WO2019126614A1 - Antibody peptide mapping and characterization using an erlic-ms/ms method - Google Patents

Abstract

Disclosed herein are methods of producing a peptide map of peptide or protein compositions, final peptide or protein drug products produced by the disclosed methods, and articles of manufacture comprising the disclosed final peptide or protein drug products. Methods of treating a subject having a disorder in which calcitonin gene related peptide (CGRP) activity is detrimental to the subject are also provided, as well as the use of ERLIC to identify antibody variants.

Description

ANTIBODY PEPTIDE MAPPING AND CHARACTERIZATION USING AN ERLIC-MS/MS METHOD

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to ET.S. Provisional Application No. 62/609,451, filed December 22, 2017, the disclosure of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] Disclosed herein are electrostatic repulsion hydrophilic interaction

chromatography (ERLIC) based methods of producing a peptide map and identifying antibody variants.

BACKGROUND

[0003] Therapeutic antibodies are playing an increased role in the pharmaceutical industry. Peptides and antibodies are much larger molecules as compared to traditional small molecule drugs and are subjected to a broad range of enzymatic and non-enzymatic reactions that can occur during expression, purification and storage, generating a number of peptide variants. The most common variants include N-terminal pyroglutamate (pE) formation, methionine (M) oxidation, asparagine (N) deamidation and aspartic (D) isomerization, C-terminal lysine (K) truncation and glycosylation. During the development of the antibody manufacturing process, sequence mutations and terminal peptide truncation can also occur. Since many variants are unavoidable, the final drug product does not have a single identity but instead contains a mixture of different variants. Some variants have been reported to cause reduced efficacy or side effects.

[0004] To evaluate the antibody manufacturing process and to characterize final antibody-based drug products, robust analytical methods, such as lot-release analytics, are required. Current techniques, however, have drawbacks including long running times, which could introduce artifacts into the sample and/or dilute the sample (thus reducing the sensitivity of the method), incompatibility with high-throughput studies, incompatible buffers for subsequent analysis of the peptides/variants, less than 100% sequence coverage, and difficulty in separating and characterizing asparagine deamidated and non-deamidated peptides.

SUMMARY

[0005] Disclosed herein are methods of producing a peptide map of a peptide or protein composition, comprising: a) digesting at least a portion of the peptide or protein composition to produce a peptide digest; b) separating the peptide digest on an electrostatic repulsion hydrophilic interaction chromatography (ERLIC) column by eluting the peptide digest from the ERLIC column in the presence of an elution gradient, wherein the elution gradient comprises a change in pH with time; and c) producing a peptide map of the peptide or protein composition from the separated peptide digest.

[0006] Also provided are final peptide or peptide drug products produced by the disclosed methods, as well as articles of manufacture comprising the disclosed final peptide or protein drug product. In some embodiments, the peptide or protein drug product is a biosimilar of fremanezumab.

[0007] Methods of treating a disorder in which calcitonin gene related peptide (CGRP) activity is detrimental to the subject are also provided. The methods comprise administering the disclosed final peptide or protein drug products to the subject.

[0008] Also disclosed is the use of ERLIC to identify antibody variants, wherein the variants include: asparagine deamidation variants; aspartic acid isomerization variants;

glycosylation variants; methionine oxidation variants; N-terminal pyroglutamate variants;

terminal peptide variants; C-terminal lysine variants; amino acid sequence mutations; or any other primary structural modifications in the antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The summary, as well as the following detailed description, is further understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosed methods, drug products, articles of manufacture, and uses, there are shown in the drawings exemplary embodiments of the methods, drug products, articles of manufacture, and uses; however, the methods, drug products, articles of manufacture, and uses are not limited to the specific embodiments disclosed. In the drawings:

[0010] FIG. 1 shows a hypothetical mechanism of tryptic peptide separation on

ERLIC.

[0011] FIG. 2A, FIG. 2B, and FIG. 2C illustrate the salt concentration gradient (FIG. 2A), pH gradient (FIG. 2B) and acetonitrile composition gradient (FIG. 2C) for gradient 1 and gradient 2, as discussed herein.

[0012] FIG. 3A and FIG. 3B illustrate representative base peak ion chromatograms of the denosumab tryptic peptides on ERLIC -MS/MS using gradient 1 (FIG. 3 A) and gradient 2 (FIG. 3B) as discussed herein.

[0013] FIG. 4A represent a 2D presentation of ERLIC-MS/MS based peptide mapping of denosumab tryptic peptides using gradient 2 (as shown in FIG. 3B) and FIG. 4B illustrates a relationship analysis of peptide retention time and pi values.

[0014] FIG. 5 illustrates extraction ion chromatograms of small tryptic peptides on method Gradient 2.

[0015] FIG. 6A, FIG. 6B, FIG. 6C, and FIG. 6D represent chromatograms and mass spectra of antibody peptides and variants. FIG. 6A illustrates exemplary extracted ion chromatograms of an N-terminal peptide of denosumab heavy chain; FIG. 6B illustrates exemplary MS2 mass spectra of an N-terminal peptide of denosumab heavy chain; FIG. 6C illustrates exemplary extracted ion chromatograms of a C-terminal peptide of denosumab heavy chain; FIG. 6D illustrates exemplary MS2 mass spectra of a C-terminal peptide of denosumab heavy chain.

[0016] FIG. 7A and FIG. 7B illustrate representative chromatograms (FIG. 7A) and mass spectra (FIG. 7B) of methionine-containing peptides H17 and its oxidized form.

[0017] FIG. 8A, FIG. 8B, and FIG. 8C illustrate the separation and analysis of non- deamidated and deamidated peptides of a peptide having the sequence

“GFYPSDIAVEWESNGQPENNYK”. FIG. 8A illustrates separation chromatograms of peptide deamidated products using Gradient 1 (top panel) and 2 (bottom panel); FIG. 8B illustrates MS1 full mass of labelled peaks with two hydrogen adduct ions (z = 2); FIG. 8C illustrates MS2 fragment ion mass spectra of labelled peaks. [0018] FIG. 9 illustrates extracted ion chromatograms of major gly copeptides in the monoclonal antibody denosumab.

[0019] FIG. 10 illustrates exemplary points of interest on a peptide map.

DETAILED DESCRIPTION

[0020] The disclosed methods, drug products, articles of manufacture, and uses may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures, which form a part of this disclosure. It is to be understood that the disclosed methods, drug products, articles of manufacture, and uses are not limited to the specific methods, drug products, articles of manufacture, and uses described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed methods, drug products, articles of manufacture, and uses.

[0021] ETnless specifically stated otherwise, any description as to a possible mechanism or mode of action or reason for improvement is meant to be illustrative only, and the disclosed methods, drug products, articles of manufacture, and uses are not to be constrained by the correctness or incorrectness of any such suggested mechanism or mode of action or reason for improvement.

[0022] Throughout this text, the descriptions refer to final peptide or protein drug products (or final drug products) and methods of generating the final drug products. Where the disclosure describes or claims a feature or embodiment associated with a final drug product, such a feature or embodiment is equally applicable to the methods of generating the final drug product. Likewise, where the disclosure describes or claims a feature or embodiment associated with a method of generating a final drug product, such a feature or embodiment is equally applicable to the final drug product.

[0023] Where a range of numerical values is recited or established herein, the range includes the endpoints thereof and all the individual integers and fractions within the range, and also includes each of the narrower ranges therein formed by all the various possible

combinations of those endpoints and internal integers and fractions to form subgroups of the larger group of values within the stated range to the same extent as if each of those narrower ranges was explicitly recited. Where a range of numerical values is stated herein as being greater than a stated value, the range is nevertheless finite and is bounded on its upper end by a value that is operable within the context of the invention as described herein. Where a range of numerical values is stated herein as being less than a stated value, the range is nevertheless bounded on its lower end by a non-zero value. It is not intended that the scope of the invention be limited to the specific values recited when defining a range. All ranges are inclusive and combinable.

[0024] When values are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value forms another embodiment. Reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise.

[0025] It is to be appreciated that certain features of the disclosed methods, drug products, articles of manufacture, and uses which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment.

Conversely, various features of the disclosed methods, drug products, articles of manufacture, and uses that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination.

[0026] Various terms relating to aspects of disclosure are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein.

[0027] As used herein, the singular forms“a,”“an,” and“the” include plural referents unless expressly stated otherwise.

[0028] The term“comprising” is intended to include examples encompassed by the terms“consisting essentially of’ and“consisting of’; similarly, the term“consisting essentially of’ is intended to include examples encompassed by the term“consisting of.”

[0029] As used herein,“peptide or protein composition” refers to a composition comprising a peptide or protein (e.g. an antibody) that has been manufactured to a stage that is suitable for characterization. For example, a peptide or protein composition may be crude extract taken from a cell culture bioreactor (the production stage), or a composition that has been purified by a process downstream from cell culture production (a drug substance), or a composition that has been stabilized in a formulation or lyophilized in a powder (a drug product). [0030] “Final peptide drug product,”“final drug product,” and“drug product” are used interchangeably and refer to a composition of peptide or protein drug (e.g. an antibody) that has been purified, formulated and packaged to a state that is ready to be placed on the market and/or provided to health care professionals and/or administered to patients.

[0031] As used herein“% hydrophobicity” refers to the % volume of an organicsolvent in the buffer. In one embodiment, the buffer is an elution buffer.

[0032] The term“comparable” as used in relation to a peptide map and a reference peptide map refers to the level of identity between the two peptide maps including the number of peaks, the height of the peaks, the width of the peaks, and/or the area of the peaks.

“Comparable” includes 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55% and 50% identity between the peptide map and the reference map and includes one or more of the following criteria:

• No new and/or no missing peaks between the two peptide maps;

• A 0% - 5% difference between the two maps when analyzed by chromatograph subtraction (a method whereby the chromatograph from the peptide/protein is “subtracted” from the chromatograph from the reference peptide/protein. If the chromatographs are the same, the subtraction will result in a“flat line” or a 0% difference);

• A 0-25% difference between the area of the most robust peaks on the peptide maps (for example, 10-15 of the most robust peaks can be selected and the area of the peaks can be calculated and compared between the maps);

• A 0-25% difference between the % modifications between the two maps (for example, the % modifications in the peptide/protein can be determined by calculating the signal intensity of the unmodified peptide/protein and the signal intensity of the modified peptide/protein using mass spectrometry and calculating the % modification. The % modification can then be compared to the % modification of the reference peptide/protein); or

• A level of comparability that satisfies and meets standards set by the US regulatory authorities, namely the Food and Drug Administration (FDA), in the event that the FDA were to assess the level of comparability during an FDA review procedure (for the avoidance of doubt, an actual review by the FDA is not required by this definition nor is it part of the disclosed methods. The FDA guidelines for assessing comparability between a peptide map and a reference peptide map can be found in Guideline ICH Q6B and Quality Considerations in Demonstrating Biosimilarity of a Therapeutic Protein Product to a Reference Product, both of which are incorporated by reference).

[0033] The terms“bottom-up characterization” or“bottom-up peptide mapping” refers to an analysis of the peptide digest.

[0034] The term“top-down,” as used in reference to an analysis strategy, refers to an analysis of the complete (undigested) peptide or protein.

[0035] The following abbreviations are used throughout the disclosure: Electrostatic repulsion hydrophilic interaction chromatography (ERLIC), mass spectrometry (MS), matrix- assisted laser desorption ionization mass spectrometry (MALDI-MS), liquid chromatography- tandem mass spectrometry (LC-MS/MS), isoelectric point (pi), and pyroglutamate (pE).

[0036] Disclosed herein are methods of producing a peptide map of a peptide or protein composition. Peptide mapping is a method used in peptide identification and/or characterization, for example, in antibody characterization. Peptide mapping plays a pivotal role in protein/peptide drug characterization. In order to generate a peptide map, the peptide or protein composition must first be digested into its constituent peptides via a chemical or enzymatic reaction. The constituent peptides are analyzed to produce a map of the original peptide or protein. Robust separation and identification of the constituent peptides then provides insight into a peptide’s full sequence information. The peptide map can be considered as the molecule’s fingerprint, and so it is an essential tool to study the primary structure of, for example, an antibody in the

biopharmaceutical industry. Due to its complexity and inherent variability, peptide mapping is generally performed in a comparative manner; for example, biosimilars can be compared to a reference or control substance, such as the innovator biologic, in a side-by-side experiment. An in-depth analysis is then required to identify minor differences in the peptide’s primary structure. The modern biopharmaceutical and protein/peptide research laboratory is tasked with providing high quality analytical results, often in high-throughput, regulated environments. Some technologies currently employed for biopharmaceutical peptide mapping are subject to high levels of irreproducibility, poor sensitivity, and high levels of time-consuming manual work.

The disclosed methods address one or more of these problems. [0037] The disclosed methods of producing a peptide map of a peptide or protein composition, comprise: a) digesting at least a portion of the peptide or protein composition to produce a peptide digest; b) separating the peptide digest on an electrostatic repulsion hydrophilic interaction chromatography (ERLIC) column by eluting the peptide digest from the ERLIC column in the presence of an elution gradient, wherein the elution gradient comprises a change in pH with time; and c) producing a peptide map of the peptide or protein composition from the separated peptide digest.

[0038] The“digesting at least a portion of the peptide or protein composition” can be performed with a protease or reducing agent that digests the peptide or protein into small peptides collectively referred to as a peptide digest. Trypsin is a protease that specifically cleaves peptides/proteins creating peptides both in the preferred mass range for mass

spectrometry (MS) sequencing and with a basic residue at the carboxyl terminus of the peptide, producing information-rich, easily interpretable peptide fragmentation mass spectra. Other proteases that may be used to digest at least a portion of the peptide or protein composition include Lys-C, ficin, pepsin, papain, Glu-C, and chymotrypsin.

[0039] In some embodiments, the peptide or protein composition is an antibody and the peptide map produced by the disclosed methods is an antibody map. Antibody maps produced from the disclosed methods can be produced, for example, as part of an antibody characterization process. In some aspects, the antibodies can be full-length antibodies comprising a heavy chain and a light chain. Alternatively, the antibody can comprise derivatives or fragments or portions of antibodies that retain the antigen-binding specificity, and also preferably substantially retain the affinity, of the full-length parent antibody molecule. For example, derivatives may comprise a single variable region (either a heavy chain or light chain variable region). Other examples of suitable antibody derivatives and fragments include, without limitation, antibodies with polyepitopic specificity, diabodies, minibodies, Fab, F(ab’)2, Fd, Fc, and Fv molecules, single chain (Sc) antibodies, single chain Fv antibodies (scFv), individual antibody light chains, individual antibody heavy chains, fusions between antibody chains and other molecules, heavy chain monomers or dimers, light chain monomers or dimers, dimers consisting of one heavy and one light chain, and other multimers. Single chain Fv antibodies may be multi-valent. Antibody derivatives, fragments, and/or portions may be recombinantly produced and expressed by any cell type, prokaryotic or eukaryotic. In some embodiments, the antibody is denosumab or fremanezumab or a biosimilar thereof.

[0040] ERLIC is a combination of ion-exchange chromatography (IEX) and hydrophilic interaction chromatography (HILIC). The retention mechanism of ERLIC is proposed in FIG. 1. ERLIC use columns with unique retention mechanisms, having a

combination of electrostatic interactions and hydrophilic interactions. For example, an ERLIC column with positively charged stationary phase can repulse basic peptides in neutral pH back to the mobile phase. Meanwhile, the mobile phase contains a high percentage of organic solvents resulting in a water layer formed on the stationary phase, which retains the polar peptides through hydrophilic interaction even if those peptides have the same charge as the stationary phase. Hydrophilic interactions are independent of electrostatic effects (the attraction and repulsion of charged bodies). Therefore, with sufficient organic solvent in the mobile phase, hydrophilic interaction can dominate the chromatography and the retention of analytes on the column.

[0041] The peptide digest is separated on an ERLIC column by eluting the peptide digest from the ERLIC column in the presence of an elution gradient, wherein the elution gradient comprises a change in pH with time. The change in pH with time can be a result of an elution buffer running through the ERLIC column. In some embodiments, the elution buffer comprises a three solvent system to produce a triple gradient that varies in pH, salt concentration and hydrophobicity.

[0042] In some embodiments, the change in pH with time comprises a reduction in pH from a high pH to a low pH. In some aspects, the high pH is greater than pH 5 and the low pH is lower than or equal to pH 5. Suitable high pHs include between pH 6 and pH 10, between pH 7 and pH 9, between pH 7 and pH 8.5, or between pH 8 and pH 8.2. In some embodiments, the high pH is between pH 8 and pH 8.2. Suitable low pHs include between pH 1 and pH 5, between pH 1 and pH 4, between pH 1.5 and pH 3.5, or between pH 2.6 and pH 2.7. In some embodiments, the low pH is between pH 2.6 and pH 2.7.

[0043] The elution gradient can further comprise a change in salt concentration with time. In some embodiments, the change in salt concentration with time comprises an increase in the concentration of a first salt, which causes a reduction in the pH with time. The elution buffer containing the first salt can have a pH of less than pH 5, less than pH 4, or less than pH 3. In some embodiments, the elution buffer containing the first salt has a pH of pH 2.5. Exemplary first salts include ammonium salts. In some aspects, the ammonium salt is ammonium formate. In some embodiments, the change in salt concentration with time comprises a decrease in the concentration of a second salt, which causes a reduction in the pH with time. The elution buffer containing the second salt can have a pH of greater than pH 4, greater than pH 5, or greater than pH 6. In some embodiments, the elution buffer containing the second salt has a pH of pH 6.8. Exemplary second salts include ammonium salts. In some embodiments, the ammonium salt is ammonium acetate. The use of ammonium salt buffer maximizes the compatibility of ERLIC with mass spectrometry (MS), which enables the subsequent characterization of peptides within an antibody tryptic digest. As used herein,“first salt” and“second salt” do not necessarily refer to a temporal relationship. Thus, in some embodiments, the change in salt concentration with time comprises an increase in the concentration of a salt, which causes a reduction in the pH with time. In other embodiments, the change in salt concentration with time comprises a decrease in the concentration of a salt, which causes a reduction in the pH with time.

[0044] The change in salt concentration with time can also, or alternatively, comprise an increase in the concentration of a first salt, which causes a reduction in pH with time, followed by a decrease in the concentration of a second salt, which causes a reduction in the pH with time.

[0045] The elution gradient can further comprise a change in hydrophobicity with time. In some embodiments, the change in hydrophobicity with time is caused by the addition of an organicsolvent that leads to a reduction in hydrophobicity from a high % hydrophobicity to a low % hydrophobicity, wherein the % hydrophobicity is the % volume of an organicsolvent in the elution buffer. The high % hydrophobicity can be greater than 50%. In some aspects, for example, the high % hydrophobicity can be between 60% to 99% hydrophobicity, between 70% to 99% hydrophobicity, or between 80% to 95% hydrophobicity. The low % hydrophobicity can be 50% or less. In some aspects, for example, the low % hydrophobicity can be between 2% to 50% hydrophobicity, between 5% to 20% hydrophobicity, or between 5% to 15%

hydrophobicity.

[0046] Suitable organic solvents include polar, dipolar, non-polar, oxygenated, hydrocarbon, and halogenated solvents. Particularly preferred organic solvents include acetonitrile, methanol, isopropanol, ethanol, tetrahydrofuran, dioxane, or a combination thereof. In some embodiments, the organic solvent is acetonitrile.

[0047] The elution buffer can be run through the ERLIC column for a time period suitable to separate the peptide digest. In some embodiments, the elution buffer can be run through the ERLIC column for a time period of 180 minutes or less. In other embodiments, the elution buffer can be run through the ERLIC column for a time period of 100 minutes or less.

For example, the elution buffer can be run through the ERLIC column for about 30 minutes to 100 minutes or about 50 minutes to 100 minutes.

[0048] In some embodiments, all of the peptide or protein composition from the separated peptide digest can be used to produce a peptide map. In some embodiments, a portion of the peptide or protein composition from the separated peptide digest can be used to produce a peptide map. Thus, in some aspects, a peptide map can be produced from at least a portion of the peptide or protein composition from the separated peptide digest.

[0049] Peptide mapping is generally a comparative procedure where the peptide map of a test sample is compared to a reference peptide map. The following are non-limiting examples of reference peptide maps:

A peptide map of a biosimilar reference peptide, such as the innovator biological drug;

An in-house working reference map, including maps of a peptide that has been prepared using the same or similar manufacturing process as the peptide to be characterised.

[0050] In some embodiments, the method can further comprise comparing the peptide map to a reference peptide map and determining if the peptide map is comparable to the reference peptide map.

[0051] None limiting examples of uses of reference peptide maps include:

to monitor the manufacturing process;

to assess consistency across lots;

to assess the quality of lots; or

during lot release.

[0052] The methods can further comprise processing at least a portion of the peptide or protein composition to produce a final drug product if the peptide map is comparable to the reference peptide map. The portion of the peptide or protein composition that is processed is a portion that was not used to produce the peptide map. Thus, in some embodiments, the disclosed methods of producing a peptide map of a peptide or protein composition comprise: a) digesting at least a portion of the peptide or protein composition to produce a peptide digest; b) separating the peptide digest on an ERLIC column by eluting the peptide digest from the ERLIC column in the presence of an elution gradient, wherein the elution gradient comprises a change in pH with time; c) producing a peptide map from at least a portion of the peptide or protein composition from the separated peptide digest; and d) processing at least a portion of the peptide or protein composition to produce a final drug product if the peptide map is comparable to a reference peptide map.

[0053] The disclosed methods can be carried out as part of a lot release analysis of the final drug product. As used herein,“lot release” refers to the disposition of the final drug product.

[0054] The disclosed methods can be carried out as part of a comparability study to ensure that product quality has not been affected by, for example, manufacturing site change, product scale up, change in drug product presentation (vial to syringe, for example), and/or changes to the manufacturing process.

[0055] In some embodiments the step of producing a peptide map of the peptide or protein composition comprises resolving the separated peptide digest using mass spectrometry.

[0056] The peptides of the peptide digest may be analysed, e.g. by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) or liquid chromatography -tandem mass spectrometry (LC-MS/MS).

[0057] The peptide maps produced by the disclosed methods may be used to analyze one or more of the following peptide characteristics:

molecular weight;

amino acid sequence;

antibody-drug conjugation;

amino acid composition;

- N-terminal degradation;

- N- and C-terminal sequencing;

post translational protein modifications, such as disulfide bridge analysis and/ or glycosylation analysis;

asparagine deamidation; aspartic acid isomerization;

glycosylation;

methionine oxidation;

- N-terminal pyroglutamate;

terminal peptide;

C-terminal lysine;

amino acid sequence mutation, such as substitution, deletion and truncation;

any other primary structural modifications in the antibody; or

any combination(s) thereof.

[0058] Global regulatory agencies, including US Food and Drug Administration (US FDA) and European Medicines Agency (EMA), have harmonized guidelines from the

International Council for Harmonisation (ICH). Guideline ICH Q6B is incorporated herein by reference. This guideline discloses the test procedures and acceptance criteria for peptide/protein drugs, and specifies the use of peptide mapping as a critical quality test procedure for drug characterization used to confirm desired product structure for lot release purposes.

[0059] Also provided herein are final peptide or protein drug products produced by the disclosed methods. In some embodiments, the final peptide or protein drug product is a biosimilar of fremanezumab.

[0060] Further provided is an article of manufacture comprising a final peptide or protein drug product, wherein the final peptide or protein drug product is a biosimilar of fremanezumab.

[0061] Also disclosed are methods of treating a subject having a disorder in which calcitonin gene related peptide (CGRP) activity is detrimental to the subject. The disclosed methods comprise administering the disclosed final peptide or protein drug products to the subject. In some embodiments, the methods of treating a subject having a disorder in which CGRP activity is detrimental to the subject can comprise: a) digesting at least a portion of a peptide or protein composition to produce a peptide digest; b) separating the peptide digest on an ERLIC column by eluting the peptide digest from the ERLIC column in the presence of an elution gradient, wherein the elution gradient comprises a change in pH with time; c) producing a peptide map of at least a portion of the peptide or protein composition from the separated peptide digest; d) processing at least a portion of the peptide or protein composition to produce a final drug product if the peptide map is comparable to a reference peptide map; and e) administering the final drug product to the subject in an amount sufficient to thereby treat the disorder.

[0062] The use of ERLIC to identify antibody variants is also provided. Suitable antibody variants include:

a. asparagine deamidation variants;

b. aspartic acid isomerization variants;

c. glycosylation variants;

d. methionine oxidation variants;

e. N-terminal pyroglutamate variants;

f. terminal peptide variants;

g. C-terminal lysine variants;

h. amino acid sequence mutations;

i. any other primary structural modifications in the antibody; or

j . any combination(s) thereof.

EXAMPLES

[0063] The following examples are provided to further describe some of the

embodiments disclosed herein. The examples are intended to illustrate, not to limit, the disclosed embodiments.

Separation and analysis of peptide variants using ERLIC

Reagents and Chemicals

[0064] The model antibody denosumab used in this study was produced by Amgen (Thousand Oaks, CA). The ERLIC column Poly WAX LP 100 x 2.1 mm, 3 pm, 300 A was purchased from PolyLC Inc. (Columbia, MD). Reagents dithiothreitol (DTT), tris-HCl buffer, ammonium formate and ammonium acetate were purchased from Thermo Fisher Scientific (Waltham, MA). Guanidine buffer and formic acid (FA) were purchased from Sigma Aldrich (St. Louis, MO). Trypsin/Lys-C mixture was purchased from Promega (Madison, WI). Zeba spin desalting columns (7K MWCO, 0.5 mL) was purchased from Thermo Fisher Scientific

(Waltham, MA). Tryptic digest

[0065] The peptide sample (350 pg) was denatured and reduced with the addition of 110 mΐ of 8 M Guanidine-HCl, pH 7.6 and 2.3 mΐ of dithiothreitol (DTT, 500 mM). The mixture was then incubated at 37 °C for half an hour. The sample was desalted and buffer exchanged into 2 M Urea 50 mM Tris-HCl, pH 7.5 using the Zeba spin desalting column. The sample was then digested by addition of 10 pg of Trypsin/Lys-C (peptide/enzyme = 35: 1) at 37 °C overnight. Finally, the sample was diluted four times with the addition of 75% acetonitrile prior to injection on the chromatography system.

ERLIC-MS/MS

[0066] ERLIC-MS/MS was performed on a Waters Acquity UPLC system coupled to a Q-Exactive plus orbitrap mass spectrometer. The autosampler was kept at 5 °C. In the process of method development, a number of method parameters were evaluated including variation of pH, salts, and ACN/H2O gradients.

[0067] Below are two representative methods with:

• solvent A: 200 mM NH4CH3CO2 (ammonium acetate) pH 6.8

• solvent B: acetonitrile

• solvent C: 200 mM NH4HCO2 (ammonium formate) pH 2.5.

Solvent A functions to increase the pH of the elution buffer. Solvent B functions to increase the hydrophobicity of the elution buffer. Solvent C functions to decrease the pH of the elution buffer and to charge the surface of the column.

[0068] Method 1 - Gradient 1 started at 5 % A, 95 % B, and 0 % C to 30 % A, 55 % B, and 15 % C in 50 min. Then the gradient was further increased to 0 % A, 10 % B, and 90 % C at 80 min and kept until 85 min. Then the gradient was decreased to the original 5 % A, 95 % B, and 0 % C at 87 min and kept to equilibrate the column for 5 min to 92 min.

Table 1. The percentage of each of solvents A, B, and C used in the elution buffer of gradient 1. The salt concentration, pH, and hydrophobicity of the gradient is shown over time.

* pH 1 is the pH value of aqueous phase (solvent A + C); pH 2 is the pH value of the total mixture (Solvent A + B + C)

[0069] The salt, pH, and acetonitrile gradients are shown in FIG. 2A-FIG. 2C, respectively.

[0070] Method 2 - Gradient 2 started from 5 % A, 95 % B, and 0 % C to 30 % A, 65 % B, and 5 % C in 65 min. Then the gradient was further increased to 0 % A, 10 % B, and 90 % C at 80 and kept until 85 min. After 85 minutes, the column is re-equilibrated to the initial salt concentration, pH, and hydrophobicity so that it is ready for a next run. The gradient was decreased to original 5 % A, 95 % B, and 0 % C at 87 min and kept to equilibrate the column for 5 min to 92 min.

Table 2. The percentage of each of solvents A, B, and C used in the elution buffer of gradient 2. The salt concentration, pH, and hydrophobicity of the gradient are shown over time.

* pH 1 is the pH value of aqueous phase (solvent A + C); pH 2 is the pH value of the total mixture (Solvent A + B + C)

[0071] After 85 minutes the column was re-equilibrated to the initial salt concentration, pH, and hydrophobicity so that it was ready for a next run. The salt, pH, and acetonitrile gradients are shown in FIG. 2A - FIG. 2C, respectively.

[0072] The MS capillary temperature was set at 320 °C with an S-lens RF level at 55. Sheath gas pressure was set at 35 psi. Auxiliary gas flow was 5 psi and heater temperature was 83 °C. The full MS setting had a resolution of 70k with a scan range of m/z 400-2000. In the MS2 setting, the top 15 ions were collected for fragmentation. The resolution was set at 17.5k with normalized collision energy of 30. Data analysis was performed on Xcalibur 4.0. Ion chromatograms of each specific peptide were extracted at 10 ppm and the identities were further confirmed through manual checking of fragment ions when necessary. The pi value of each peptide was estimated based on isoelectric point calculator (IPC), a web service providing accurate estimation of peptide and peptide pi values. The values used in this study were the measured IPC peptide pi values.

Separation Method Development

[0073] Because ERLIC has a complex mechanism in terms of retaining peptides, a number of parameters including pH, salt, and a hydrophobicity gradient were developed to separate a mixture of peptides from an antibody tryptic digest. The antibody used in the study was denosumab, which when digested with trypsin results in greater than 59 peptides.

[0074] In this study, ERLIC was coupled with a high speed mass spectrometer which served as a powerful second dimensional separation. In order to be compatible with the mass spectrometer, the method development initially started with an acetonitrile/water gradient containing 0.1 % formic acid, but the basic peptides were poorly separated. This was likely due to the basic peptides being positively charged in a neutral or acidic environment and repulsed from the stationary phase. [0075] Subsequently, the method was modified with increased pH and the addition of ammonium salt, with gradients of acetonitrile/water and a constant 200 mM ammonium formate buffer at pH 4.5. Under this condition, basic peptides were better separated. However, with the addition of salt and an increased pH value, the acidic peptides were bound tightly with the column through electrostatic interaction and detected as late eluting peaks. In some cases, acidic peptides with low pi values were bound too strong to be eluted from the column.

[0076] After a series of developments, a finely tuned method with both a pH and H2O/ACN gradient, plus the addition of ammonium salt, was developed. Representative chromatograms are shown in FIG. 3A and FIG. 3B. The pH gradient was achieved by gradually increasing the ratio of ammonium acetate (pH 6.8) to ammonium formate (pH 2.2). Gradient 2 shown in FIG. 3B, with a shallower pH and H2O/ACN gradient, resulted in a better separation profile.

Peptide Analysis

[0077] As shown in the FIG. 4A, the tryptic peptides were clearly separated on the ERLIC -MS/MS in two-dimensional scale. In this ERLIC-MS/MS analysis, each tryptic peptide was successfully identified by accurate mass, some of which were further confirmed by their MS/MS fragment ions when necessary. The sequence coverage by ERLIC was 100% for both the light and heavy chains. This is an improvement as compared to traditional RPLC methods which do not typically achieve 100% sequence coverage. The peptide identities are also labeled in FIG. 4A according to sequence. Detailed sequence information, measured masses, and retention times of both light chain and heavy chain peptides are provided in Table 3 and Table 4, below. Since the ERLIC column readily retained small peptides and even single amino acids with sufficient method development, theoretically it should retain the entirety of tryptic digests of monoclonal antibodies with 100% sequence coverage. As shown in FIG. 5, the small tryptic peptides of denosumab were successfully extracted from the ERLIC-MS/MS data and confirmed by their accurate masses. Comparatively, RPLC-MS/MS based peptide mapping generally voids small hydrophilic peptides resulting in sequence coverages of less than 100%. The results herein clearly demonstrate ERLIC-MS/MS as a potentially powerful tool in the bottom-up

characterization of monoclonal antibodies for sequence coverage analysis, fingerprint analysis, and other sequence based analyses. [0078] It was hypothesized that there would be a relationship between the retention time on ERLIC and the peptide pi value and hydrophilicity. As shown in FIG. 4B, a correlation between peptides with relatively lower pi values and longer retention times was clearly evident. This can be explained by the electrostatic interactions between the peptides and the column, as basic peptides with high pi values are repulsed from the stationary phase with less binding and reduced retention times. Peptide retention time on ERLIC would also be affected by peptide hydrophilicity, which would explain why peptides with close pi values have different retention time, as less hydrophilic peptides elute earlier. This observed relationship indicated that peptide retention pattern on ERLIC would be predictive of peptide sequence. The potential for retention time prediction would serve as supportive information to further confirm the peptide identities deduced from mass spectra fragments and reduce the false identification of peptides during peptide characterization.

[0079] The peptides which did not elute in the prior art RPLC-MS/MS method are small and hydrophilic. However, they were successfully eluted and characterised by the method disclosed herein.

Peptide Variant Analysis

[0080] A number of enzymatic and non-enzymatic variants/modifications on the antibody are considered as critical quality attributes (CQA) in a biologic’s development. Further investigation of these variants is usually performed on RPLC-MS/MS for in-depth

characterization. As shown in Table 5, the major charge variant peptides including terminal peptides, methionine-oxidized peptides, asparagine deamidated peptides, and glycopeptides, were successfully identified based on their molecular ions and fragment ions on ERLIC-MS/MS. All results correlated well with data from RPLC-MS/MS based peptide mapping (data not shown).

Table 5. The characterization of major variant peptides of denosumab

Mod. % = modification %

Terminal Peptides

[0081] N-terminal pyroglutamate (pE) formation is one of the many post-translational modifications commonly observed during monoclonal antibody manufacture and storage. Both N-terminal glutamine and glutamic acid can spontaneously cyclize to form pyroglutamate in vitro , with glutamic acid conversion to pE observed at a slower rate than for glutamine.

Formation of pE from glutamine results in loss of one amine group and can generate an acidic variant in iCIEF (imaged capillary isoelectric focusing) or CEX (cation exchange

chromatography). The N-termini of both the light chain and heavy chain of denosumab have glutamic acid (E). On the light chain, the peptide retention time decreased from 49.4 min to 43.5 min after the conversion from E to pE. The measured pE formation percentage was 0.16 %. On the heavy chain, the peptide retention time decreased from 47.4 min to 41.6 min (FIG. 6A and FIG. 6B) after the conversion from E to pE, and pE formation percentage was 0.06%. The decreased retention time is likely due to decreased peptide hydrophilicity and a higher peptide pi value, as pyroglutamate is less hydrophilic and less acidic than glutamic acid.

[0082] C-terminal lysine variation is also commonly observed in biopharmaceutical monoclonal antibodies. Although this modification does not affect drug bioactivity, it can contribute to the formation of charge variants and is generally monitored to demonstrate manufacturing consistency. The C-terminal peptide SLSLSPGK has a pi value of 10.04 and a retention time of 33.0 min (FIG. 6C and FIG. 6D). After lysine truncation, the peptide had a decreased retention time of 28.6 min with a lower pi value of 5.974, contradicting the general principle discussed before where peptides with higher pi values have relatively shorter retention times. This observed phenomenon indicated that a terminal lysine/arginine can play a role in the observed retention time on ERLIC. The C-terminal end of the peptide would be attracted to the stationary phase due to less electrostatic repulsion than for the N-terminal of the peptide. It could also be explained by the decreased hydrophilicity after the loss of lysine. The measured percentage of C-terminal lysine truncation by integrated EIC intensity was 99.03 %. The measured variant’s percentages of terminal peptides correlate well with the RPLC-MS based method (data not shown).

Methionine Oxidation

[0083] A number of amino acids such as methionine, cysteine, tryptophan, and lysine have the potential to be oxidized by reactive oxygen species. Among them, methionine is often the most susceptible residue to be oxidized, especially when exposed on the surface of the antibody with more accessibility to solvents and reactive oxygen species. Methionine oxidation can lead to decreased therapeutic effects and antibody stability. In this study, ERLIC-MS/MS was successfully applied for the characterization of methionine oxidation. Based on previous studies, methionine residues M85, M106, M253, and M398 were prone to oxidation. As shown in FIG. 7A - FIG. 7B, representative peptide H17-DTLMISR and its oxidized form were successfully extracted from the total ion chromatogram and further confirmed by their accurate molecular ions and MS2 fragment ions. After methionine oxidation, the peptide retention time of H17 increased from 40.1 min to 44.5 min. As shown in Table 3, this retention time shift was also consistently observed in other methionine peptides, which may be explained by the increased peptide hydrophilicity after oxidation. The percentages of methionine oxidation measured by ERLIC-MS/MS were similar to the data from traditional RPLC-MS/MS (data not shown).

Deamidation

[0084] Deamidation of asparagine (Asn, N) to aspartic acid (Asp, D) and/or isoaspartic residues (iso Asp, isoD) is a common non-enzymatic degradation pathway of monoclonal antibodies. Deamidation can introduce negative charge to the antibody and affect antibody shelf- life if not formulated appropriately. If the deamidation site is located in the complementarity determining region (CDR), it can significantly reduce target binding affinity and therapeutic effects. Therefore it is important to accurately measure the percentage of deamidation formation and monitor its changes during different stages of monoclonal antibody production and storage. Characterization of deamidated asparagine on a peptide molecule is challenging and the most common and efficient way is to apply a bottom-up peptide mapping method by RPLC-MS/MS. However, as peptide size increases, the peptide hydrophobicity differences between deamidated and non-deamidated peptides become very narrow, resulting in separation challenges by RPLC- MS/MS. In addition, the molecular weight of a deamidated peptide is only ~ 1 Dalton heavier than the non-deamidated peptide and overlap of isotopic peaks can further complicate the analysis. Therefore a second dimension separation on a mass spectrometer can be challenging.

[0085] Method development for RPLC-MS/MS generally requires significant effort and fine-tuning. For example, the tryptic peptide“GFYPSDIAVEWESNGQPENNYK” (also known as PENNY peptide), located in the Fc constant region of the monoclonal antibody, has been widely studied as it appears in the majority of monoclonal antibody drugs approved or in development. Even after years of research on the PENNY peptide using RPLC-MS/MS, there is still a need for a more robust analytical method for its characterization. The PENNY peptide has three asparagine residues: N385, N390, and N391. N385 and N390 are prone to deamidate and each residue can potentially form three deamidated products (succinimide intermediate, isoaspartic acid, and aspartic acid). Combined with the inherent difficulties of deamidation separations on RPLC and MS, it can be difficult to fully characterize the PENNY peptide with a high level of accuracy and assurance. In this study, ERLIC demonstrated superior capability in the characterization of deamidated peptides of a monoclonal antibody. As shown in FIG. 8A, robust baseline separation of Asp and isoAsp deamidated products (Peaks 4, 5 and 6) from the non-deamidated peptide (Peak 3) was readily achieved using Gradient 1 (top panel). Gradient 2 (bottom panel) had a steeper pH gradient during the peptide elution window, which led to decreased resolution. As shown in FIG. 8B, the mass profiles of deamidated peptides (Peaks 4, 5 and 6) are ~ 1 Dalton heavier than the original peptide (Peak 3). The site of deamidation was also identified by analysis of MS2 fragment ions and the major fragment ions were labelled accordingly in FIG. 8C. Peaks 4 and 5 were identified as deamidation at position N390 as they contained the signature fragment ion y6 with m/z 765, corresponding to fragment

[PEDNYK+H]⁺ or [PEisoDNYK+H]⁺. Peak 6 was identified as deamidation at position N385 by the bl4 ion [GFYPSDIAVEWESD+H]⁺ or [GFYPSDIAVEWESisoD+H]⁺. It has been reported that there are three major deamidated products in ³⁸⁵isoAsp, ³⁹⁰ Asp and ³⁹⁰isoAsp, which correlated well with the data presented in this study. It was reported that the isoAsp peptide was more acidic and eluted later than the Asp peptide on the ERLIC column; therefore Peak 4 was tentatively assigned as ³⁹⁰ Asp and Peak 5 as ³⁹⁰isoAsp.

[0086] In the Gradient 1 method, the retention time differences were sufficiently large between non-deamidated Peak 3 (37.23 min) and deamidated Peak 4 (40.9 min), Peak 5 (43.7 min), and Peak 6 (45.0 min). Each deamidated peak was also well separated from each other.

The ERLIC method was also successfully applied for the monitoring of succinimide intermediate ³⁹⁰Suc (Peak 1) and ³⁸⁵Suc (Peak 2) formation from the same analysis. The mass shift was approximately 17 Daltons lower as shown in the mass spectra due to the loss an ammonium molecule (FIG. 8B). The identification and differentiation was based on detection of signature peptide y6, which is m/z 747 [PESucNYK+H]⁺ for Peak 1 and m/z 764 [PENNYK+H]⁺ for Peak 2. As shown in FIG. 8A, the succinimide intermediates Peak 1 & Peak 2 eluted earlier than the original peptide, which is due to the decreased hydrophilicity after the formation of the succinimide ring and the loss of an amine group. For characterization of deamidation, ERLIC- MS/MS required less time for method development, resulting in superior separation capacity, than for the RPLC-MS/MS method. The demonstrated capabilities suggest the potential uses of ERLIC as a new tool for the characterization of biologic therapeutics deamidation. Glvcosylation

[0087] Glycosylation increases monoclonal antibody heterogeneity by the incorporation of a number of different glycans on the heavy chain. It is one of the more complex and prominent forms of peptide post-translational modification. Glycosylation can affect monoclonal antibody structural stability, biological activity, immunogenicity, and pharmacokinetics. The characterization of antibody glycosylation can be divided into the following three analytical strategies: top-down (intact peptide), bottom -up (gly copeptide), and released glycan. The bottom-up peptide mapping based characterization is also typically performed on RPLC-MS/MS. The most common glycopeptide on monoclonal antibodies is H20 (²⁹⁴EEQFNSTFR³⁰²) with different glycans attached at N298. ERLIC-MS/MS was also used for glycopeptide analysis and demonstrated similar capability as RPLC-MS/MS in the characterization of tryptic peptide H20 (²⁹⁴EEQFNSTFR³⁰²) with different glycans attached. As shown in FIG. 9, the detected glycopeptides (GOF-GlcNAc, GO, GOF, G1F, G2F, and M5) were successfully extracted from the total ion chromatogram by their accurate masses. The percentage of each glycoform was estimated by the integrated intensity from each extracted ion chromatogram. The most abundant glycopeptide species were GOF (64.6 %) and G1F (14.6 %). The results correlated well with results from the intact analysis, released glycan analysis, and RPLC-MS/MS based glycopeptide characterization (data not shown). Interestingly, the glycopeptide retention times followed the order: [GOF-GlcNAc] (70.7 min) < [GO] (70.8 min) < [GOF] (70.9 min) < [G1F] (71.1 min) < [G2F] (71.3 min). This observation suggested that the increasing size of the glycans on the glycopeptide increased the retention time on the ERLIC column. This could be explained by the increased hydrophilicity from the addition of extra sugar moieties. Similar to RPLC, the major glycoforms all elute in a narrow retention time window, but can be easily resolved on the second dimension mass spectrometer. The glycopeptide identities were determined based on their accurate masses and isotopic mass profiles and fragment ions. The results clearly demonstrated that this ERLIC-MS/MS method can be applied for glycopeptide characterization.

Conclusions

[0088] In this study, a novel one dimensional ERLIC-MS/MS based peptide mapping method was developed and successfully applied for the characterization of monoclonal antibodies. As an example, the method was successfully applied for the characterization of the IgG2 monoclonal antibody denosumab, including full sequence coverage, terminal peptides, and variant analysis (including methionine oxidation, asparagine deamidation, and glycopeptides). Compared to reversed phase LC-MS/MS methods, ERLIC demonstrated unique advantages. For example, due to its unique separation mechanism, ERLIC-MS/MS was successfully applied for identification of all peptides from a trypsin digest, including small peptides and single amino acids, with 100% sequence coverage of both the heavy chain and light chain. The relationship between peptide retention time and pi values was observed with inverse correlation between retention times and pi values. The most common charge variants (i.e., C-terminal pyroglutamate formation, N-terminal lysine truncation, and asparagine deamidation) were successfully characterized by ERLIC-MS/MS with results similar to those obtained from traditional RPLC- MS/MS. For the separation and characterization of asparagine deamidated peptides, the disclosed methods demonstrated superior performance to RPLC-MS/MS, which is known to be challenging. Furthermore, ERLIC demonstrated superior capability in the separation of deamidated and non-deamidated peptides as demonstrated by analysis of the PENNY peptide in the Fc region. Finally, the ERLIC-MS/MS method was successfully utilized for the analysis of methionine oxidation and glycosylation demonstrating broad utility for characterizing multiple product variant attributes in a single run. The developed method can be used alone for peptide- mapping based characterization of monoclonal antibodies, or as an orthogonal method to complement the RPLC-MS/MS method. This study extends the traditional applications of ERLIC from being a trapping/fractioning column to biologic therapeutics characterization. The ERLIC-MS/MS method can enhance biologic therapeutics analysis with more reliability and confidence for bottom-up peptide mapping characterization.

[0089] Those skilled in the art will appreciate that numerous changes and modifications can be made to the preferred embodiments of the invention and that such changes and modifications can be made without departing from the spirit of the invention. It is, therefore, intended that the appended claims cover all such equivalent variations as fall within the true spirit and scope of the invention.

[0090] The disclosures of each patent, patent application, and publication cited or described in this document are hereby incorporated herein by reference, in its entirety. EMBODIMENTS

[0091] The following list of embodiments is intended to complement, rather than displace or supersede, the previous descriptions.

Embodiment 1. A method of producing a peptide map of a peptide or protein composition, comprising:

a) digesting at least a portion of the peptide or protein composition to produce a peptide digest;

b) separating the peptide digest on an electrostatic repulsion hydrophilic interaction chromatography (ERLIC) column by eluting the peptide digest from the ERLIC column in the presence of an elution gradient, wherein the elution gradient comprises a change in pH with time; and

c) producing a peptide map of the peptide or protein composition from the separated peptide digest.

Embodiment 2. The method of embodiment 1, wherein the change in pH is a result of an elution buffer running through the ERLIC column.

Embodiment 3. The method of embodiment 1 or 2, further comprising comparing the

peptide map to a reference peptide map and determining if the peptide map is comparable to the reference peptide map.

Embodiment 4. The method of embodiment 3, further comprising processing at least a portion of the peptide or protein composition to produce a final peptide or protein drug product if the peptide map is comparable to the reference peptide map.

Embodiment 5. The method of embodiment 4, wherein the method is carried out as part of a lot release analysis of the final peptide or protein drug product. Embodiment 6. The method of any one of the previous embodiments, wherein the step of producing a peptide map of the peptide or protein composition comprises resolving the separated peptide digest using mass spectrometry.

Embodiment 7. The method of any one of the previous embodiments, wherein the change in pH with time comprises a reduction in pH from a high pH to a low pH.

Embodiment 8. The method of embodiment 7, wherein the high pH is greater than pH 5 and wherein the low pH is lower than or equal to pH 5.

Embodiment 9. The method of embodiment 8, wherein the high pH is between pH 6 and pH 10, between pH 7 and pH 9, between pH 7 and pH 8.5, or between pH 8 and pH 8.2.

Embodiment 10. The method of embodiment 9, wherein the high pH is between pH 8 and pH 8.2.

Embodiment 11. The method of embodiment 8, wherein the low pH is between pH 1 and pH 5, between pH 1 and pH 4, between pH 1.5 and pH 3.5, or between pH 2.6 and pH 2.7.

Embodiment 12. The method of embodiment 11, wherein the low pH is between pH 2.6 and pH 2.7.

Embodiment 13. The method of any one of the previous embodiments, wherein the elution gradient further comprises a change in salt concentration with time.

Embodiment 14. The method of embodiment 13, wherein the change in salt concentration with time comprises an increase in the concentration of a first salt in an elution buffer, which causes a reduction in the pH with time. Embodiment 15. The method of embodiment 14, wherein the elution buffer containing the first salt has a pH of less than pH 5, less than pH 4, or less than pH 3.

Embodiment 16. The method of embodiment 15, wherein the elution buffer containing the first salt has a pH of pH 2.5.

Embodiment 17. The method of any one of embodiments 14-16, wherein the first salt is an ammonium salt.

Embodiment 18. The method of embodiment 17, wherein the ammonium salt is ammonium formate.

Embodiment 19. The method of embodiment 13, wherein the change in salt concentration with time comprises a decrease in the concentration of a second salt in an elution buffer, which causes a reduction in the pH with time.

Embodiment 20. The method of embodiment 19, wherein the elution buffer containing the second salt has a pH of greater than pH 4, greater than pH 5, or greater than pH 6.

Embodiment 21. The method of embodiment 20, wherein the elution buffer containing the second salt has a pH of pH 6.8.

Embodiment 22. The method of any one of embodiments 19-21, wherein the second salt is an ammonium salt.

Embodiment 23. The method of embodiment 22, wherein the ammonium salt is ammonium acetate.

Embodiment 24. The method of embodiment 13, wherein the change in salt concentration with time comprises an increase in the concentration of a first salt, which causes a reduction in pH in time, followed by a decrease in the concentration of a second salt, which causes a reduction in the pH with time.

Embodiment 25. The method of any one of the previous embodiments, wherein the elution gradient further comprises a change in hydrophobicity with time.

Embodiment 26. The method of embodiment 25, wherein the change in hydrophobicity with time is caused by the addition of an organicsolvent that leads to a reduction in hydrophobicity from a high % hydrophobicity to a low % hydrophobicity, wherein the % hydrophobicity is the % volume of an organicsolvent in the elution buffer.

Embodiment 27. The method of embodiment 26, wherein the high % hydrophobicity is greater than 50%.

Embodiment 28. The method of embodiment 27, wherein the high % hydrophobicity is between 60% to 99% hydrophobicity, between 70% to 99% hydrophobicity, or between 80% to 95% hydrophobicity.

Embodiment 29. The method of embodiment 26, wherein the low % hydrophobicity is 50% or less.

Embodiment 30. The method of embodiment 29, wherein the low % hydrophobicity is between 2% to 50% hydrophobicity, between 5% to 20% hydrophobicity, or between 5% to 15% hydrophobicity.

Embodiment 31. The method of any one of embodiments 26-30, wherein the organic

solvent is acetonitrile, methanol, isopropanol, ethanol, tetrahydrofuran, dioxane, or a combination thereof.

Embodiment 32. The method of any one of embodiments 2-31, wherein the elution buffer is run through the ERLIC column for 180 minutes or less, or 100 minutes or less. Embodiment 33. The method of embodiment 32, wherein the elution buffer is run through the ERLIC column for about 30 minutes to 100 minutes or about 50 minutes to 100 minutes.

Embodiment 34. A final peptide or protein drug product produced by the method of any one of the previous embodiments.

Embodiment 35. The final peptide or protein drug product of embodiment 34, wherein the peptide or protein drug product is a biosimilar of fremanezumab.

Embodiment 36. An article of manufacture comprising the final peptide or protein drug product of embodiment 34 or 35.

Embodiment 37. A method of treating a subject having a disorder in which calcitonin gene related peptide (CGRP) activity is detrimental to the subject, comprising administering the final peptide or protein drug product of embodiment 34 or 35 to the subject.

Embodiment 38. Use of ERLIC to identify antibody variants, wherein the variants are: a) asparagine deamidation variants;

b) aspartic acid isomerization variants;

c) glycosylation variants;

d) methionine oxidation variants;

e) N-terminal pyroglutamate variants;

f) terminal peptide variants;

g) C-terminal lysine variants;

h) amino acid sequence mutations;

i) any other primary structural modifications in the antibody; or

j) any combination thereof.

Claims

What is claimed:

1. A method of producing a peptide map of a peptide or protein composition, comprising: a) digesting at least a portion of the peptide or protein composition to produce a peptide digest;

b) separating the peptide digest on an electrostatic repulsion hydrophilic interaction chromatography (ERLIC) column by eluting the peptide digest from the ERLIC column in the presence of an elution gradient, wherein the elution gradient comprises a change in pH with time; and

c) producing a peptide map of the peptide or protein composition from the separated peptide digest.

2. The method of claim 1, wherein the change in pH is a result of an elution buffer running through the ERLIC column.

3. The method of claim 1 or 2, further comprising comparing the peptide map to a reference peptide map and determining if the peptide map is comparable to the reference peptide map.

4. The method of claim 3, further comprising processing at least a portion of the peptide or protein composition to produce a final peptide or protein drug product if the peptide map is comparable to the reference peptide map.

5. The method of claim 4, wherein the method is carried out as part of a lot release analysis of the final peptide or protein drug product.

6. The method of any one of the previous claims, wherein the step of producing a peptide map of the peptide or protein composition comprises resolving the separated peptide digest using mass spectrometry.

7. The method of any one of the previous claims, wherein the change in pH with time comprises a reduction in pH from a high pH to a low pH.

8. The method of claim 7, wherein the high pH is greater than pH 5 and wherein the low pH is lower than or equal to pH 5.

9. The method of claim 8, wherein the high pH is between pH 6 and pH 10, between pH 7 and pH 9, between pH 7 and pH 8.5, or between pH 8 and pH 8.2.

10. The method of claim 9, wherein the high pH is between pH 8 and pH 8.2.

11. The method of claim 8, wherein the low pH is between pH 1 and pH 5, between pH 1 and pH 4, between pH 1.5 and pH 3.5, or between pH 2.6 and pH 2.7.

12. The method of claim 11, wherein the low pH is between pH 2.6 and pH 2.7.

13. The method of any one of the previous claims, wherein the elution gradient further comprises a change in salt concentration with time.

14. The method of claim 13, wherein the change in salt concentration with time comprises an increase in the concentration of a first salt in an elution buffer, which causes a reduction in the pH with time.

15. The method of claim 14, wherein the elution buffer containing the first salt has a pH of less than pH 5, less than pH 4, or less than pH 3.

16. The method of claim 15, wherein the elution buffer containing the first salt has a pH of pH 2.5.

17. The method of any one of claims 14-16, wherein the first salt is an ammonium salt.

18. The method of claim 17, wherein the ammonium salt is ammonium formate.

19. The method of claim 13, wherein the change in salt concentration with time comprises a decrease in the concentration of a second salt in an elution buffer, which causes a reduction in the pH with time.

20. The method of claim 19, wherein the elution buffer containing the second salt has a pH of greater than pH 4, greater than pH 5, or greater than pH 6.

21. The method of claim 20, wherein the elution buffer containing the second salt has a pH of pH 6.8.

22. The method of any one of claims 19-21, wherein the second salt is an ammonium salt.

23. The method of claim 22, wherein the ammonium salt is ammonium acetate.

24. The method of claim 13, wherein the change in salt concentration with time comprises an increase in the concentration of a first salt, which causes a reduction in pH in time, followed by a decrease in the concentration of a second salt, which causes a reduction in the pH with time.

25. The method of any one of the previous claims, wherein the elution gradient further comprises a change in hydrophobicity with time.

26. The method of claim 25, wherein the change in hydrophobicity with time is caused by the addition of an organicsolvent that leads to a reduction in hydrophobicity from a high % hydrophobicity to a low % hydrophobicity, wherein the % hydrophobicity is the % volume of an organicsolvent in the elution buffer.

27. The method of claim 26, wherein the high % hydrophobicity is greater than 50%.

28. The method of claim 27, wherein the high % hydrophobicity is between 60% to 99% hydrophobicity, between 70% to 99% hydrophobicity, or between 80% to 95%

hydrophobicity.

29. The method of claim 26, wherein the low % hydrophobicity is 50% or less.

30. The method of claim 29, wherein the low % hydrophobicity is between 2% to 50% hydrophobicity, between 5% to 20% hydrophobicity, or between 5% to 15%

hydrophobicity.

31. The method of any one of claims 26-30, wherein the organic solvent is acetonitrile, methanol, isopropanol, ethanol, tetrahydrofuran, dioxane, or a combination thereof.

32. The method of any one of claims 2-31, wherein the elution buffer is run through the ERLIC column for 180 minutes or less, or 100 minutes or less.

33. The method of claim 32, wherein the elution buffer is run through the ERLIC column for about 30 minutes to 100 minutes or about 50 minutes to 100 minutes.

34. A final peptide or protein drug product produced by the method of any one of the previous claims.

35. The final peptide or protein drug product of claim 34, wherein the peptide or protein drug product is a biosimilar of fremanezumab.

36. An article of manufacture comprising the final peptide or protein drug product of claim 34 or 35.

37. A method of treating a subject having a disorder in which calcitonin gene related peptide (CGRP) activity is detrimental to the subject, comprising administering the final peptide or protein drug product of claim 34 or 35 to the subject.

38. Use of ERLIC to identify antibody variants, wherein the variants are: a) asparagine deamidation variants;

b) aspartic acid isomerization variants;

c) glycosylation variants;

d) methionine oxidation variants;

e) N-terminal pyroglutamate variants;

f) terminal peptide variants;

g) C-terminal lysine variants;

h) amino acid sequence mutations;

i) any other primary structural modifications in the antibody; or j) a combination thereof.