tetrabodies, minibodies, domain antibodies (dAbs), such as single domain antibodies (sdAbs), VFv fragments, and the like. H H and V NAR Antibody fragments as defined above are known in the art, including fragments, single chain antibodies, bispecific, trispecific, tetraspecific or multispecific antibodies formed from antibody fragments or antibodies, including but not limited to Fab-Fv or Fab-Fv-Fv constructs. References:

number

[0067] material Ready-to-use batches of UCB in-house developed products were obtained: null (non-product-expressing) harvest cell culture fluid (HCCF), upstream HCCF of a genetically engineered dihydrofolate reductase-deficient (DG44) CHO cell line producing recombinant full-length IgG antibody, and the corresponding purified mAb. Tris-HCl 1M (Tris-HCl, pH 8.0) buffer, sodium deoxycholate (SDC), Pierce High pH Reverse-Phase Peptide Fractionation Kit, trypsin / Lys-C protease mix, dithiothreitol (DTT), NabProtein A plus spin columns, NabProtein L plus spin columns, triethylammonium bicarbonate (TEAB), iodoacetamide (IAM), dimethyl sulfoxide (DMSO), 3K Da and 100K Da protein concentrators PES MWCO, and Acclaim PepMap 100 C18 (100 Å, 3 μm, 1 mm × 150 mm) columns were purchased from Thermo Scientific (Waltham, MA, USA). Quan-Recovery vials, MassPREP bovine serum albumin (BSA, SwissProt P02769) digestion standard, MassPREP alcohol dehydrogenase (ADH, SwissProt P00330) digestion standard, MassPREP phosphorylase b (PYGM, SwissProt P00489) digestion standard, MassPREP enolase (ENO1, SwissProt P00924) digestion standard, RapiGest SF detergent, and an ACQUITY UPLC CSH C18 column (130 Å, 1.7 μm, 1 mm × 150 mm) were purchased from Waters (Milford, MA, USA). Sodium caprylate, 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 purchased from Biosolve (Valkenswaard, The Netherlands).

[0068] Sample pretreatment mAb normal digestion A 1 mg mAb sample was diluted with 50 mM TEAB and denatured with 0.1% RapiGest. The sample was then reduced with 10 mM DTT at 60°C for 60 minutes and carboxymethylated with 20 mM IAM for 30 minutes in the dark at room temperature. The alkylated sample was then digested with trypsin / Lys-C at a 1:20 enzyme-to-protein ratio and incubated overnight at 37°C. The reaction was stopped with 1% TFA, held for 20 minutes, and centrifuged. The supernatant was acidified and dried using a centrifugal vacuum concentrator. The dried sample was dissolved in formic acid (FA), acetonitrile (ACN), and water (0.1:2:98, v / v / v), spiked with the four MassPREP proteins, and transferred to a Quan-recovery vial for LC-MS / MS analysis.

[0069] mAb native digestion A modified native digestion method from Huang et al. [8] was used. 1 mg of mAb sample was diluted with 50 mM TEAB. The sample was then digested overnight at 37°C with a trypsin / Lys-C protease mix (1:500 ratio). Disulfide bonds were subsequently reduced with 3 mM DTT and incubated at 90°C for 10 min. The resulting digest was centrifuged at 15,000 × g for 2 min. The supernatant was acidified and dried using a centrifugal vacuum concentrator. The dried sample was dissolved in FA, ACN, and water (0.1:2:98, v / v / v), and the four MassPREP proteins and RTC mixture were added. The sample was transferred to a Quan-recovery vial for LC-MS / MS analysis.

[0070] mAb affinity removal - conventional and native digestion Protein A and Protein L beaded agarose resins were used. For each, 10 mg of mAb sample was diluted in loading buffer (50 mM Tris-HCl, pH 7.0). The resin was first equilibrated, and then the sample was applied. The flow-through was collected. To study the effect of wash solutions on HCP recovery during affinity removal, 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 NaCl, pH 5.5), and wash 3 (50 mM Tris-HCl, 0.1 M sodium caprylate, 0.3 M arginine, pH 8.0). The flow-through and washes from each experiment were pooled together and buffer-exchanged into 50 mM TEAB using a 3 KDa MWCO concentrator. Buffer-exchanged samples were either denatured or directly digested with RapiGest according to the normal or native digestion protocols described above.

[0071] HCCF and mAb offline high pH fractionation Aliquots of HCCF (null and upstream) filtered through 0.2 μm syringe filters, as well as purified mAb samples, were all diluted with 50 mM TEAB and then denatured, reduced, carboxymethylated, digested, acidified, and centrifuged according to the previously described standard digestion protocol. The supernatants were fractionated using an offline high-pH fractionation kit. Eight fractions were collected from each sample using an eluate containing 0.1% triethylamine and a gradient of ACN ranging from 5% to 50%. The fractions were then dried in vacuo, reconstituted with 0.1% FA, spiked with the four MassPREP proteins and RTC mixture, and transferred to Quan-recovery vials for LC-MS / MS analysis.

[0072] Reversed-phase (RP)-LC-MS / MS for HCP analysis Peptides were separated at 65°C on an Acclaim PepMap 100 C18 column using a Thermo Fisher Scientific Ultimate 3000 UHPLC system equipped with a microLC device. Samples were injected at a flow rate of 50 μl / 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 enhance peptide ionization efficiency and convergence of ion current to less charged states. To improve peak capacity, a multistep gradient from 1% to 40% B was applied for 160 min, followed by a 5-min column wash at 80% B and a 10-min column 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 a DDA with the following settings: MS scans had an m / z range of 360-1300, a full-width at half-maximum (FWHM) resolution of 120K, an automatic gain control (AGC) target of 1E6, and a maximum injection time (MIT) of 200 ms. The top 40 most abundant ions were selected from each MS scan and subjected to isotope exclusion processing. MS / MS was performed on ions with a charge state greater than 5000 counts in the +2 to +4 range. Higher-energy collisional dissociation (HCD) was performed at 28% normalized energy, followed by ion trap analysis. A 2 m / z isolation window was used, and the dynamic exclusion duration was set to automatic. MS / MS scans had a resolution of 15K (FWHM), an AGC target of 2E5, and a MIT of 50 ms.

[0073] Building an in-house database MS / MS data from the high-pH fractionated samples were searched against a customized database containing 78,119 protein sequences, including all entries for Critecutulus griseus from Uniprot.org (including Swiss-Prot and TrEMBL annotations), mAb heavy and light chains, and common contaminants. The de novo sequencing-assisted database search hybrid program Byos v4.1 (Protein Metrics, San Carlos, CA) was used for the search. Search parameters included a mass tolerance of 10 ppm for precursors and 20 ppm for fragments, semispecific peptide termini with no more than two missed cleavages, an automated peptide score cutoff, and a 2% protein false positive rate (FDR) cutoff. Cysteine ​​carboxymethylation was considered a fixed modification, methionine or tryptophan oxidation a common variable modification, and asparagine deamidation a rare variable modification. A maximum of one common modification and one rare modification were allowed. Protein hits were adjusted to a |Log Prob| (base 10 logarithm of the protein p-value) score at least 2.0 lower than the top decoy protein score, and PSM filtering was postponed until after protein assembly. Common protein contaminants were excluded, and only doubly and triply charged peptides were considered. A total of 4380 proteins were identified and integrated to construct an in-house database. The sequences of these proteins were used to create a FASTA-formatted database.

[0074] Peptide and protein identification using in-house databases MS / MS data from the analyzed samples were searched by Byos against an in-house database (4380 protein sequences), immunoglobulin G-binding protein A sequences, four standard protein digests from MassPREP (P02769, P00330, P00489, and P00924), mAb heavy and light chains, and common contaminants. Search parameters included a mass tolerance of 10 ppm for precursors and 20 ppm for fragments, fully specific peptide termini with no more than two missed cleavages, an automated peptide score cutoff, and a protein FDR cutoff of 2%. Oxidation of methionine or tryptophan was considered a common variable modification. Asparagine deamidation, pyroglutamate formation of N-terminal glutamine, N-terminal acetylation, and serine dehydration were considered rare variable modifications. A maximum of one common modification and one rare modification were allowed. Protein hits were adjusted to a |Log Prob| score at least 2.0 lower than the top decoy score. PSM filtering was postponed until after protein assembly. Common protein contaminants were excluded, and only doubly and triply charged peptides were considered. Further peptide / protein filtering was performed by eliminating peptides with fewer than six amino acids and all single-spectrum protein hits. Features missing in some replicates but correctly assigned in others were inferred by Byos, thereby reducing missing values ​​and improving subsequent differential abundance analysis.

[0075] Hi3 quantification All peptides that passed the identification filter were exported along with their respective MS1 XIC peak areas. All intensities were normalized by dividing by the total intensity per replicate and multiplying by the average of the total intensities of the three replicates. Prior to Hi3 LFQ measurement, all modified peptides and peptides with a coefficient of variation greater than 25% across triplicates were excluded. A common signal response curve was created by plotting the sum of the average MS1 intensities of the top three peptides for all four MassPREP proteins against their concentrations (units: femtomoles)

[10] . A linear curve fit was performed and used to estimate the individual HCP abundance (units: moles) based on the sum of the average intensities of the top three peptides for each identified HCP. The amount of each identified HCP was estimated (units: ng / mg) using its molecular weight and the initial mAb amount.

[0076] result The MS / MS data of purified mAbs analyzed by the method of the present invention were searched against an in-house HCP database (4,380 protein sequences) and a global HCP database (78,119 protein sequences) containing all entries for C. griseus. A 28% increase in the number of identified HCP protein groups (Figure 3) and a 16% increase in unique peptides was observed when using the in-house database. This significant improvement in scoring sensitivity achieved by using a low-redundancy database allows for more comprehensive and high-quality HCP profiling.

Claims

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

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

3. The method of claim 1 or 2, wherein the recombinant protein is a recombinant antibody.

4. The method according to any one of claims 1 to 3, wherein the affinity chromatography matrix is ​​selected from Protein A, Protein G or Protein L.

5. 3. 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 claims 1 to 5, wherein the trypsin digestion is carried out using a mixture of trypsin and lysine-C protease.

7. The method according to any one of claims 1 to 6, 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 claims 1 to 7, wherein the reversed-phase liquid chromatography is ultra-high performance liquid chromatography.

9. The method according to any one of claims 1 to 8, wherein the reversed phase liquid chromatography is carried out under heating conditions, preferably at 45°C to 65°C.

10. The method according to any one of claims 1 to 9, wherein the reversed-phase liquid chromatography is carried out using a C18 column.

11. 11. The method of any one of claims 1 to 10, wherein the charge enhancing agent comprises dimethyl sulfoxide (DMSO), meta-nitrobenzyl alcohol (m-NBA), ortho-nitroanisole (o-NA), ethylene carbonate (EC), propylene carbonate (PC), or sulfolane.

12. The method according to any one of claims 1 to 11, wherein said gradient of charge enhancer is 5% to 0.9%, preferably 1.5% to 0.9% DMSO.

13. The method of any one of claims 1 to 12, wherein the mass analysis is performed using data-dependent acquisition.

14. The method of any one of claims 1 to 13, wherein the HCP database is based on an HCP database derived from a host cell line used to produce the antibody.

15. The method according to any one of claims 1 to 14, wherein the antibody sample is a purified or partially purified antibody sample.

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