EA047087B1 - MULTIPLEX QUANTITATIVE DETERMINATION OF POST-TRANSLATIONAL MODIFICATIONS OF PROTEINS USING TANDEM MASS TAGS - Google Patents

Description

Link to sequence list

Incorporated herein by reference is the sequence listing filed in machine-readable form as file 10693WO01-Sequence.txt, created on January 26, 2021, 1462 bytes in size.

Technical field

The present invention relates to biopharmaceuticals and relates to multiplex quantitation of post-translational modifications of proteins, such as therapeutic monoclonal antibodies, using tandem mass tags (TMT).

State of the art

Since their first approval by the US Food and Drug Administration (FDA) in 1986, recombinant monoclonal antibodies (mAbs) have become one of the fastest growing classes of biotherapeutics for the treatment of a variety of human diseases in the drug pipeline and market. biopharmaceuticals due to their high specificity, long half-life in the bloodstream, ability to induce an effector response in immune cells, and fewer side effects compared to small molecule drugs. To date, approximately 80 IgG mAb-based drugs have been approved by the FDA and the European Medicines Agency (EMA), and more than 70 are in late-stage development.

IgG mAbs are covalent heterotetrameric proteins with a molecular weight of approximately 150 kDa, consisting of two identical heavy and light chains that are covalently linked by multiple disulfide bonds to form a Y-shaped structure. Due to their large size and structural complexity, mAbs are subject to a wide range of post-translational modifications (PTMs), such as Fc glycosylation, methionine oxidation (Met), asparagine deamidation (Asn), aspartic acid cyclization/isomerization (Asp), N-terminal glutamine cyclization (Gln) or glutamate (Glu), C-terminal lysine (Lys) clipping, non-enzymatic glycosylation of Lys and trisulfide bond, during cell culture, purification, drug formulation and storage. PTMs may be a major source of structural heterogeneity and play an important role in modulating the physicochemical properties of mAbs. Some PTMs, such as glycosylation, deamidation and oxidation, depending on the location of the affected residues (e.g. in complementarity determining regions (CDRs)), may even have detrimental effects on stability, function, immunogenicity and pharmacokinetics/pharmacodynamics, which are generally considered critical quality attributes (CQAs) of antibodies that should be closely monitored during drug development (Wang et al., J. Pharm. Sci., 2007, 96, 1-26; Manning et al., Pharm Res. 2010, 27, 544- 557). Oxidation of two conserved Met residues located at the interface of heavy chain constant domains 2 (CH2) and 3 (CH3) in most IgG antibodies can reduce thermostability (Houde et al., Mol. Cell. Proteomics., 2010, 9, 1716-1728 ), protein A binding (Bertolotti-Ciarlet et al., Mol. Immunol., 2009, 46, 1878-1882), FcRn binding (Zhang et al., Anal. Chem., 2014, 86, 3468-3475) and period half-life of IgG antibodies from the bloodstream (Wang et al., Mol. Immunol., 2011, 48, 860866), while oxidation of Met or tryptophan (Trp) and deamidation of Asn in solvent-exposed CDRs could potentially affect antigen binding and activity (Wei et al., J. Pharm. Sci., 2009, 98, 3509-3521; Yan et al., J. Pharm. Sci., 2009, 98, 3509-3521). N-linked glycosylation at the conserved CH2 heavy chain Asn in the Fc region of antibodies is also important for maintaining mAb structure and stability and in some cases may regulate downstream effector functions such as complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) via modulating binding to FCY receptors (Jennewein, M.F., Alter, G., Trends in Immunology., 2017, 38, 358-372). Therefore, it is important to characterize and monitor levels of PTMs, especially those considered CQAs, during the development, production and storage of therapeutic antibodies to ensure product quality and determine any potential impact on ultimate safety and efficacy.

Peptide mapping by liquid chromatography-tandem mass spectrometry (LC-MS) is used in the biopharmaceutical industry to characterize therapeutic antibodies at the molecular level (Beck et al., Anal. Chem., 2013, 85, 715-736; Sandra, K. et al., J. Chromatogr. A., 2014, 1335, 81-103). This method uses a bottom-up technique involving enzymatic (i.e. typically trypsin) digestion of proteins under non-reducing (peptide mapping under non-reducing conditions) or reducing (peptide mapping under reducing conditions) conditions, followed by separation of the resulting peptides and analysis using ultraviolet (UV) ) detection and/or mass spectrometry (MS), which provides the benefits of assessing the structural integrity maintained by interchain and intrachain disulfide bonds, confirming the amino acid sequence of a protein, and providing site-specific quantitation of post-translational and chemical modifications that may occur during production, processing or storage (Mouchahoir, T., Schiel, J.E., Anal. Bioanal. Chem., 2018, 410, 2111-2126).

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In the LC-MS-based peptide mapping method for PTM analysis, site-specific PTMs are quantified from the MS1 spectra of enzymatic hydrolysates by calculating the ratio of the selected ion chromatogram (EIC) peak area of the modified PTM-containing peptide to the sum of the EIC peak areas of the corresponding native peptide and modified peptide. Although an optimized peptide mapping workflow can provide high sequence coverage and allow efficient characterization of multiple features (e.g., different site-specific PTMs) of an antibody sample in a single LC-MS run, thanks to the rapid development of LC-MS instrumentation and bioinformatics software , this traditional label-free approach for relative quantitation of site-specific PTMs requires sample preparation and mass spectrometry data collection for individual samples, which can be time consuming, making it difficult to meet the ever-increasing demands for monoclonal antibody characterization during drug development. Recent advances in peptide mapping technology have largely focused on improving the efficiency of sample preparation, such as the development of an automated system to increase throughput or an in-line digestion system to reduce sample preparation time (Richardson et al., Anal. Biochem., 2011, 411, 284-291; Cao et al., J. Pharm. Sci., 2019, 108, 3540-3549; and Mao et al., mAbs. 2019, 11, 767-778), however, when the sample size increases, The instrument runtime required to acquire LC/MS data from samples also increases linearly, which not only limits the overall efficiency of the peptide mapping workflow for protein characterization, but can also lead to large variability in PTM quantitation due to instrument fluctuations associated with time and temperature, hardware and software failures of the device or instability during sample storage in an automatic sampler during LC-MS analysis of hydrolysates during peptide mapping.

Brief description of the invention

In one aspect of the present invention, there is provided a method for quantifying multiple quality attributes of multiple samples in a single mass spectrometry (MS) run, comprising contacting two or more samples with a digestion solution under conditions sufficient to digest the two or more samples, wherein each sample is digested separately, and the digestion solution is a buffer solution containing no Tris buffer; contacting each of the two or more digested samples with a specific tandem mass tag (TMT) labeling reagent under conditions sufficient to label peptides in each of the two or more digested samples using the specific TMT labeling reagent; quenching peptide labeling in each of the two or more digested samples; combining equal volumes of two or more labeled digest samples into a single pooled sample solution; and analyzing a solution of one pooled sample using targeted mass spectral analysis, thereby allowing multiple quality attributes of two or more samples to be quantified in a single mass spectrometry (MS) run.

In some embodiments, the plurality of quality indicators include post-translational modification (PTM).

In some embodiments, the PTM includes one or more of deamidation, oxidation, glycation, disulfide bond formation, N-terminal pyroglutamate formation, C-terminal lysine removal, and glycosylation.

In some embodiments, the PTM includes glycosylation.

In some embodiments, quantifying multiple quality metrics in a single MS run includes quantifying PTMs by quantifying the relative abundance of PTMs from selected peak areas of the resulting reporter ion generated in the target mass spectra.

In some embodiments, the method further includes contacting each of the two or more digested samples with a small molecule additive before contacting each of the two or more digested samples with a specific TMT labeling reagent.

In some embodiments, the low molecular weight additive is selected from the group consisting of BOC-Y-OH, p-cresol, hydroxyphenylacetic acid (HPAA), hydroxybenzoic acid (HBA), acetaminophen, and p-aminobenzoic acid (PABA).

In some embodiments, the small molecule additive is PABA.

In some embodiments, the peptides are glycopeptides.

In some embodiments, the glycopeptides are derived from a monoclonal antibody.

In some embodiments, the monoclonal antibody is of the IgG1, IgG2, IgG3, IgG4, or mixed isotype.

In some embodiments, the two or more samples represent 2 to 16 samples.

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In some embodiments, the method further includes obtaining two or more samples for analysis.

In some embodiments, the method further includes preparing two or more samples for digestion before contacting the two or more samples with a digestion solution under conditions sufficient to digest the two or more samples.

In some embodiments, preparing two or more samples prior to digestion includes bringing each of the two or more samples into contact with a denaturing and reducing solution under conditions that allow denaturation and reduction of the sample; and contacting each of the two or more denatured and reduced samples with an alkylating solution under conditions that alkylate the sample.

In some embodiments, analyzing a solution of one pooled sample using targeted mass spectral analysis includes applying one pooled sample to a separation column and performing targeted mass spectral analysis of the eluted components of the sample.

In some embodiments, the separation column is a liquid chromatography column.

In some embodiments, performing targeted mass spectral analysis of eluted sample components involves using electrospray ionization to generate charged ions from the eluted sample components and measuring the generated charged ions.

In one aspect of the invention, a method for quantifying post-translational modifications (PTMs) of multiple samples in a single mass spectrometry (MS) run includes contacting two or more samples with a digestion solution under conditions sufficient to digest the two or more samples, where each sample is digested separately , and the digestion solution is a buffer solution not containing Tris buffer; contacting each of the two or more digested samples with a low molecular weight additive; contacting each of the two or more digested samples with a specific tandem mass tag (TMT) labeling reagent under conditions sufficient to label peptides in each of the two or more digested samples using the specific TMT labeling reagent; quenching peptide labeling in each of the two or more digested samples; combining equal volumes of two or more labeled digest samples into a single pooled sample solution; and analyzing a solution of one pooled sample using targeted mass spectral analysis, thereby allowing the PTMs of two or more samples to be quantified in a single MS run.

In some embodiments, the PTM includes one or more of deamidation, oxidation, glycation, disulfide bond formation, N-terminal pyroglutamate formation, C-terminal lysine removal, and glycosylation.

In some embodiments, PTMs include glycosylation.

In some embodiments, quantitating PTMs includes quantifying the relative abundance of PTMs from selected peak areas of the resulting reporter ion generated in the target mass spectra.

In some embodiments, the low molecular weight additive is selected from the group consisting of BOC-Y-OH, p-cresol, hydroxyphenylacetic acid (HPAA), hydroxybenzoic acid (HBA), acetaminophen, and p-aminobenzoic acid (PABA).

In some embodiments, the small molecule additive is PABA.

In some embodiments, the peptides are glycopeptides.

In some embodiments, the glycopeptides are derived from a monoclonal antibody.

In some embodiments, the monoclonal antibody is of the IgG1, IgG2, IgG3, IgG4, or mixed isotype.

In some embodiments, the two or more samples represent 2 to 16 samples.

In some embodiments, the method further includes obtaining two or more samples for analysis.

In some embodiments, the method further includes preparing two or more samples for digestion before contacting the two or more samples with a digestion solution under conditions sufficient to digest the two or more samples.

In some embodiments, preparing two or more samples prior to digestion includes contacting each of the two or more samples with a denaturing and reducing solution under conditions that allow denaturation and reduction of the sample; and contacting each of the two or more denatured and reduced samples with an alkylating solution under conditions that alkylate the sample.

In some embodiments, analyzing a solution of one combined sample with

- 3 047087 using targeted mass spectral analysis involves applying one pooled sample to a separation column and performing targeted mass spectral analysis of the eluted components of the sample.

In some embodiments, the separation column is a liquid chromatography column.

In some embodiments, performing targeted mass spectral analysis of eluted sample components involves using electrospray ionization to generate charged ions from the eluted sample components and measuring the generated charged ions.

In various embodiments, any of the features or components of the embodiments discussed above or later in the present invention can be combined, and such combinations are within the scope of the present invention. Any particular value discussed above or hereinafter in the present invention may be combined with another related value discussed above or hereinafter in the present invention to define a range in which said values represent the upper and lower limits of the range, and such ranges and all values falling within such ranges are within the scope of the present invention. Each of the values discussed above or later in the present invention may be specified with a deviation of 1, 5, 10 or 20%. For example, a concentration of 10 mM may be reported as 10 ± 0.1 mM (1% deviation), 10 ± 0.5 mM (5% deviation), 10 ± 1 mM (10% deviation), or 10 ± 2 mM (20% deviation). Other embodiments will become apparent upon reading the following detailed description of the invention.

Brief description of graphic materials

In fig. 1A and 1B show the effect of NCE (35-70) on the reporter ion content of two representative TMT-labeled peptides in mAb-A (Fig. 1A) and on the relative quantitation of different types of PTMs in mAb-A along with the PTM quantification results obtained with using the traditional approach (Fig. 1B). In fig. 1B, the order of the columns from left to right for each site-specific PTM is as follows: control (no TMT), NCE 35, NCE 40, NCE 50, NCE 55, NCE 60, NCE 70.

In fig. 2A and 2B show the effect of NCE (35-100) on the quantification of Lys glycosylation in mAb-B (Fig. 2A) and the effect of NCE (27-100) on the quantification of pyroglutamate at the N-terminal Glu in mAb-B (Fig. 2B). Black error bars: standard deviation of PTM percentages in two parallel analyses. The order of the columns in Fig. 2A (from left to right): control (no TMT), NCE 35, NCE 40, NCE 50, NCE 55, NCE 60, NCE 70, NCE 80, NCE 90 and NCE 100. Column order in FIG. 2B (from left to right): control (no TMT), NCE 27, NCE 30, NCE 35, NCE 40, NCE 50, NCE 55, NCE 60, NCE 70, NCE 80, NCE 90 and NCE 100.

In fig. 3A and 3B show the effect of NCE (35-80) on the quantification of trisulfide bonds in IgG1 mAb-C (Fig. 3A) and IgG4 mAb-D (Fig. 3B), along with quantitative results obtained using the traditional approach.

In fig. 4A shows the evaluation of the relative quantitation of four PTMs in mAb-E in six TMT channels at a ratio of 1:1:1:1:1:1. Pooled TMT-labeled samples were obtained from three determinations and analyzed using LC-MS/MS in two parallel analyses. Boxplots show the mean (larger dot), the 25th to 75th percentile (box), and the 5th to 95th percentile (whiskers). PTM percentages calculated using the traditional approach are indicated by dotted lines.

In fig. 4B shows an assessment of the reproducibility of the relative quantitation of four PTMs in mAb-E in six TMT channels at a ratio of 1:1:1:1:1:1. Pooled TMT-labeled samples were obtained from three determinations and analyzed using LC-MS/MS in two parallel analyses. Boxplots show the mean (dot), 25th and 75th percentiles (box), and 5th and 95th percentiles (whiskers). PTM percentages calculated using the traditional approach are indicated by dotted lines.

In fig. Figure 5 shows the sensitivity estimate for the relative quantitation of four PTMs in mAb-A in six TMT channels at a ratio of 1:2:4:8:16:32. Pooled TMT-labeled samples were obtained from three determinations and analyzed using LC-MS/MS in two parallel analyses. Boxplots show the mean (dot), 25th and 75th percentiles (box), and 5th and 95th percentiles (whiskers). PTM percentages calculated using the traditional approach are indicated by dotted lines.

In fig. Figure 6 shows the quantification of PTMs in forced-degraded mAb-A samples using a targeted MS/MS approach (solid bars) and a conventional approach (striped bars). Black error bars: standard deviation of PTM percentages in two parallel analyses. The column order from left to right for each site-specific PTM is: mAb-A-S0 (no TMT), mAb-A-S0 (TMT-126), mAb-A-S1 (no TMT), mAb-A-S1 ( TMT-127), mAb-A-S2 (without TMT), mAb-A-S2 (TMT-128), mAb-A-S3 (without TMT),

- 4 047087 mAb-A-S3 (TMT-129), mAb-A-S4 (without TMT), mAb-A-S4 (TMT-130), mAb-A-S5 (without TMT), mAb-A-S5 (TMT-131).

In fig. 7A and 7B show PTM quantification of samples to assess the comparability of mAb-Fs manufactured at different manufacturing sites using a targeted MS/MS approach (solid bars) and a conventional approach (striped bars). In fig. 7A shows PTMs with levels >2.5%, and FIG. 7B shows PTMs with levels <2.5%. Black error bars: standard deviation of PTM percentages in two parallel analyses. The order of the columns is from left to right for each site-specific PTM for both Figs. 7A and 7B is the following: mAb-F-P1 (no TMT), mAb-F-P1 (TMT-128), mAb-F-P2 (no TMT), mAbF-P2 (TMT-129), mAb-F -P3 (without TMT), mAb-F-P3 (TMT-130), mAb-F-P4 (without TMT), mAb-F-P4(TMT-131).

In fig. Figure 8 shows the quantification of trisulfide bonds in standard samples to evaluate mAb-E trisulfide bonds using a targeted MS/MS approach (solid bars) and a conventional approach (striped bars). Trisulfide mAb-E standards with varying levels of trisulfide bonds were prepared by mixing the H2S-exposed sample and the reference standard sample in various ratios. mAb-E-TS0 (100:0), mAb-E-TS1 (75:25), mAb-E-TS2 (50:50), mAb-E-TS3 (25:75) and mAb-E-TS4 ( 0:100).

In fig. 9 shows the effect of various small molecule additives during TMT labeling on the relative abundance of TMT-labeled fragments with different TMT tags and different isoforms for the WQQG peptide (residues 1-4 SEQ ID NO: 3) and TTPP peptide (residues 1-4 SEQ ID NO: 4 ). The TMT labeling sites for each fragment were highlighted (in bold). Boc-Y: H-(tert-butoxycarbonyl)-tyrosine; HPAA: 4-hydroxyphenylacetic acid; HBA: 4-hydroxybenzoic acid; PABA: 4-aminobenzoic acid.

In fig. 10 shows the structure of an exemplary tandem mass label and exemplary tandem mass label reagents.

In fig. 11 shows the chemical structures of exemplary anti-labeling reagents BOC-Y-OH, p-cresol, hydroxyphenylacetic acid (HPAA), hydroxybenzoic acid (HBA), acetaminophen and/or p-aminobenzoic acid (PABA).

Detailed Description of the Invention

While referring to the description of the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, since these methods and conditions may vary. It is also understood that the terminology used in the present invention is intended to describe particular embodiments only and is not intended to be limiting, since the scope of the present invention is limited only by the appended claims. Any embodiments or features of embodiments may be combined with each other, and such combinations are clearly within the scope of the present invention.

Unless otherwise specified, all technical and scientific terms used in the present invention have the meaning commonly understood by one skilled in the art to which this invention relates. In the context of the present invention, the term "approximately" when used in relation to a particular given numerical value means that the value may differ from the given value by no more than 1%. For example, in the context of the present invention, the expression approximately 100 includes 99 and 101, and all values in between (eg, 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to those described in the present invention may be used in the practice or testing of the present invention, preferred methods and materials are described below. The contents of all patents, applications and non-patent publications mentioned in this specification are incorporated herein by reference in their entirety.

Abbreviations used in the present invention.

ACN: acetonitrile;

ADCC: antibody dependent cellular cytotoxicity;

Asn: asparagine;

AUC: area under the curve;

Boc-Y: N(tert-butoxycarbonyl)-tyrosine;

CDC: complement dependent cytotoxicity;

CDR: complementarity determining region;

CQA: Critical Quality Attributes;

CV: coefficient of variation;

EIC: selected ion chromatogram;

ELISA: enzyme-linked immunosorbent assay;

ESI-MS: electrospray ionization mass spectrometry;

FA: formic acid;

FDA: Food and Drug Administration;

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FLR: fluorescent detection;

HBA: 4-hydroxybenzoic acid;

HC: heavy chain;

HILIC: hydrophilic interaction liquid chromatography;

HPAA: 4-hydroxyphenylacetic acid;

IgG: immunoglobulin G;

LC: light chain;

LC-MS: liquid chromatography with tandem mass spectrometry;

mAb: monoclonal antibody;

Met: methionine;

MS: mass spectrometry;

MW: molecular weight;

NCE: normalized collision energy;

PABA: 4-aminobenzoic acid;

PK: pharmacokinetics;

PROCA: procainamide;

PQA: product quality indicator;

PTM: post-translational modification;

RP-LC-MS/MS: reversed-phase liquid chromatography with tandem mass spectrometry;

SPE: solid phase extraction;

TCEP-HCl: tris(2-carboxyethyl)phosphine hydrochloride;

TFA: trifluoroacetic acid;

TMT: tandem mass tag;

UV: Ultraviolet.

Definitions.

The term antibody in the context of the present invention refers to immunoglobulin molecules consisting of four polypeptide chains: two heavy (H) chains and two light (L) chains linked by disulfide bonds (i.e., full-length antibody molecules), as well as their multimers (for example, IgM) or their antigen-binding fragments. Each heavy chain consists of a heavy chain variable region (HCVR or _VH ) and a heavy chain constant region (consisting of _CH1 , CH2 and CH3 domains). In various embodiments, the heavy chain may be of the IgG isotype. In some cases, the heavy chain is selected from IgG1, IgG2, IgG3 or IgG4. In some embodiments, the heavy chain is of the IgG1 or IgG4 isotype, optionally including a chimeric hinge region of the IgG1/IgG2 or IgG4/IgG2 isotype. Each light chain consists of a light chain variable region (LCVR or VL) and a light chain constant region ( _CL ). In the VH and _VL regions, regions of hypervariability can be further distinguished, called complementarity determining regions (CDRs), which are interspersed with more conserved regions, called framework regions (FR). Each V _H and V _L consists of three CDRs and four FRs, located from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term antibody includes reference to both glycosylated and non-glycosylated immunoglobulins of any isotype or subclass. The term antibody includes antibody molecules produced, expressed, generated or isolated by recombinant methods, such as antibodies isolated from a host cell transfected to express the antibody. For an overview of antibody structure, see Lefranc et al., IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains, 27(1) Dev., Comp. Immunol., 55-77 (2003); and M. Potter, Structural correlates of immunoglobulin diversity, 2(1), Surv. Immunol. Res., 27-42 (1983).

The term antibody also covers a bispecific antibody that includes a heterotetrameric immunoglobulin that can bind to several different epitopes. One half of a bispecific antibody, which includes one heavy chain and one light chain and six CDRs, binds to one antigen or epitope, and the other half of the antibody binds to another antigen or epitope. In some cases, a bispecific antibody may bind the same antigen but different epitopes or non-overlapping epitopes. In some cases, both halves of a bispecific antibody have identical light chains while maintaining dual specificity. Bispecific antibodies are generally described in US Patent Application Publication No. 2010/0331527 (December 30, 2010).

The term antigen-binding portion of an antibody (or antibody fragment) refers to one or more antibody fragments that retain the ability to specifically bind an antigen. Examples of binding fragments encompassed by the term antigen-binding portion of an antibody include (i) a Fab fragment—a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragment - a divalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of one arm of the antibody; (v) dAb fragment (Ward et al.

- 6 047087 (1989), Nature, 241:544-546), consisting of a VH domain; (vi) dedicated CDR; and (vii) scFv, consisting of two Fv fragment domains, VL and VH, connected by a synthetic linker to form a single protein chain in which the VL and VH regions are paired to form monovalent molecules. Other forms of single chain antibodies, such as diabodies, are also covered by the term antibody (see, for example, Holliger et al. (1993), 90, PNAS U.S.A., 6444-6448; and Poljak et al. (1994), 2, Structure, 1121-1123).

In addition, antibodies and antigen binding fragments thereof can be produced using standard recombinant DNA technologies well known in the art (Sambrook et al., 1989). Methods for producing human antibodies in transgenic mice are also known in the art. Using VELOCIMMUNE® technology (see, for example, US Pat. No. 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method for producing monoclonal antibodies, high-affinity chimeric antibodies to the desired antigen are first isolated, having a human variable region and a murine constant region. VELOCIMMUNE® technology involves the creation of a transgenic mouse whose genome contains human heavy and light chain variable regions operably linked to endogenous mouse constant region loci such that the mouse produces an antibody containing the human variable region and the mouse constant region in response to antigenic stimulation. DNA encoding the antibody heavy and light chain variable regions is isolated and operably linked to DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in a cell capable of expressing a fully human antibody.

The term human antibody is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human mAbs of the invention may contain amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or somatic mutation in vivo), for example, in the CDR and, in particular, in CDR3. However, the term human antibody in the context of the present invention is not intended to include mAbs in which CDR sequences derived from the germ line of other mammalian species (eg, mouse) have been grafted onto human FR sequences. The term includes antibodies produced recombinantly in the body of a non-human mammal or in the cells of a non-human mammal. The term is not intended to include antibodies isolated from or obtained from a human subject.

In the context of the present invention, the term subject refers to an animal, preferably a mammal, more preferably a human, in need, for example, of alleviating, preventing and/or treating a disease or disorder.

Post-translational modification (PTM) refers to the covalent modification of proteins after protein biosynthesis. Post-translational modifications can occur on amino acid side chains or at the C or N termini of the protein. Examples of post-translational modifications of antibodies include deamidation, oxidation, glycation, disulfide bond formation, N-terminal pyroglutamate formation, C-terminal lysine removal, and glycosylation.

The term tandem mass tag (TMT) is a chemical tag used for the quantification and identification of biological macromolecules such as proteins, peptides and nucleic acids based on mass spectrometry (MS). TMT belongs to a family of reagents called isobaric mass tags. They provide an alternative to gel- or antibody-based quantitation, but can also be used in combination with these and other methods. In addition to aiding in protein quantification, TMT tags can also improve the detection sensitivity of certain highly hydrophilic analytes, such as phosphopeptides, in RP-HPLC-MS assays. Currently, six varieties of TMT are commercially available (Thermo Fisher Scientific, Altham, MA, USA): TMTzero - core structure without isotopic substitution; TMTduplex - isobaric pair of mass markers with one isotopic substitution; TMTsixplex - isobaric set of six mass markers with five isotopic substitutions; TMT 10-plex - a set of 10 isotopic mass tags that use the TMTsixplex reporter region but an isotope of a different element to create a mass difference of 0.0063 Da, TMTpro - a 16 plex version with a reporter and mass normalizer different from the original TMT, and TMTpro Zero. The tags contain four regions, namely the mass reporter region (M), the cleavable linker region (F), the mass normalization region (N), and the protein reacting group (R). The chemical structures of all tags are identical, but each contains isotopes substituting at different positions, so that the mass reporter and mass normalization regions have different molecular masses in each tag. Collectively, the regions of the M-F-N-R labels have the same overall molecular weight and structure, so when chromatographic or electrophoretic

- 7 047087 separation and in one-step MS mode, molecules labeled with different labels are indistinguishable. In MS/MS fragmentation, sequence information is obtained from fragmentation of the peptide backbone, and quantitation data is simultaneously obtained from tag fragmentation yielding mass reporter ions.

In the context of the present invention, the term glycopeptide/glycoprotein is a modified peptide/protein during or after its synthesis with covalently linked carbohydrates or glycan. In certain embodiments, the glycopeptide is derived from a monoclonal antibody, for example, from a protease digest of the monoclonal antibody.

In the context of the present invention, the term glycan is a compound containing one or more sugar units, which typically include glucose (Glc), galactose (Gal), mannose (Man), fucose (Fuc), N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) and N-acetylneuraminic acid (NeuNAc) (Frank Kjeldsen et al., Anal. Chem., 2003, 75, 2355-2361). The glycan moiety in a glycoprotein such as a monoclonal antibody is an important feature in determining its function or cellular localization. For example, a certain monoclonal antibody is modified with a certain glycan moiety.

In the context of the present invention, the term sample refers to a mixture of molecules that contains at least a molecule of an analyte, for example a glycopeptide, for example derived from a monoclonal antibody, which is subjected to manipulation in accordance with the methods of the invention, including, for example, separation, analysis, extraction , concentration or profiling.

In the context of the present invention, the terms assay or assay are used interchangeably and refer to any of various methods for separating, detecting, isolating, purifying, solubilizing, detecting and/or characterizing molecules of interest (eg, peptides). Examples include, but are not limited to, solid phase extraction, solid phase microextraction, electrophoresis, mass spectrometry such as ESI-MS (electrospray ionization mass spectrometry), SPE HILIC (hydrophilic interaction liquid chromatography with solid phase extraction), or MALDI-MS (mass - matrix-assisted laser desorption/ionization spectrometry), liquid chromatography, e.g. high performance, e.g. reverse phase, normal phase or size exclusion, ion vapor liquid chromatography, liquid-liquid extraction, e.g. accelerated liquid extraction, supercritical fluid extraction, microwave extraction, membrane extraction, Soxhlet extraction, precipitation, clarification, electrochemical detection, staining, elemental analysis, Edmund degradation, nuclear magnetic resonance, infrared spectral analysis, flow injection analysis, capillary electrochromatography, UV detection and combinations thereof.

The term profiling in the context of the present invention refers to any of various analytical methods that are used in combination to determine the content, composition or characteristic ratio of proteins, such as a peptide, in a sample.

Contacting in the context of the present invention involves bringing together at least two substances in solution or solid phase.

Targeted mass spectrometry in the context of the present invention is a mass spectrometry method that uses multiple tandem mass spectrometry steps (MC _n with n=2 or 3) for ions of a certain mass (m/z) at a certain time. The m/z and time values are defined in the inclusion list derived from the previous analysis.

Tandem mass spectrometry, also known as MS/MS or MS2, is an instrumental analysis technique in which two or more mass spectrometers are coupled together using an additional reaction step to increase their ability to analyze chemical samples. Typically, tandem MS is used for the analysis of biomolecules such as proteins and peptides. The molecules of a single sample are ionized, and the first spectrometer (denoted MS1) separates these ions by their mass-to-charge ratio (often referred to as m/z or m/Q). Ions with a specific m/z ratio coming from MS1 are selected and then cleaved into smaller fragment ions, for example, by collision-induced dissociation, ion-molecular reaction, or photodissociation. These fragments are then introduced into a second mass spectrometer (MS2), which in turn separates the fragments by their m/z ratio and detects them. The fragmentation step allows ions that have very similar m/z ratios to be identified and separated in conventional mass spectrometers.

General description.

Peptide mapping coupled with liquid chromatography-tandem mass spectrometry (LC-MS) has become a key analytical method for quantifying quality attributes (e.g., post-translational modifications (PTMs)) of monoclonal antibodies during drug development. However, the traditional label-free approach to relative quantification of PTMs requires a large amount of operating time.

- 8 047087 instrument for collecting LC-MS data from individual digested samples, which limits the effectiveness of the peptide mapping method, especially given the ever-increasing need for protein characterization.

Thus, there is a need for methods for characterizing proteins with increased efficiency. The disclosed invention satisfies this need.

The present invention discloses a novel approach based on tandem mass tag (TMT) coupled with targeted mass spectrometry for multiplexed site-specific quantitation of PTM proteins, including monoclonal antibodies. This new method builds on the studies reported herein, in which the inventors unexpectedly discovered that this approach allows simultaneous quantification of quality metrics (eg, PTMs) for multiple samples in a single LC-MS run. Specifically, using this method, multiple samples of digested antibodies are chemically labeled with variants of tandem mass tags, pooled in equal volume, and then analyzed using targeted mass spectrometry. Differentially labeled peptides are indistinguishable in the full MS spectrum of intact peptides due to the identical molecular structure and mass of each variant, but each labeled peptide variant produces a unique reporter ion in the MS/MS spectrum when fragmented within the mass spectrometer, thus allowing the peptide to be distinguished in different samples and displaying the peptide content of the corresponding sample. The relative abundance of PTMs in each sample is quantified from the selected peak areas of the resulting reporter ion generated from the corresponding differentially labeled native and modified peptides in the target MS/MS spectra, allowing the simultaneous quantification of multiple quality metrics (e.g., PTMs) for multiple samples, e.g. , up to 16-plex detection, driven by the current ability to multiplex the commercially available TMT reagent in a single LC-MS run, resulting in significant reductions in data acquisition time and run-to-run variation in PTM quantitation results.

In some embodiments, the method includes producing TMT peptides. In some embodiments, sample preparation includes contacting the sample with a denaturing and reducing solution under conditions that ensure denaturation and reduction of the sample; bringing the denatured and reduced sample into contact with the alkylating solution under conditions that ensure alkylation of the sample; bringing the alkylated sample into contact with a digestion solution under conditions that permit digestion of the sample and TMT labeling; and bringing the digested sample into contact with the quenching solution under conditions that stop the specimen from digesting. The resulting TMT peptides can then be analyzed, for example, using LC-MS.

In some embodiments, bringing the sample into contact with the digestion solution under conditions that allow digestion involves following a standard digestion protocol, except that it is carried out in a non-Tris buffer, such as sodium phosphate buffer (PBS). , pH from 7.5 to 8. In some embodiments, the pH of the PBS solution is adjusted before adding the TMT reagent to the digestion solution. Tandem mass tag (TMT) kits are reagents that readily react with peptides (N-terminus and lysine, any basic NH2); have equivalent molecular weight (same mass of MC1); and generate different MS2 reporter fragment ions due to the distribution of heavy atoms around each tag. Exemplary TMTs are shown in FIG. 10 and are commercially available from Thermo Fisher Scientific (Waltham, MA, USA). Although commercially available TMTs are known to react only with NH ₂ groups (N-terminus, lysine), the present inventors have discovered that TMTs can also react significantly with OH groups (tyrosine, threonine, serine). This reactivity was challenging because it complicated integration by cleaving each peptide into multiple forms, reducing the signal of each peak, and leading to excessive TMT labeling. The method disclosed in the present invention overcomes these limitations by adding a low molecular weight additive at a high concentration (for example, 100 mol low molecular weight additive/mol peptide), which allows TMT to still react rapidly with the CI ₂ groups of the peptides, but redirects excess TMT away from the peptides, which allows most TMT peptides to exist in one form and be more easily integrated. In some examples, the low molecular weight additive is BOC-Y-OH, p-cresol, hydroxyphenylacetic acid (HPAA), hydroxybenzoic acid (HBA), acetaminophen, and/or p-aminobenzoic acid (PABA). The chemical structures of these reagents are shown in Fig. 11. In some embodiments, the method includes adding a low molecular weight additive to the hydrolyzate sample before adding each TMT reagent. Each TMT reagent, such as commercially available TMT reagents from Thermo Fisher Scientific, is dissolved in ACN, added to each protein sample, and allowed to incubate, e.g., at room temperature for 1 hour. All reactions are quenched, e.g., quenched to pH less than 4 before pooled into one sample and then evaluated using LC-MS. In some embodiments, the method includes preparing a sample

- 9 047087 for sample injection for LC and its evaluation by performing targeted MS2.

The method disclosed in the present invention can be used with peptide mapping under reducing (% PTM) and non-reducing (% trisulfide bonds) conditions. In various embodiments, for peptide mapping under non-reducing conditions, an overlabeling inhibitor such as PABA is not required because non-reducing peptides are not subject to over-labeling.

In some embodiments, the sample contains peptides. For example, the sample includes peptides with PTMs. In some embodiments, the peptides are glycopeptides, such as glycopeptides derived from a monoclonal antibody. In some embodiments, the monoclonal antibody is of the IgG1, IgG2, IgG3, IgG4, or mixed isotype.

In some embodiments, the sample is a monoclonal antibody and the digestion solution contains one or more proteases, such as trypsin. In some examples, the method is used to characterize/analyze glycopeptides, such as glycopeptides derived from a monoclonal antibody, such as an antibody that has been digested by one or more proteases. For example, the methods can be used to characterize the glycosylation of proteins, such as monoclonal antibody (mAb) therapeutics. In certain embodiments, samples at any intermediate stage may be concentrated, diluted, desalted, or the like.

In various embodiments, the separation column is a liquid chromatography (LC) separation column. Liquid chromatography, including HPLC, can be used to analyze peptides, including monoclonal antibodies. Various types of liquid chromatography can be used to study these structures, including anion exchange chromatography, reverse phase HPLC, size exclusion chromatography, high performance anion exchange chromatography and normal phase (NP) chromatography, including Nf-VeLC (see, for example, Alpert et al ., J. Chromatogr. A, 676:191-202 (1994). Hydrophilic interaction chromatography (HILIC) is a variant of NF-HPLC that can be performed on partially aqueous mobile phases, allowing the separation of peptides, carbohydrates, nucleic acids and many proteins in normal phase. The elution order for HILIC is from least polar to most polar, as opposed to the order in reverse phase HPLC. HPLC can be performed, for example, on an HPLC system from Waters (eg, Waters 2695 Alliance HPLC system), Agilent, Perkin Elmer, Gilson, etc.

In some embodiments, the LC-MS/MS analysis is performed using an ACQUITY UPLC peptide BEH C18 column. The column temperature can be maintained at a constant level throughout the chromatography run, for example, by using a commercial column heater. In some embodiments, the column is maintained at a temperature of from about 18°C to about 70°C, for example, from about 30°C to about 60°C, from about 40°C to about 50°C, for example, at about 20°C , approximately 25°C, approximately 30°C, approximately 35°C, approximately 40°C, approximately 45°C, approximately 50°C, approximately 55°C, approximately 60°C, approximately 65°C or approximately 70°C . In some embodiments, the column temperature is approximately 40°C. In some embodiments, the analysis time may be from about 15 minutes to about 240 minutes, such as from about 20 minutes to about 70 minutes, from about 30 minutes to about 60 minutes, from about 40 minutes to about 90 minutes, from about 50 minutes to about 100 minutes, about 60 minutes to about 120 minutes, about 50 minutes to about 80 minutes. After LC, the eluent is then subjected to MS/MS analysis.

In some embodiments, for example, for targeted or untargeted LC-MS/MS analysis, an aliquot of each TMT-labeled sample is separated on an ACQUITY UPLC peptide BEH C18 column. The eluent is then electrosprayed and analyzed using a Q-Exactive Plus hybrid HCD mass spectrometer used for peptide fragmentation in MS/MS experiments. A target list containing trigger m/z, z, target retention time window, and collision energy of TMT-labeled native and modified peptides is loaded into the inclusion list to drive MS/MS analysis of precursors detected in MS1 survey scans. The peptide is then determined and the PTM is identified. In some embodiments, the percentage of each PTM is calculated using the peak area in the selected ion chromatogram (EIC) of the modified peptide relative to the sum of the peak areas of the modified and native peptides.

Examples

The following examples are provided to provide those skilled in the art with complete disclosure and description of how to make and use the methods of the invention and are not intended to limit the scope of what the inventors believe they have created.

- 10 047087 invention. Efforts have been made to ensure accuracy with respect to the numerical parameters used (e.g. quantities, temperature, etc.), but the possibility of some experimental errors and deviations should be considered. Unless otherwise noted, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, room temperature is approximately 25°C, and pressure is at or near atmospheric pressure.

Example 1. Materials and reagents.

All human IgG1 and IgG4 monoclonal antibodies (mAb-A, mAb-B, mAb-C, mAb-D, and mAb-E) in this study were manufactured by Regeneron (Tarrytown, NY). Samples for assessing the stability of mAb-A were created by incubating a control sample under various stress conditions and labeled as mAb-A-S0 (control), mAb-A-S1 (T=45°C, t=28 days), mAb-A -S2 (T=25°C, t=6 months) and mAb-A-S3 (T=5°C, t=24 months), respectively. The mAb-B comparability samples were manufactured at four different manufacturing sites and are designated mAb-B-P1, mAbB-P2, mAb-B-P3, and mAb-B-P4, respectively. Trisulfide mAb-E standards with varying levels of trisulfide bonds were prepared by mixing the H ₂ S-exposed sample and the reference standard sample in various ratios, and are designated mAb-E-TS0 (100:0), mAb-E-TS1 ( 75:25), mAb-E-TS2 (50:50), mAb-E-TS3 (25:75) and mAb-E-TS4 (0:100), respectively. Peptide H-glycosidase F Rapid (PNGαse F Rapid) with 5 times the amount of PNGαse F Rapid buffer was purchased from New England Biolabs Inc. (Ipswich, Massachusetts). Glacial acetic acid (purity >99%), iodoacetamide, monobasic sodium phosphate, dibasic sodium phosphate, 4-aminobenzoic acid (PABA), 4-hydroxybenzoic acid (HBA), 4-hydroxyphenylacetic acid (HPAA) and N-(tert-butoxycarbonyl )-Tyrosine (Boc-Y) were purchased from Sigma (St. Louis, MO, USA). Sequencing grade trypsin and Asp-N were purchased from Promega (Madison, WI). Isobaric reagents tandem mass tags (TMT), tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl), trifluoroacetic acid (TFA, sequencing grade), and acetonitrile (Optima, LC/MS grade) were purchased from Thermo Fisher Scientific ( Waltham, Massachusetts). 1.0 M Tris-HCl UltraPure (pH 7.5) was purchased from Invitrogen Life Technologies (Carlsbad, CA), and high purity water was purchased from Milli-Q system (Bedford, MA).

Peptide mapping of mAbs under reducing conditions. Peptide mapping under reducing conditions was performed to quantify levels of PTMs (e.g., N-terminal Gln/Glu cyclization, Met oxidation, Asn deamidation, Asp cyclization/isomerization, C-terminal Lys clipping, Lys glycation, and Fc glycosylation) for samples to determine stability mAb-A and samples to determine the comparability of mAb-B. 500 μg of each sample was buffer exchanged to 5 mM acetic acid to remove TMT-reactive compounds (e.g., histidine, Tris-HCl) and then denatured and reconstituted in 5 mM acetic acid in the presence of 5 mM TCEP-HCl at 80°C in for 10 minutes. After denaturation and reduction, each sample was diluted in 100 mM PBS (pH 8.0) containing 8 M urea and alkylated with iodoacetamide for 30 min in the dark at room temperature. After alkylation, each sample was further diluted with 50 mM PBS (pH 8.0) to reduce the urea concentration to less than 1 M. For trypsin digestion, each diluted sample was incubated with trypsin at an enzyme to substrate ratio of 1:20 (w/w) at 37°C for 4 h. For Asp-N digestion, each diluted sample was incubated with Asp-N at an enzyme to substrate ratio of 1:50 (w/w) at 37°C for 4 h. To obtain a deglycosylated sample for glycosylation quantification, an aliquot of each trypsin-digested sample was further incubated with PNGase F (1 mU/μg protein) for an additional 1 h at 37°C to remove N-linked glycans at the peptide level. After digestion with trypsin, Asp-N, or PNGase F, each digested sample was divided into two equal aliquots. One aliquot was quenched with 10% TFA to stop trypsin digestion and subjected to online LC-MS analysis, while the other aliquot was retained for the subsequent TMT labeling procedure.

Tryptic peptide mapping of unreduced mAbs. Peptide mapping under non-reducing conditions was performed to quantify the levels of trisulfide bonds for trisulfide standards mAb-C, mAb-D and mAb-E. 500 μg of each sample was buffer exchanged with 100 mM PBS (pH 7.5) to remove TMT-reactive compounds (e.g., histidine, Tris-HCl) and then denatured in 8 M urea containing 1.0 mM iodoacetamide at 100 mM PBS, pH 7.5 at 50°C for 10 min in the dark. After denaturation, 100 mM PBS (pH 7.5) was added to dilute the urea concentration 5-fold. Each sample was then digested with trypsin at an enzyme to substrate ratio of 1:20 (w/w) at 37°C for 4 hours. After trypsin digestion, each digested sample was divided into two equal aliquots. One aliquot was quenched with 10% TFA to stop trypsin digestion and subjected to online LC-MS analysis, while the other aliquot was retained for the subsequent TMT labeling procedure.

TMT labeling of reduced and unreduced mAb hydrolysates. TMT6-plex reagents were dissolved in 41 μL of acetonitrile according to the manufacturer's protocol (Thermo Scientific).

- 11 047087

An aliquot of each digested sample (100 μg) for peptide mapping experiments under reducing and nonreducing conditions was incubated with 41 μl of TMT label dissolved in acetonitrile in the presence of an overlabeling control reagent (PABA, HBA, HPAA, or Boc-Y) (100 mol reagent /mol protein) for 1 h at ambient labeling temperature. The labeling reaction was stopped by adding 10% TFA. TMT-labeled digests of mAb-A stability samples, mAb-B comparability samples, and mAb-E trisulfide standards were pooled in equal amounts, respectively, and then subjected to LC-MS/MS analysis.

LC-MS/MS analysis. For untargeted LC-MS/MS analysis, an aliquot (approximately 8 μg) of each digest was injected onto an ACQUITY UPLC peptide BEH C18 column (Waters, 2.1 x 150 mm, 1.7 μm particle size, 130 A pore size). Peptides were eluted with a linear gradient that increased from 0.1% mobile phase B to 35% mobile phase B over 75 min (mobile phase A: 0.05% TFA in water; mobile phase B: 0.045% TFA in acetonitrile) at speed flow 0.25 ml/min at column temperature 40°C. The eluent was then electrosprayed and analyzed using a Q-Exactive Plus hybrid high-energy collision dissociation (HCD) mass spectrometer used for peptide fragmentation in MS/MS experiments. The device operated in positive ion mode and was configured with the following data acquisition parameters: resolution MS1=70000; AGC (Automatic Gain Control) target MS1=1x10 ⁶ ; maximum input time MC1=50 ms; scanning range MS1=400-2000 m/z; resolution MS2=17500; target AGC MS2=1x10 ⁵ ; maximum input time=100 ms; TopN=5; isolation window=4.0 m/z; normalized collision energy=27; vacancy rate=10%; peptide match=preferred; exclude isotopes=on; dynamic exception=10s.

For targeted LC-MS/MS analysis, an aliquot of each TMT-labeled sample (approximately 8 μg) was separated on an ACQUITY UPLC peptide BEH C18 column (Waters, 2.1 x 150 mm, 1.7 μm particle size, 130 A pore size) with a longer linear gradient that increased from 0.1% mobile phase B to 40% mobile phase B over 150 min (mobile phase A: 0.05% TFA in water; mobile phase B: 0.045% TFA in acetonitrile) at speed flow 0.25 ml/min at column temperature 40°C. The eluent was then electrosprayed and analyzed using a Q-Exactive Plus hybrid HCD mass spectrometer used for peptide fragmentation in MS/MS experiments. A target list containing trigger m/z, z, target retention time window, and collision energy of TMT-labeled native and modified peptides was loaded into the inclusion list to drive MS/MS analysis of precursors detected in MS1 survey scans. The device operated in positive ion mode and was configured with the following data acquisition parameters: resolution MS1=17500; target AGC MS1=1x10 ⁶ ; maximum input time MC1=50 ms; scanning range MS1=400-2000 m/z; resolution MS2=17500; target AGC MS2=1x10 ⁵ ; maximum input time=100 ms; fixed first mass=100; isolation window=2.0 m/z; normalized collision energy=27-100.

Data analysis. Peptide detection and PTM identification were performed using Byonic (Protein Metrics Inc., San Carlos, CA; see Bern et al., Curr. Protoc. Bioinformatics., 2012, 40, 13.30.1-12.20.14) and verified manually. In a label-free approach for relative quantification of site-specific PTMs, selected ion chromatograms in MS1 spectra were generated based on the m/z of the first isotopic peak of both the native peptide and the modified peptide, and the areas of the selected peaks were integrated using skyline-daily (MacCoss Lab , University of Washington, Washington; see MacLean et al., Bioinformatics., 2010, 26, 966-968).

The percentage of each PTM was calculated using the peak area in the selected ion chromatogram (EIC) of the modified peptide relative to the sum of the peak areas of the modified and native peptides. In a targeted MS/MS approach for relative quantification of site-specific PTMs, selected ion chromatograms in MS/MS spectra were generated based on the reporter ion m/z of both the native peptide and the modified peptide, and the areas of the selected peaks were integrated using skyline-daily (MacCoss Lab, University of Washington, Washington). The percentage of each PTM was calculated using the EIC peak area of the reporter ion from the modified peptide relative to the sum of the reporter ion peak areas from the modified and native peptides.

Example 2: HCD Collision Energy Optimization for PTM Quantification.

TMT peptides are labeled with isobaric amine-reactive tags that generate quantitative information upon collisional activation. TMT reporter ions are formed by cleavage of the amide bond due to collision energy to form a cluster of ions between 126 and 131 m/z in the low mass region of tandem mass spectra (Thompson et al., Anal. Chem., 2003, 75, 1895-1904 ). In the TMT-based approach for PTM quantification, the reporter ion generated in the target MS/MS spectra of the native peptide and the modified peptide is used to calculate the percentage of PTM. TMT reporter ion content,

- 12 047087 generated in the MS/MS spectra correlates with the normalized collision energy (NCE) in the HCD cell applied to the TMT-labeled peptide. The effect of NCE(35-70) on the reporter ion content of TMT-labeled peptides in mAb-A was examined as shown in FIG. 1A. Reporter ion intensities were maximum when NCE was increased to 50 or 55, and then decreased as NCE was further increased to 70. However, an NCE of 35 tends to provide extensive fragmentation information for peptide identification, and a higher NCE provides less information about fragmentation as NCE increases. For example, when NCE was increased to 70, only a small amount of fragmentation information was retained in the MS/MS spectra.

Quantification of different types of PTMs in mAb-A using reporter ion generated in target MS/MS spectra of native and modified peptides at different NCEs, investigated for Met oxidation, Asn deamidation, Asp cyclization/isomerization, C-terminal Lys clipping and glycosylation Fc, and compared with PTM quantification using a traditional MS1-based approach. It was noted that there was no significant difference in the percentages of different PTMs at NCE 35–70, and the percentages of PTMs were comparable to those determined using the traditional approach where the first isotopic peak of native and modified precursor peptides in MS1 survey scans was used to calculate the percentage ( see Figure 1B), demonstrating the feasibility of a targeted MS/MS approach for PTM quantification. The results also show that the changing trend of reporter ion abundance at different NCEs is similar for native and modified peptides. To maximize the sensitivity of quantifying rare PTMs while maintaining satisfactory fragmentation information for peptide sequence confirmation, NCE 55 was selected to quantify Met oxidation, Asn deamidation, Asp cyclization/isomerization, C-terminal Lys clipping, and Fc glycosylation.

Cyclization of N-terminal Gln or Glu in the light or heavy chain to form pyroglutamate (PyroE or PyroQ) is a major type of N-terminal modification (Liu et al., mAbs, 2014, 6, 1145-1154). However, this modification inhibits the reaction of the TMT labeling reagent with the N-terminal amino group by blocking the N-terminus of the peptide, limiting the ability of this multiplexing approach to quantify this modification. To overcome this limitation, N-terminal peptides from mAb-A containing Gln or Glu at the N-terminus as well as a lysine residue at the C-terminus were examined. In the native peptide, both the N-terminal amino group and the ε-amino group of the lysine side chain can be labeled with TMT tags, however, in the modified peptide, only the ε-amino group of the lysine side chain can be labeled due to the blocked N-terminal amino group. To evaluate the feasibility of using a reporter ion generated from native and modified peptides for this PTM quantification, the percentage of pyroglutamate at different NCEs (27–100) was calculated and compared with the value obtained from the traditional approach. As shown in FIG. 2B, this approach provided the most comparable result at NCE 35 compared to the traditional approach (NCE 35 also for PyroQ). Reduced percentages were observed at higher and lower NCEs, and at NCE 27 and NCE 55, the percentage of pyroglutamate was only ∼50% of the value obtained with the traditional approach. Therefore, NCE 35 was selected for the quantification of pyroglutamate.

Quantification of Lys glycation in mAb-B and mAb-C (at different levels) using a reporter ion was also investigated. Glycation is a non-enzymatic process that can lead to the modification of primary amines by reducing sugars (eg, glucose, fructose). Lysine residues are particularly susceptible to glycation in the protein with an increase in side chain mass of 162.05 Da and create charge heterogeneity by changing the primary amine side chain from basic to neutral (Liu et al., mAbs, 2014, 6, 1145-1154).

Because Lys glycation inhibits trypsin cleavage of the peptide bond at glycated Lys sites, the result is a peptide with glycated Lys residues of a different length compared to a peptide having native Lys residues. Therefore, peptide mapping with Asp-N cleavage was performed to analyze the glycation products at the Lys site. Like the N-terminal Gln/Glu pyroglutamate, glycation can alter the physical properties of the lysine residue, rendering the lysine residue unreactive with the TMT reagent and therefore leading to different amounts of TMT marks on the native peptide and the glycated peptide. Investigation of the effect of different NCEs (35–100) on the quantification of this PTM showed that different percentages were also obtained at different NCEs, with higher NCE yielding higher glycation percentages. A percentage comparable to quantitation with the traditional approach can be achieved by increasing the NCE to 90-100 (Fig. 2). The observed strong dependence on NCE for quantification of pyroglutamate and glycation PTMs may be due to inconsistent trends in reporter ion abundance with different NCEs between the native peptide and the modified peptide. For example, the reporter ion content was maximum at different NCEs of 55 and 70 for

- 13 047087 native peptide and glycosylated peptide, respectively. Therefore, to achieve comparable quantification to the traditional approach, NCE 90 was selected to quantify Lys glycation. It should be noted that due to the lack of sufficient fragmentation information, a preliminary analysis with a lower NCE value (e.g., 35) can be used to confirm the peptide sequence and lysine modification.

Trisulfide is also a common modification that has been found in all recombinant IgG subclasses. Trisulfide bonds are often present between light and heavy chain peptides in monoclonal antibodies (Gu et al., 2010, 400, 89-98). This trisulfide modification occurs by incorporating a sulfur atom into the disulfide bond between the heavy and light chains (Cys-SS-Cys +H ₂ S+[O]^Cys-SSS-Cys+H ₂ O), which leads to an increase in mass by 31.97 Da. The presence of trisulfide bonds has been reported to have no effect on thermal stability as well as antigen binding and potency. However, levels of trisulfide bonds can vary from batch to batch and from process to process, likely due to variations in trace H2S levels in production bioreactors, so levels of trisulfide bonds are routinely monitored in monoclonal antibody samples during cell culture process development. To develop a multiplex method for the quantification of trisulfide bonds, quantitative results at different NCEs were examined for the monoclonal antibodies mAb-C (IgG1) and mAb-D (IgG4), respectively. Interestingly, the percentage of trisulfide bonds showed a clear linear relationship with NCE for both Mab-C and mAb-D, as shown in Fig. 3A and 3B. When the NCE was increased to 55, the % trisulfide bonds determined using this approach were comparable to the conventional approach for both IgG1 and IgG4 antibodies, and therefore it was selected as the optimized NCE for trisulfide bond quantification.

Example 3: Low molecular weight additives to prevent over-labeling with TMT reagent.

It has been reported that in addition to the N-terminal amino group and ε-amino group of the lysine side chain, TMT labeling of the lysine side chain can also occur at tyrosine (Tyr), threonine (Thr) and serine (Ser) as a result of an off-target reaction with the hydroxyl group in these amino acids (Zecha et al., Mol. Cell. Proteomics., 2019, 18, 1468-1478). This excess TMT labeling may reduce the sensitivity of PTM quantification in this approach by separating the native peptide and modified peptide responses into multiple fragments labeled with different amounts of TMT tag. For example, the inventors observed this when quantifying a PTM from a tryptic peptide (SEQ ID NO: 3, WQQGNVFSCSVMHEALHNHYTQK) located in the conserved CH3 domain of the IgG heavy chain containing a methionine residue susceptible to oxidation. Following TMT tagging, a mixture of fragments with different TMT tags (up to 6) was observed, and multiple isoforms were observed for compounds containing 3, 4, and 5 TMT tags. In the table presented in Fig. 9, the relative abundance of WQQG peptide fragments (residues 1-4 of SEQ ID NO: 3) with different TMT tags and with different isoforms is summarized. The highly heterogeneous TMT labeling of this peptide distributes the total abundance into as many as 15 different forms with relative abundances ranging from 0.2 to 22.0%, which poses a major challenge to quantify Met oxidation in this peptide, especially when the level is low, using the approach , based on target MS/MS.

To inhibit TMT overlabeling, various ratios of TMT to protein (w/w) ranging from 8:1 to 1:1 were initially examined. Reducing the amount of TMT reagent can minimize overlabeling, but may also increase underlabeling of the peptide. For example, the efficiency of labeling target peptides can be reduced from more than 90 to ~35% when changing the ratio from 8:1 to 1:1. Alternatively, various hydroxyl-containing small molecules (PABA, HBA, HPAA, and Boc-Y) introduced into digests during TMT labeling were evaluated as competing reagents for these nontarget amino acid residues that react with excess TMT reagent and inhibit overlabeling. As shown in FIG. 9, the addition of these small molecules did inhibit the overlabeling of TMT peptides to a certain extent. For peptide WQQG (residues 1-4 SEQ ID NO: 3), although fragments containing 3 TMT tags are still an overlabeled product, the multiple isoforms of the 3 TMT-tagged peptide converged into a single dominant form, accounting for more than 50% of the total the content of all fragments related to this peptide. The formation of fragments containing 5 and 6 TMT tags was significantly suppressed; instead, there was an increase in TMT 2 and 3 labeling. A similar inhibitory effect could be observed for another TTPP peptide: addition of small molecules suppressed the formation of 3 TMT-labeled product from 26 to <7% and increased the expected 2 TMT-labeled product to ∼94%. Although all four small molecules demonstrated the ability to inhibit TMT overlabeling, PABA was selected as the most suitable competing reagent given its higher hydrophilicity and lower molecular weight compared to other small molecules, which is less likely to interfere with target peptides and introduce background signal mass- 14 047087 spectrometry.

Example 4: Reproducibility and sensitivity of multiplex PTM quantitation using TMT.

To evaluate the reproducibility and sensitivity of this targeted MS/MS approach for PTM quantification, six aliquots were taken from each tryptic digest of a mAb-A sample from three preparations (three determinations) with a known ratio of 1:1:1:1 :1:1 and 1:2:4:8:16:32 and then labeled with 6-plex TMT tags. Three pooled samples of each ratio were analyzed in duplicate (two parallel runs) using LC-MS/MS. Four representative PTMs (Lys glycosylation, Asn deamidation, Asp cyclization/isomerization, and Met oxidation) with levels ranging from 0.6 to 3.3% (percentages calculated based on the traditional approach) were selected to evaluate the quantification reproducibility and sensitivity of this approach . The integrated peak areas of the selected reporter ion chromatograms of native and modified peptides were used to calculate the percentage of four PTMs in each TMT channel. In fig. 4A shows the evaluation of the relative quantitation of four PTMs in mAb-E in six TMT channels at a ratio of 1:1:1:1:1:1. In fig. 4B shows an assessment of the reproducibility of the relative quantitation of four PTMs in mAb-E in six TMT channels at a ratio of 1:1:1:1:1:1.

In fig. Figure 5 shows the average percentages and RSD of four PTMs quantified in six TMT channels at a ratio of 1:2:4:8:16:32, corresponding to sample loading amounts of 0.15, 0.30, 0.60, 1.2, 2.4 and 4.8 mcg, respectively. The average percentages and relative standard deviations (RSD) of the four PTMs quantified at different loading amounts ranged from 0.6-0.9 to 3.3-29.0% for Lys glycosylation, from 1.6-1.9 to 3.6-7.2% for Asn deamidation, from 2.4-2.6 to 3.4-7.3% for Asp cyclization/isomerization and from 3.3-3.5 to 3.6-9. 4% for Met oxidation. Overall, the average percentages of these PTMs quantified at different loading amounts were comparable to those calculated using the traditional approach (loading amount approximately 5.0 μg), even for rare PTMs such as Lys glycosylation (approximately 0. 6%) examined in this study. Although, when the loading amount was 0.15 μg, a large deviation was observed with an RSD of 29.0% (>15%) to quantify this low level of Lys glycation, which could be due to the very low content of the reporter ion generated from the modified peptide at NCE 90; All RSDs of PTM quantitation with higher levels or higher loading amounts were within 15%, demonstrating the high sensitivity of this approach for multiplex PTM quantitation at levels as low as 1.0% when the sample loading amount is only approximately 0 .15 mcg.

Example 5: Multiplex PTM Quantitation Using Sample TMT to Assess mAb-A Stability.

Samples for mAb-A stability assessment were analyzed using this targeted MS/MS approach. A total of four samples, including a control sample, were digested with trypsin under reducing conditions and then labeled with 4-plex TMT reagents (channels 126, 127, 128, 129, 130, and 131). The pooled sample was analyzed using LC-MS/MS to quantify PTM. Individual digested samples not labeled with TMT were also analyzed using a traditional approach for PTM quantification. In fig. Figure 6 summarizes the quantitative results of multiplex PTM determination for six samples using this approach, as well as the quantification results using the traditional approach for comparison. Comparable results for PTM quantification were observed with both approaches. An effect of temperature was observed on the levels of oxidation at Met258 and Met434, deamidation at Asn390, and cyclization/isomerization at Asp286, with samples incubated at 45 and 25°C exhibiting higher levels than the control sample and the sample incubated at 5°C. In addition, the sample incubated at 45°C for 28 days exhibited slightly higher levels of these PTMs than the sample incubated at 25°C for 6 months. There was no significant difference in levels when comparing the control sample with the sample incubated at 5°C for 24 months. For oxidation, deamidation, and cyclization/isomerization at other sites, their levels were comparable in all four samples, regardless of the exposure conditions tested in this study. This case study demonstrates the potential of this approach to quantify differences in PTM levels in antibody samples.

Example 6: Multiplex PTM quantitation using TMT for samples to assess mAb-B comparability.

This example illustrates the quantification of PTM levels in samples to assess mAb-F comparability to demonstrate comparable quality of monoclonal antibodies produced at different processing sites. A total of four samples for evaluation

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Claims (17)

comparators were digested with trypsin under reducing conditions and then labeled with 4-plex TMT reagents (channels 128, 129, 130, and 131). The pooled sample was analyzed using LC-MS/MS to quantify PTM. Individual digested samples not labeled with TMT were also analyzed using a traditional approach for PTM quantification. Quantitative PTM results for the four comparability samples obtained using both approaches are summarized in FIG. 7A and 7B. Two approaches were used to quantify the comparability of PTM percentages, and all PTMs, including Met oxidation, Asn deamidation, Asp cyclization and isomerization, and C-terminal lysine, showed comparable levels in four mAb-B samples produced at different processing sites. In fig. 7A shows PTMs with levels >2.5%, and FIG. 7B shows PTMs with levels <2.5%. Example 7: Multiplex PTM Quantitation Using TMT for Standard Samples to Evaluate Trisulfide Linkages of mAb-E. Trisulfide bond standards were obtained to construct a calibration curve to monitor trisulfide bond levels in process samples during cell culture process development. In this example, the authors used a targeted MS/MS approach to quantify the levels of trisulfide bonds in five standard samples to evaluate the trisulfide bonds of mAb-E, prepared by mixing an H2S-exposed sample with a reference standard sample in various 100 ratios: 0, 75:25, 50:50, 25:75 and 0:100 respectively. Five standard samples for trisulfide bond assessment were digested with trypsin under non-reducing conditions and then labeled with 5-plex TMT reagents (channels 126, 127, 128, 129, and 130). The pooled sample was analyzed using LC-MS/MS to quantify trisulfide bonds. Individual digested samples not labeled with TMT were also analyzed using the traditional approach for comparison. As shown in FIG. 8, the levels of trisulfide bonds in standard samples determined using this approach were 14.9, 11.1, 7.5, 3.2 and 0.1%, respectively. These results were in good agreement with values calculated from mixture ratios of exposed and reference standards and were also comparable to values obtained from the traditional approach, with corresponding trisulfide bond levels of 15.2, 11.7, 7.8 , 3.2 and 0.1%, respectively. The disclosed TMT-based multiplex approach is compatible with current peptide mapping workflows for PTM quantification with minimal changes to the sample preparation procedure, but significantly reduces data acquisition time in mass spectrometry, especially when analyzing a large set of samples. The present invention demonstrates that by tuning NCE from 35 to 90, it is possible to quantify different types of PTMs using reporter ion generated from native and modified peptides in target MS/MS spectra, achieving percentages comparable to those in the traditional approach. This approach has been shown to provide excellent reproducibility and sensitivity for PTM quantification with levels as low as 1.0% even with small sample loadings. The multiplexing functionality of this approach enhances the analytical capabilities of LC/MS-based biopharmaceutical protein characterization by quantifying the quality of multiple samples in a single LC/MS run, and also reduces the run-to-run variability in PTM quantification that can be encountered with individual analysis of samples using LC-MS with a traditional approach, which improves the quality of mass spectrometric data for accurate and reproducible quantification of PTMs. Overall, as demonstrated in the present invention by the example of the analysis of monoclonal antibody samples in various case studies, the developed approach provides more an effective method compared to the traditional approach to better meet the ever-increasing needs for characterization of monoclonal antibodies at different stages of drug development. The scope of the present invention is not limited to the specific embodiments described in the present invention. Indeed, various modifications of the present invention in addition to those described herein will be apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to be within the scope of the appended claims. CLAIM 1. A method for quantifying multiple quality attributes of multiple samples in a single mass spectrometry (MS) run, comprising bringing two or more samples into contact with a digestion solution under conditions sufficient to digest two or more samples, wherein each sample is digested separately and digested - 16 047087 pouring solution is a buffer solution that does not contain Tris buffer; contacting each of the two or more digested samples with a small molecule additive before contacting each of the two or more digested samples with a specific tandem mass tag (TMT) labeling reagent, wherein the small molecule additive is selected from the group consisting of BOC-Y-OH , p-cresol, hydroxyphenylacetic acid (HPAA), hydroxybenzoic acid (HBA), acetaminophen and p-aminobenzoic acid (PABA); contacting each of the two or more digested samples with a specific TMT labeling reagent under conditions sufficient to label peptides in each of the two or more digested samples using the specific TMT labeling reagent; quenching peptide labeling in each of the two or more digested samples; combining equal volumes of two or more labeled digest samples into a single pooled sample solution; and analyzing a solution of one pooled sample using targeted mass spectral analysis, thereby allowing multiple quality attributes of two or more samples to be quantified in a single MS run. 2. The method of claim 1, wherein the plurality of quality indicators include post-translational modification (PTM). 3. The method of claim 2, wherein the PTM comprises one or more of deamidation, oxidation, glycation, disulfide bond formation, N-terminal pyroglutamate formation, C-terminal lysine removal, and glycosylation. 4. The method according to claim 3, where the PTM includes glycosylation. 5. The method according to any one of claims 2 to 4, wherein the quantification of multiple quality indicators in a single MS run includes quantification of PTMs by quantifying the relative abundance of PTMs from the areas of selected peaks of the resulting reporter ion generated in the target mass spectra. 6. The method according to claim 1, where the low molecular weight additive is PABA. 7. Method according to any one of claims 1 to 6, wherein the peptides are glycopeptides. 8. The method of claim 7, wherein the glycopeptides are derived from a monoclonal antibody. 9. The method according to claim 8, where the monoclonal antibody is of the IgG1, IgG2, IgG3, IgG4 or mixed isotype. 10. Method according to any one of paragraphs. 1-9, where two or more samples represent from 2 to 11 samples. 11. The method according to any one of claims 1 to 10, further comprising obtaining two or more samples for analysis. 12. The method of any one of claims 1 to 11, further comprising preparing two or more samples for digestion before contacting the two or more samples with a digestion solution under conditions sufficient to digest the two or more samples. 13. The method of claim 12, wherein preparing the two or more samples prior to digestion comprises contacting each of the two or more samples with a denaturing and reducing solution under conditions that ensure denaturation and reduction of the sample; and contacting each of the two or more denatured and reduced samples with an alkylating solution under conditions that alkylate the sample. 14. The method according to any one of claims 1 to 13, wherein analyzing a solution of one pooled sample using targeted mass spectral analysis includes applying one pooled sample to a separation column and performing targeted mass spectral analysis of the eluted components of the sample. 15. The method of claim 14, wherein the separation column is a liquid chromatography column. 16. The method of claim 14 or 15, wherein performing targeted mass spectral analysis of eluted sample components comprises using electrospray ionization to generate charged ions from the eluted sample components and measuring the generated charged ions. 17. A method for quantifying post-translational modifications (PTMs) of multiple samples in a single mass spectrometry (MS) run, comprising bringing two or more samples into contact with a digestion solution under conditions sufficient to digest two or more samples, where each sample is digested separately and the digestion solution is a buffer solution containing no Tris buffer; contacting each of two or more digested samples with a low molecular weight additive, wherein the low molecular weight additive is selected from the group consisting of BOC-Y-OH, p-cresol, hydroxyphenylacetic acid (HPAA), hydroxybenzoic acid (HBA), acetaminophen and p-aminobenzoic acids (PABA); bringing each of two or more digested samples into contact with a specific tandem mass tag (TMT) labeling reagent under conditions sufficient to label the peptides in each of the two or more digested samples using the specific tag reagent -