Method for preparing trypsin-resistant polypeptides for mass spectrometric analysis - Patents.com

Abstract

The methods disclosed herein involve preparing and detecting polypeptides using neutrophil elastase, such as human neutrophil elastase. The polypeptides are optionally denatured, reduced, and / or alkylated before undergoing a first digestion. A second digestion involves the use of neutrophil elastase, such as human neutrophil elastase. The prepared fragments are then analyzed by chromatography, electrophoresis, or spectrometry, or a combination of these methods. The methods are particularly useful for preparing therapeutic polypeptides for analysis, particularly polypeptides that are not easily cleaved, such as some bispecific T-cell engager (BiTE®) molecules.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 642,444, filed March 13, 2018, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application is filed with a sequence listing in electronic format. The sequence listing, provided as a file entitled "A-2151-WO-PCT sequence listing_ST25.txt", was created on March 12, 2019 and is 261 KB in size. The information in the electronic sequence listing is incorporated herein by reference in its entirety.

[0003] The subject matter presented herein relates to the field of polypeptide analysis. In particular, the subject matter presented herein relates to the preparation of a sample for detecting a polypeptide contained therein, such as a therapeutic polypeptide. The method disclosed herein uses sequential digestions in the preparation of the polypeptide, the final digestion including the use of neutrophil elastase. [Background technology]

[0004] Multi-attribute methods (MAMs) rely on enzymatic digestion of molecules followed by mass spectrometry-based characterization and quantification of key attributes. Modifications in complementarity determining regions (CDRs) are of particular concern as they may affect the potency and / or safety of the molecule. Trypsin, which cleaves at the C-terminus of lysine and arginine residues, is typically utilized as the enzyme of choice for peptide mapping and MAMs for a number of reasons, including the high digestion specificity and frequency of occurrence of lysine and arginine residues; trypsin digestion also typically results in peptides with a basic residue on the C-terminus that have an optimal length for mass spectrometry-based analysis. However, many of the bispecific T cell engager (BiTE®) molecules currently under development at Amgen contain a conserved α-CD3 domain with two CDRs immediately flanked by a long linker region. One of these CDRs (HGNFGNSYISYWAY) contains two asparagine residues that may be susceptible to deamidation and a tryptophan residue that may be susceptible to oxidation. This region of the molecule does not contain residues amenable to trypsin digestion (Figure 1). Thus, attributes of interest within the CDR domains cannot be monitored by MAM due to the large size of this peptide (approximately 8 kDa), the difficulty of separating modified versions of the peptide by chromatography, poor recovery of the peptide and / or the general challenges associated with ionizing and interpreting mass spectrometry measurements corresponding to peptides of this size. Furthermore, this linker region does not have residues that are sensitive to commonly used secondary proteases such as Asp-N, Lys-C and Glu-C. Although chymotrypsin cleaves at tryptophan residues, the use of this enzyme was not pursued due to both the low specificity of this enzyme and the possibility that oxidation at tryptophan residues could affect the quantification of this attribute. Similarly, many BiTE® molecules also have shorter linker peptides (approximately 5-7 kDa) immediately adjacent to the target-specific CDRs that are similarly difficult to monitor using trypsin digestion. For example, a potential CDR aspartic acid isomerization site is located within the 5.7 kDa linker peptide.Taken together, these attributes can have a significant impact on targeting and / or CD3 binding, which necessitates MAM-based surveillance. Summary of the Invention [Means for solving the problem]

[0005] In a first aspect, there is provided herein a method of preparing a polypeptide for analysis comprising cleaving a polypeptide in a sample in a first digestion, wherein the cleaving produces at least two fragments of the polypeptide, which at least two fragments of the polypeptide are subsequently digested with neutrophil elastase, and analyzing the sample after digesting the at least two fragments of the polypeptide with neutrophil elastase.

[0006] In a second aspect, provided herein is a method of preparing a polypeptide for analysis, comprising the steps of cleaving a polypeptide in a sample in a first digestion, where the cleaving produces at least two fragments of the polypeptide; dividing the sample containing the at least two fragments into a first aliquot and a second aliquot; digesting the at least two fragments of the first aliquot with neutrophil elastase; and analyzing the first and second aliquots. In some sub-aspects, the first and second aliquots are combined after digesting the at least two polypeptide fragments with neutrophil elastase; in such a case, the first and second aliquots may contain approximately equal amounts of the polypeptide.

[0007] In both the first and second aspects, the step of cleaving the polypeptide in the first digestion comprises proteolytic or chemical cleavage. In those sub-aspects that comprise proteolytic cleavage, such cleavage is performed by a protease, where the protease has an activity different from neutrophil elastase. In some sub-aspects, the protease is selected from the group consisting of trypsin, endoproteinase Glu-C, endoproteinase Arg-C, pepsin, chymotrypsin, chymotrypsin B, Lys-N protease, Lys-C protease, Glu-C protease, Asp-N protease, pancreatopeptidase, carboxypeptidase A, carboxypeptidase B, proteinase K and thermolysin, and combinations thereof. In some sub-embodiments of these first and second aspects, the protease is selected from the group consisting of trypsin, Asp-N and Glu-C, and combinations thereof; in still other sub-embodiments, the protease is trypsin. However, when chemical cleavage is used to fragment polypeptides in the sample in the first and second aspects, such cleavage is performed by chemicals such as chemicals selected from the group consisting of cyanogen bromide, 2-nitro-5-thiocyanobenzoic acid, hydroxylamine, and BNPS-skatole, and combinations thereof.

[0008] Furthermore, the polypeptides may be denatured and / or reduced and / or alkylated prior to cleaving the polypeptides in the sample in the first digest. The neutrophil elastase may be, for example, human neutrophil elastase (EC 3.4.21.37). When analyzing the sample, the analysis may include at least one technique selected from the group consisting of chromatography, electrophoresis, spectroscopy or spectrometry, and combinations thereof. For example, if the technique for analyzing the sample includes chromatography, the chromatographic technique can be selected from the group consisting of gas chromatography, liquid chromatography, high performance liquid chromatography, ultra-high performance liquid chromatography, size exclusion chromatography, ion exchange chromatography, affinity chromatography, expanded adsorption fluidized bed chromatography, reversed phase chromatography, hydrophobic interaction chromatography, and combinations thereof; if the technique is electrophoretic, the electrophoretic technique can be selected from the group consisting of gel electrophoresis, free-flow electrophoresis, electrofocusing, isotachophoresis, affinity electrophoresis, immunoelectrophoresis, reverse electrophoresis, and capillary electrophoresis, capillary zone electrophoresis, and combinations thereof; and if the technique is spectroscopy, the spectroscopy technique can be selected from mass spectrometry, ultraviolet spectroscopy, visible spectroscopy, fluorescence spectroscopy, and ultraviolet-visible spectroscopy, and combinations thereof. In certain sub-embodiments, the technique includes liquid chromatography-mass spectrometry; in other cases, the technique includes capillary zone electrophoresis coupled with mass spectrometry.

[0009] In a third aspect, disclosed herein is a method of preparing a polypeptide for analysis, comprising the steps of denaturing, reducing, and alkylating the polypeptide; digesting the polypeptide with trypsin to generate trypsin-cleaved polypeptide fragments; dividing the trypsin-cleaved polypeptide fragments into a first aliquot and a second aliquot; digesting the trypsin-cleaved polypeptide fragments of the first aliquot with neutrophil elastase; combining the first aliquot and the second aliquot in a ratio of about 1:1; and analyzing the combined aliquot. The neutrophil elastase can be, for example, human neutrophil elastase (EC 3.4.21.37). When analyzing a sample, the analysis can include at least one technique selected from the group consisting of chromatography, electrophoresis, spectroscopy, spectrometry, and combinations thereof. For example, if the technique for analyzing the sample includes chromatography, the chromatographic technique can be selected from gas chromatography, liquid chromatography, high performance liquid chromatography, ultra high performance liquid chromatography, size exclusion chromatography, ion exchange chromatography, affinity chromatography, expanded adsorption fluidized bed chromatography, reversed phase chromatography, hydrophobic interaction chromatography, and combinations thereof; if the technique is electrophoretic, the electrophoretic technique can be selected from the group consisting of gel electrophoresis, free-flow electrophoresis, electrofocusing, isotachophoresis, affinity electrophoresis, immunoelectrophoresis, reverse electrophoresis, and capillary electrophoresis, capillary zone electrophoresis, and combinations thereof; and if the technique is spectroscopy, the spectroscopy (or spectrometry) technique can be selected from mass spectrometry, ultraviolet spectroscopy, visible spectroscopy, fluorescence spectroscopy, and ultraviolet-visible spectroscopy, and combinations thereof. In certain sub-embodiments, the technique includes liquid chromatography-mass spectrometry; in other cases, the technique includes capillary zone electrophoresis coupled with mass spectrometry.

[0010] In a fourth aspect, disclosed herein is a method of preparing a polypeptide for analysis, comprising the steps of preparing a sample comprising the polypeptide, applying a filter having a molecular weight cutoff to the sample, digesting the sample on the filter with a first protease; digesting the sample on the filter with a second protease; and analyzing the sample; wherein the second protease is neutrophil elastase; and the first protease is a method different from the second protease. In this aspect, the first protease can comprise a protease selected from the group consisting of trypsin, Asp-N and Glu-C; further, the polypeptide can be denatured and / or reduced and / or alkylated prior to digesting the polypeptide with the first polypeptide. The neutrophil elastase can be, for example, human neutrophil elastase (EC 3.4.21.37). When analyzing the sample, the analysis can include at least one technique selected from the group consisting of chromatography, electrophoresis, spectroscopy, spectrophotometry, and combinations thereof. For example, if the technique for analyzing the sample includes chromatography, the chromatographic technique may be selected from gas chromatography, liquid chromatography, high performance liquid chromatography, ultra high performance liquid chromatography, size exclusion chromatography, ion exchange chromatography, affinity chromatography, expanded adsorption fluidized bed chromatography, reversed phase chromatography, hydrophobic interaction chromatography, and combinations thereof; if the technique is electrophoretic, the electrophoretic technique may be selected from the group consisting of gel electrophoresis, free-flow electrophoresis, electrofocusing, isotachophoresis, affinity electrophoresis, immunoelectrophoresis, reverse electrophoresis, and capillary electrophoresis, capillary zone electrophoresis, and combinations thereof; and if the technique is spectroscopy or spectrometry, the spectroscopy (or spectrometry) technique may be selected from mass spectrometry, ultraviolet spectroscopy, visible spectroscopy, fluorescence spectroscopy, and ultraviolet-visible spectroscopy, and combinations thereof.In certain sub-embodiments, the technique comprises liquid chromatography-mass spectrometry; in other cases, the technique comprises capillary zone electrophoresis coupled to mass spectrometry. The filter can have, for example, a molecular weight cutoff of 30 kDa.

[0011] In yet a fifth aspect, disclosed herein is a method of preparing a polypeptide for analysis by liquid chromatography, capillary zone electrophoresis, or mass spectrometry, comprising the steps of: providing a sample comprising the polypeptide; applying the sample to a 30 kDa MWCO filter; capturing the sample on the filter to produce a retentate; denaturing the sample retentate on the filter; reducing the sample retentate on the filter; alkylating the sample on the filter; digesting the sample on the filter with a first protease; filtering the sample through the same filter and retaining the filtrate; digesting the sample on the filter with a second protease; filtering the sample through the same filter into the previously retained filtrate; quenching active proteases in the filtrate with guanidine; and analyzing the filtrate by liquid chromatography, capillary zone electrophoresis, or mass spectrometry; wherein the second protease is neutrophil elastase, and the first protease is different from the second protease. In such embodiments, the neutrophil elastase can be human neutrophil elastase (EC 3.4.21.37).

[0012] In these five embodiments, the method may be performed at least in part by automation: in some sub-embodiments, addition and exchange of solutions is handled by automated liquid handling equipment or robots.

[0013] Furthermore, in these five aspects, the methods disclosed herein may be used to prepare and analyze therapeutic polypeptides. The therapeutic polypeptides may be selected from the group consisting of antibodies or antigen-binding fragments thereof, derivatives of antibodies or antibody fragments, and fusion polypeptides. Examples of antibodies and antibody constructs include infliximab, bevacizumab, cetuximab, ranibizumab, palivizumab, abagovomab, abciximab, actoxumab, adalimumab, afelimomab, afutuzumab, alacizumab, alacizumab pegol, ald518, alemtuzumab, alirocumab, altumomab, amatuximab, anatumomab mafenatox, anrukinzumab, apolizumab, arcitumomab, acelizumab, altinumab, atlizumab, atolimumab, thrombin ... Silizumab, bapineuzumab, basiliximab, bavituximab, bectumomab, belimumab, benralizumab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bivatuzumab, bivatuzumab mertansine, blinatumomab, brosozumab, brentuximab vedotin, briakinumab, brodalumab, canakinumab, cantuzumab mertansine, caplacizumab, capromab pendetide, carlumab, catumaxomab, cc49, cedelizumab, sertoli Zumab pegol, cetuximab, sitatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, conatumumab, crenezumab, cr6261, dacetuzumab, daclizumab, darotuzumab, daratumumab, demcizumab, denosumab, detumomab, dorlimomab aritox, drozitumab, durigotumab, dupilumab, eclumeximab, eculizumab, edovacomab, edrecolomab, efalizumab, efungumab, elotuzumab , ersilimomab, enabatuzumab, enlimomab pegol, enokizumab, enoticumab, encituximab, epitumomab situxetan, epratuzumab, erenumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evolocumab, exibirimab, fanolesomab, faralimomab, farletuzumab, fasinumab, fbta05, felvizumab, fezakinumab, ficlatuzumab, figitumumab, framvotumab, fontolizumab, foralumab,Foravirumab, fresolimumab, furanumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, gevokizumab, girentuximab, glembatumumab vedotin, golimumab, gomilikimab, gs6624, ibalizumab, ibritumomab tiuxetan, icrucumab, igovomab, imciromab, imgatuzumab, inlacumab, indatuximab ravtansine, infliximab, intetumumab, inolimomab, inotuzumab ozogamicin, ipilimumab, iratumumab, itri uzumab, ixekizumab, keliximab, labetuzumab, lebrikizumab, remaresomab, lerdelimumab, lexatumumab, ribivirumab, ligelizumab, lintuzumab, lirilumab, lorvotuzumab mertansine, lucatumumab, rumiliximab, mapatuzumab, maslimomab, mavrilimumab, matuzumab, mepolizumab, metelimu- mab, milatuzumab, minletumomab, mitumomab, mogamulizumab, morolimumab, motavizumab, moxetumomab passudotox, muromonab-cd3, nacolomab butafenatox, namirumab, naptumomab estab Fenatox, narutuzumab, natalizumab, nebacumab, necitumumab, nerelimomab, nesbacumab, nimotuzumab, nivolumab, nofetumomab merpentan, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab, onartuzumab, oportuzumab monatox, oregovomab, olticumab, otelixizumab, oxelumab, ozanezumab, ozoralizumab, pasivaximab, palivizumab, panitumumab, panobacumab, palsatuzumab, pascolizumab, pateclizumab, patoc Ritumab, Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab, Pidilizumab, Pintumomab, Placlumab, Ponezumab, Priliximab, Pritumumab, PRO140, Quilizumab, Racotumomab, Ladolezumab, Rafivirumab, Ramucirumab, Ranibizumab, Raxibacumab, Regavirumab, Reslizumab, Rilotumumab, Rituximab, Lobatumumab, Loredumab, Romosozumab, Rontalizumab, Rovelizumab, Ruplizumab, Samalizumab, Sarilumab, Satumomabpendetide, Secukinumab, Sevirumab, Sibrotuzumab,Sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, subizumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptox, tefibazumab, terimomab alitox, tenatumomab, tefibazumab, teneliximab, teplizumab, teprotumumab, tezepelumab, TGN1412, tremelimumab, ticilimumab, tildrakizumab, tigatuzumab, TNX-6 50, tocilizumab, toralizumab, tositumomab, tralokinumab, trastuzumab, TRBS07, tregalizumab, tucotuzumab celmoleukin, tuvilumab, ublituximab, urelumab, urtoxazumab, ustekinumab, bapaliximab, batelizumab, vedolizumab, veltuzumab, beparimomab, besencumab, visilizumab, volociximab, borsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, diralimumab, zolimomab alitox, and antibodies thereof shown in Table H. In yet other sub-embodiments, the therapeutic polypeptide is a glycoprotein, a CD polypeptide, a HER receptor polypeptide, a cell adhesion polypeptide, a growth factor polypeptide, an insulin polypeptide, an insulin-related polypeptide, a coagulation polypeptide, a coagulation-related polypeptide, albumin, IgE, a blood group antigen, a colony stimulating factor, a receptor, a neurotrophic factor, an interferon, an interleukin, a viral antigen, a lipoprotein, calcitonin, glucagon, atrial natriuretic factor, a pulmonary surfactant, tumor necrosis factor α and β, enkephalinase, mouse gonadotropin-related peptide, tides, DNAses, inhibins, activing, integrins, protein A, protein D, rheumatoid factors, immunotoxins, bone morphogenetic proteins, superoxide dismutase, surface membrane polypeptides, decay accelerating factors, AIDS envelopes, transport polypeptides, homing receptors, addressins, regulatory polypeptides, immunoadhesins, myostatin, TALL polypeptides, amyloid polypeptides, thymic stromal lymphopoietin, RANK ligand, c-kit polypeptides, TNF receptors and angiopoietins and biologically active fragments thereof,or a variant thereof.

[0014] In some cases, the therapeutic polypeptide is a BiTE® (Bispecific T Cell Engager). [Brief description of the drawings]

[0015] [Figure 1] Figure 1 shows the α-CD3 domain conserved among multiple BiTE® molecules (canonical and half-life extended). Trypsin digestion produces an 8 kDa peptide containing two complementarity determining regions (CDRs) with multiple hotspots. [Diagram 2] FIG. 1 shows enzyme specificity for human neutrophil elastase. P1 corresponds to the C-terminus of one amino acid and P1' corresponds to the N-terminus of the following residue. Relative font size corresponds to the probability of cleavage at that particular residue (adapted from the MEROPS database; (Rawlings et al., 2014)). [Diagram 3] FIG. 1 shows extracted ion chromatograms (EICs) of unstressed and forced-deamidated (pH 8.5, 50° C., t=3 days) samples of BiTE®-1 showing unmodified and deamidated species after forced deamidation. [Figure 4] Tandem mass spectrometry (MSMS) of the three deamidated species observed in Figure 3. The diagnostic b3 ion is shifted +0.9838 Da in peak 3 compared to peaks 1 and 2. This mass shift corresponds to deamidation. [Diagram 5] FIG. 1 shows a schematic diagram highlighting the differences between a single digest for BiTE® molecules, a conventional MAM, and a MAM approach incorporating a 1:1 mix of samples digested with trypsin and samples digested with trypsin followed by neutrophil elastase. [Figure 6]FIG. 1 shows the experimental design used to evaluate the efficacy and robustness of Protocol 3 (for details, see Examples below). [Figure 7] FIG. 1 shows a schematic diagram of the oversample preparation outlined in Protocol 4 (see, e.g., Examples below for further details). [Figure 8] Figure 1 shows trypsin specificity of protocol 4 for BiTE®-1, BiTE®-2 and BiTE®-3. Specificity was first calculated by MassAnalyzer by specifying a stringent trypsin cleavage in the search parameters. The data was then re-searched using a cleavage at the C-terminus of residues KRVITAL (low specificity). Trypsin specificity was the total area of ​​peptides identified using the stringent trypsin search divided by the total area of ​​peptides identified using the KRVITAL cleavage search. This value was expressed as a percentage. [Figure 9] Figure 1 shows the percent of total area corresponding to the 8 kDa linker peptide for BiTE®-1, BiTE®-2, and BiTE®-3. Data was searched by MassAnalyzer using truncations at the C-terminus of residues KRVITAL (low specificity). The total area of ​​peptides corresponding to the 8 kDa linker peptide was divided by the total area of ​​all peptides identified using the KRVITAL truncation search. This value is expressed as a percentage. The percent of total area corresponding to the 8 kDa linker peptide is higher for BiTE®-2 because, as a canonical BiTE® molecule, it does not contain an Fc domain added for half-life extension. [Figure 10] Figure 1 shows an overlay of total ion chromatograms (TIC) of replicate injections of a BiTE®-3 sample prepared using Protocol 3. Injections were prepared from the same vial and separated by approximately 4 days. [Figure 11]Figure 13 shows a TIC overlay of replicate injections of a BiTE®-3 sample prepared using Protocol 4. Injections were prepared from the same vial and separated by approximately 4 days. [Figure 12] Figure 1 shows a TIC overlay comparing BiTE®-2 samples prepared in triplicate using protocols 4 and 5. Two peaks (arrows) observed in four samples from all three protocols are consistent with nonspecific alkylation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] To address the issue of cleavage-resistant polypeptides for MAM analysis, we tested the cleavage site and specificity for a number of commercially available enzymes. Human neutrophil elastase (HNE; EC 3.4.21.37, also known as elastase-2, human leukocyte elastase (HLE), myeloid serine protease, medullasin, and PMN elastase) is reported to cleave at the C-terminus of valine, isoleucine, threonine, alanine, and leucine residues (Figure 2; (Doucet and Overall, 2011; Rawlings et al., 2014)); however, this enzyme also cleaves nonspecifically, a disadvantage that decreases in frequency as substrate length decreases (Stein et al., 1987). Given the amino acid sequence of a target polypeptide, digestion with HNE can serve as a means to monitor regions of interest within a polypeptide, aside from the tendency of HNE to cleave polypeptides nonspecifically.

[0017] For example, we observed that when bispecific T cell engager (BiTE®) molecules are digested by HNE alone, the resulting peptides are highly numerous and low in abundance (due to non-specific cleavage). For example, FIG. 1 shows the α-CD3 domain (canonical and half-life extended) conserved among multiple BiTE® molecules. Trypsin digestion produces an 8 kDa peptide that contains two complementarity determining regions (CDRs) with multiple hot spots that are difficult to identify and characterize using conventional mass spectrometry (MS)-based experiments. This peptide also lacks residues that are susceptible to cleavage by other commonly used enzymes, such as Glu-C and Asp-N.

[0018] The inventors further unexpectedly observed that following on-filter trypsin digestion, both the conserved 8 kDa linker peptide and the molecule-specific linker peptide of the BiTE® molecule were also retained by 30 kDa molecular weight cut-off (MWCO) filters, regardless of whether the filter membrane was composed of regenerated cellulose or polyethersulfone. Enhancing the specificity of HNE by first digesting with a first protease that is not HNE, and exploiting the unexpected retention of the linker peptide resulting from the first protease digestion by the 30 kDa MWCO (or other cut-off) filter, enabled the development of novel, robust methods for characterization of BiTE® molecules (and other polypeptides) and quantification of key attributes.

[0019] A description of a novel sample preparation method is disclosed that uses a first digestion step followed by digestion with HNE.

[0020] definition Both the general description above and the detailed description below are exemplary and explanatory only and are not limiting. The use of the singular includes the plural unless specifically stated otherwise. The use of "or" means "and / or" unless specifically stated otherwise. The use of the term "comprising" and other forms such as "comprises" and "includes" is not limiting. Terms such as "element" or "component" include both elements and components containing one unit and elements and components containing two or more subunits unless specifically stated otherwise. The use of the term "moiety" can include a portion or the entire moiety. When a numerical range is mentioned, such as 1 to 5, all intervening values ​​are expressly included, such as 1, 2, 3, 4, and 5, and fractions thereof such as 1.5, 2.2, 3.4, and 4.1.

[0021] "About" or "to" when modifying an amount (e.g., "about" 3 mM) means that variations around the modified amount can occur. These variations can occur by a variety of means, including typical measuring and processing procedures, inadvertent error, purity of the components, etc.

[0022] "Comprising" and "comprising" are intended to mean that the method includes the recited elements, but does not exclude other unrecited elements. The terms "consisting essentially of" and "consisting essentially of," when used in conjunction with the methods disclosed herein, include the recited elements and exclude unrecited elements that would alter the basic nature of the method, but do not exclude other unrecited elements. The terms "consisting of" and "consisting of," when used to define a method, exclude substantial method steps. Embodiments defined by each of these transition terms are within the scope of the present disclosure.

[0023] "Coupled" means directly as well as indirectly related. For example, a device or process may be directly related to another device or process, or these devices or processes may be indirectly related to each other, for example, through another device or process.

[0024] "Protein," "peptide," and "polypeptide" are used interchangeably to refer to a chain of amino acids in which each amino acid is connected to the next by a peptide bond.

[0025] "Denaturation," "denaturing," "denaturing," and the like refer to a process in which polypeptides at least partially lose the quaternary, tertiary, and secondary structure found in the polypeptides in their native state by applying an external stress or reagent (i.e., a denaturing agent).

[0026] "Denaturing agent" refers to any substance, composition, energy, or condition capable of denaturing a polypeptide. Examples of denaturing agents include strong acids or bases, inorganic salts, organic solvents, radiation, chaotropic agents, or heat, or combinations thereof.

[0027] "Denatured polypeptide", "denatured protein" and the like refer to a polypeptide having its secondary, tertiary and / or quaternary structure altered from a native polypeptide. A polypeptide may be fully denatured or partially denatured. "Non-denatured polypeptide" or "non-denatured protein" (and similar terms) refer to a polypeptide that maintains its secondary, tertiary and, where applicable, quaternary structure. "Natural polypeptide" or "natural protein" (and the like) refer to a polypeptide found in nature, having any primary reference sequence listed in the UniProt Knowledgebase database (The UniProt, 2017) or the UniGene database (Pontius et al., 2003).

[0028] "Reduced polypeptide" or "reduced protein" (and similar terms) means a polypeptide in which at least one of its interchain or intrachain disulfide bonds has been broken. Such bonds may form, for example, between reduced thiol groups, such as those available on cysteine ​​residues.

[0029] "Alkylated polypeptide" or "alkylated protein" (and the like) refers to a polypeptide to which an alkyl group has been transferred. In practice, polypeptides are often alkylated on thiol groups (e.g., as available on cysteine ​​residues) to prevent reduced thiols from forming or from reforming disulfide bonds or crosslinks after reduction.

[0030] "Digestion" and the like, in the context of a polypeptide, refers to the fragmentation of the polypeptide into two or more fragments, which fragmentation may be mediated by another agent, chemical, or enzyme.

[0031] "Proteolytic cleavage," "proteolytic digestion," and the like, refer to the cleavage of a polypeptide by breaking peptide bonds within the polypeptide, thus generating fragments. Proteolytic cleavage can be mediated by enzymes.

[0032] "Protease," "peptidase," "proteolytic cleaving enzyme," "endoproteinase," "proteinase," and the like refer to an enzyme, a polypeptide or fragment thereof that catalyzes the hydrolysis of peptide bonds. Protease includes variants of the amino acid sequences of known proteases that catalyze peptide bond hydrolysis, even if such catalytic activity is reduced in the variant.

[0033] Enzyme-catalyzed reactions are classified according to the Enzyme Commission numbering system. Proteases catalyze peptide bond hydrolysis, so they are classified as aminopeptidases in EC 3.4.11, dipeptidases in EC 3.4.13, dipeptidyl peptidases and tripeptidyl peptidases in EC 3.4.14, peptidyl-dipeptidases in EC 3.4.15, serine-type carboxypeptidases in EC 3.4.16, metallocarboxypeptidases in EC 3.4.17, and cytosine-type carboxypeptidases in EC 3.4.18. The EC 3.4 class includes endoproteinases, endoproteinases of unknown catalytic mechanism, EC 3.4.99, endoproteinases of unknown catalytic mechanism, EC 3.4.19, omega-peptidases, serine proteases, EC 3.4.21, cysteine ​​proteases, EC 3.4.22, aspartic endopeptidases, EC 3.4.23, metallopeptidases, EC 3.4.24, threonine endopeptidases, EC 3.4.25, and endoproteinases of unknown catalytic mechanism, EC 3.4.99. Specific examples of proteases include trypsin (EC 3.4.21.4), endoproteinase Asp-N (EC 3.4.24.33), and endoproteinase Glu-C (EC 3.4.21.19).

[0034] Elastases (except those classified as EC 3.4.21.70) are proteases that hydrolyze elastin, the elastic fiber polypeptide of the extracellular matrix. There are eight human genes that code for elastases (Table A):

[0035] [Table 1]

[0036] [Table 2]

[0037] "Neutrophil elastase" (sometimes called elastase-2, leukocyte elastase, myeloid serine protease, medullasin, or PMN elastase) is an elastase secreted by neutrophils and macrophages during inflammation that destroys bacteria. Human neutrophil elastase (HNE) was first described in 1968 (Janoff and Scherer, 1968). HNE was shown to differ from pancreatic elastase in that soybean trypsin inhibitor and salivary kallikrein inhibitor inhibit HNE but not pancreatic elastase activity. HNE maintains activity at physiological salt concentrations, whereas pancreatic elastase activity is reduced. HNE is at least 10-fold more resistant to serum elastase inhibitors than pancreatic elastase (as defined by Janoff and Scherer, 1968). Finally, HNE is also less pH-tolerant than pancreatic elastase (Janoff and Scherer, 1968).

[0038] Sinha et al. (Sinha et al., 1987) first reported the amino acid sequence of HNE and demonstrated that the polypeptide has only 43% sequence homology with porcine pancreatic elastase. The UniProte database for UniProtKB-P08246 (ELNE_HUMAN) (The UniProt, 2017) describes ELANE as encoding HNE as a 267 amino acid precursor polypeptide consisting of a signal peptide (residues 1-27), a propeptide domain (residues 28-29) and a mature polypeptide (residues 30-267). Within the mature polypeptide, a peptidase S1 domain (residues 30-247) is present. Disulfide bonds can be found forming between residues 55 and 71, 151 and 208, 181 and 187 and 198-223. The asparagine residues at positions 88, 124 and 174 can be glycosylated (N-linked). The sequence for HNE is shown in Table B, where the signal peptide is in bold, the propeptide domain is single underlined, the mature polypeptide is in italics, and the S1 peptidase domain is double underlined, italics and bold.

[0039] [Table 3]

[0040] HNE is capable of hydrolyzing nearly all polypeptide members of the extracellular matrix, including several types of collagen, fibronectin, proteoglycans, heparin and cross-linked fibrin.

[0041] HNE cleaves peptide bonds in which the P1 residue is a small alkyl group. The substrate specificity is summarized in Figure 2.

[0042] "Chemical cleavage" in the context of generating fragments of a polypeptide means fragmentation of a polypeptide with a chemical. A "chemical" is a non-proteolytic substance or compound. A chemical can be organic or inorganic.

[0043] "Molecular weight cutoff" or "MWCO" refers to the MWCO of a membrane being an expression of the membrane selectivity for solute molecules of various molecular weights (MW), where the MW values ​​(expressed in Daltons (Da)) are derived from solute molecules that result in 90% rejection when a range of different MW solutes are filtered in a target solvent (which is water for most liquid-based pressure-driven membrane applications), where rejection is determined by Equation (1):

number

[0044] MWCO is determined experimentally using dextrans, polyethylene glycols, and proteins of various molecular weights to evaluate the MWCO of a membrane or filter. Rejection depends on a number of solutes and process parameters such as solute type, concentration, hydrodynamics, pressure, temperature, and pH. MWCO measurements are usually performed in separate experiments using different solutes, each with a given MW.

[0045] For the low range of MW (below approximately 10 kDa), polyethylene glycols (PEGs), n-alkanes and oligostyrenes can be used in solute inhibition assays. For the high range of MW (above approximately 10 kDa), dextrans and sugars are often used (Rohani et al., 2011).

[0046] method Disclosed herein is a method of digesting a polypeptide in a sample with a first protease and a second protease, where the first protease generates at least two fragments of the polypeptide, which are subsequently digested by a second protease; and analyzing the sample after digestion with the second protease, where the second protease is neutrophil elastase; and the first protease is different from the second protease. Other steps in the method may include dividing the sample into two aliquots after digesting the polypeptide in the sample with the first protease, and then digesting the polypeptide in one of the two aliquots with the second protease. These aliquots may then be combined before analysis. The polypeptide in the sample may be denatured and / or reduced and / or alkylated before digestion with the first protease. Analysis may include chromatography, electrophoresis, spectrometry, and combinations thereof.

[0047] In some embodiments, the method includes applying a sample to a filter having a MWCO before digesting the polypeptides in the sample on the filter with a first protease, digesting the polypeptides in the sample with a second protease, and analyzing the sample. Such embodiments can further incorporate a filtering step. The MWCO filter, in some embodiments, retains a significant proportion of polypeptides and polypeptide fragments, even when the size of these polypeptides or polypeptide fragments has a MW significantly smaller than the MWCO of the filter.

[0048] Method Steps Polypeptide Denaturation In some embodiments, the polypeptides prepared and analyzed according to the methods disclosed herein are denatured.

[0049] Polypeptides can be denatured using a variety of art-recognized techniques and denaturants. In some embodiments, multiple denaturants are used together, either simultaneously or sequentially. For example, the denaturants of SDS and heat can be combined to denature a polypeptide.

[0050] Denaturation of proteins can be performed by any means that disrupts quaternary, tertiary, or secondary polypeptide structure, for example, the use of chaotropes such as urea and denaturing detergents (e.g., sodium dodecyl sulfate (SDS)), heat, reducing agents, and agents that inactivate reactive thiol groups to block disulfide reformation. The pH of the polypeptide-containing sample can also be manipulated to promote denaturation. These components are often used together to effectively unravel polypeptide chains.

[0051] Additional examples of chaotropes, in addition to urea, include n-butanol, ethanol, guanidine chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, and thiourea. Urea is preferred in most cases.

[0052] Detergents are classified according to the form of hydrophilic group: anionic, cationic, nonionic and zwitterionic. Anionic and cationic detergents are more likely to be denaturing, examples of which include: SDS, sodium cholate, sodium deoxycholate, sodium glycolate, sodium taurocholate, sodium taurodeoxycholate, N-lauroyl sarcosine, lithium dodecyl sulfate (anionic) and hexadecyltrimethylammonium bromide (CTAB) and trimethyl(tetradecyl)ammonium bromide (TTAB) (cationic). In some cases zwitterionic detergents may be useful, examples of which include amidosulfobetaine-14 (ASB-14), amidosulfobetaine-16 (ASB-16), C7Bz0, CHAPS, CHAPSO, EMPIGEN® BB, 3-(N,N-dimethyloctylammonio)propanesulfonate inner salt (SB3-8), d(decyldimethylammonio)propanesulfonate inner salt (SB3-10), etc. Anionic detergents are preferred, especially SDS.

[0053] A denaturing agent can be heat (for most polypeptides), e.g., elevated temperatures of 30° C. or greater. Denaturing agents include stirring. In some embodiments, essentially or substantially low or no salt can denature a polypeptide.

[0054] The denaturant can be a solvent, such as ethanol or other alcohol.

[0055] Steps for denaturing polypeptides have been extensively studied and reported; see, for example, (Tanford, 1968) for details. Those skilled in the art know how to denature a polypeptide given the properties of the polypeptide and the numerous denaturing agents to choose from.

[0056] Polypeptide reduction A reduced polypeptide is a polypeptide that has been exposed to reducing conditions sufficient to reduce reducible residues in the polypeptide structure, such as cysteine. If the reduced polypeptide contains a thiol group or a sulfur-containing residue, the thiol group in the reduced polypeptide is reduced. A reduced polypeptide that contains a cysteine ​​residue has the sulfur atom of the cysteine ​​residue reduced, which may be designated as "-SH". A reduced polypeptide may be a disulfide bond-containing polypeptide. A disulfide bond-containing polypeptide may be reduced by exposure to reducing conditions that induce one or more disulfide bonds (disulfide bridges) in the disulfide bond-containing polypeptide to break.

[0057] A "reducing agent," "reductant," or "reducer" is an element or compound that loses (or donates) electrons to another chemical species in an oxidation-reduction chemical reaction. Reducing agents allow disulfide groups to become reactive by generating thiol (-SH) groups. Common polypeptide reducing agents are shown in Table C.

[0058] [Table 4]

[0059] In some embodiments, the denaturation and reduction of the polypeptide are carried out simultaneously, hi other embodiments, the denaturation and reduction of the polypeptide are carried out in separate steps.

[0060] One of skill in the art knows how to reduce a polypeptide given the nature of the polypeptide and the reducing agent selected.

[0061] Alkylation of polypeptides contained in a sample "Deactivating reactive thiol groups" refers to blocking free thiol groups in a polypeptide to prevent undesired thiol-disulfide exchange reactions. Alkylating agents are substances that induce the replacement of hydrogen with an alkyl group.

[0062] The alkylation of free cysteines, which frequently occurs after their reduction, prevents the formation and reformation of disulfide bonds that would otherwise form between the free thiols of cysteine ​​residues. Commonly used alkylating agents include n-ethylmaleimide (NEM), iodoacetamide (IAA) and iodoacetic acid. Other suitable alkylating agents include dithiobis(2-nitro)benzoic acid; acrylamide; 4-vinylpyridine; nitrogen mustards, such as chlorambucil and cyclophosphamide; cisplatin; nitrosoureas, such as carmustine, lomustine and semustine; alkyl sulfonates, such as busulfan; ethyleneimines, such as thiotepa; and triazines, such as decarbazine. Those skilled in the art are aware of the reagents that can be used to protect sulfhydryl groups and the methods of using such reagents.

[0063] Digestion of Polypeptides The methods disclosed herein include cleaving a polypeptide of interest in a sample in a first digest, the cleaving producing at least two fragments of the polypeptide. Any method of fragmenting a polypeptide may be used, provided that at least two fragments of the polypeptide are produced; further, provided that complete degradation of the polypeptide, e.g., into single amino acids, is not desired.

[0064] In such digestion steps, a convenient means of cleavage includes using a protease (wherein the protease is not neutrophil elastase). Any protease can be used as long as such protease cleaves the polypeptide into at least two fragments. The rationale behind this first digestion prior to digestion with neutrophil elastase is to present the polypeptide fragments to neutrophil elastase (such as human neutrophil elastase) where neutrophil elastase has increased specificity compared to when presented with the intact polypeptide.

[0065] In some embodiments, a mixture of two or more proteases may be used. In other embodiments, the first digestion may include multiple sequential digestions. For example, a first digestion is performed with a first protease, and then in a subsequent reaction, a second digestion is performed with a second protease (wherein neither protease is neutrophil elastase). The second (or subsequent) digestion is completed to improve the specificity of the neutrophil elastase used in the step of further cleaving the polypeptide with neutrophil elastase.

[0066] Examples of useful proteases (but not all inclusive) include trypsin, endoproteinase Glu-C, endoproteinase Arg-C, pepsin, chymotrypsin, chymotrypsin B, Lys-N protease, Lys-C protease, Glu-C protease, Asp-N protease, pancreatopeptidase, carboxypeptidase A, carboxypeptidase B, proteinase K, and thermolysin. In some embodiments, combinations of these proteases are used. In some embodiments, trypsin alone is used.

[0067] These and other proteases, including their peptide bond selectivities and EC numbers, are listed in Table D, which is adapted from (Unspecified, 2007). The sources listed for each protease are for illustration only; many of these proteases are commercially available.

[0068] In some embodiments, a protein:protease ratio (w / w) of 10:1, 20:1, 25:1, 50:1 or 100:1 can be used. In some embodiments, the ratio is 20:1. In some embodiments, the neutrophil elastase used is at a concentration of about 100 ng / mL to 1 mg / mL, or about 100 ng / mL to 500 μg / mL, or about 100 ng / mL to 100 μg / mL, or about 1 μg / mL to 1 mg / mL, or about 1 μg / mL to 500 μg / mL, or about 1 μg / mL to 100 μg / mL, or about 10 μg / mg to 1 mg / mL, or about 10 μg / mg to 500 μg / mL, or about 10 μg / mg to 100 μg / mL. In some embodiments, the digestion step is for 10 minutes to 48 hours, or 30 minutes to 48 hours, or 30 minutes to 24 hours, or 30 minutes to 16 hours, or 1 hour to 48 hours, or 1 hour to 24 hours, or 1 hour to 16 hours, or 1 hour to 8 hours, or 1 hour to 6 hours, or 1 hour to 4 hours. In some embodiments, the digestion step is incubated at a temperature of 20°C to 45°C, or 20°C to 40°C, or 22°C to 40°C, or 25°C to 37°C. A person skilled in the art can select appropriate conditions (buffer, incubation time, amount of protease, volume, etc.) since in vitro protease digestion is clearly understood in the art.

[0069] [Table 5]

[0070] [Table 6]

[0071] In some embodiments, the first digestion is performed using a chemical. Particularly useful chemicals include chemicals that cleave polypeptides in a site-specific manner. Such chemicals include cyanogen bromide (CNBr; carbononitridic bromide), which cleaves C-terminally of methionine residues; 2-nitro-5-thiocyanobenzoate (NTCB), which cleaves N-terminally of cysteine ​​residues; asparagine-glycine dipeptides, which can be cleaved using hydroxylamine; formic acid, which cleaves at aspartic acid-proline (Asp-Pro) peptide bonds, and BNPS-skatole (3-bromo-3-methyl-2-(2-nitrophenyl)sulfanylindole), which cleaves C-terminally of tryptophan residues. Those skilled in the art will know how to select appropriate variables, including polypeptide concentration, chemical concentration, incubation time and temperature, etc. See, for example, further (Crimmins et al., 2001; Li et al., 2001; Tanabe et al., 2014).

[0072] In some embodiments, the first digestion can include multiple sequential digestions, where at least one of such sequential digestions includes using chemicals such as CNBr, NTCB, hydroxylamine, formic acid, and BNPS-skatole. For example, a first digestion is performed with a chemical that cleaves the polypeptide into at least two fragments, and then in a subsequent reaction, a second digestion is performed with a protease (where the protease is not neutrophil elastase), or vice versa. The second (or subsequent) digestion is completed to improve the specificity of the neutrophil elastase used in the step of further cleaving the polypeptide with neutrophil elastase.

[0073] In the methods disclosed herein, the subsequent digestion is carried out with neutrophil elastase. In some embodiments, the neutrophil elastase is human.

[0074] In some embodiments, protein:neutrophil elastase ratios (w / w) of 10:1, 20:1, 25:1, 50:1 or 100:1 can be used. In some embodiments, the ratio is 20:1. In some embodiments, the neutrophil elastase used is at a concentration of about 100 ng / mL to 1 mg / mL, or about 100 ng / mL to 500 μg / mL, or about 100 ng / mL to 100 μg / mL, or about 1 μg / mL to 1 mg / mL, or about 1 μg / mL to 500 μg / mL, or about 1 μg / mL to 100 μg / mL, or about 10 μg / mg to 1 mg / mL, or about 10 μg / mg to 500 μg / mL, or about 10 μg / mg to 100 μg / mL. In some embodiments, the digestion step is for 10 minutes to 48 hours, or 30 minutes to 48 hours, or 30 minutes to 24 hours, or 30 minutes to 16 hours, or 1 hour to 48 hours, or 1 hour to 24 hours, or 1 hour to 16 hours, or 1 hour to 8 hours, or 1 hour to 6 hours, or 1 hour to 4 hours. In some embodiments, the digestion step is incubated for 35 minutes. In some embodiments, the digestion step is incubated at a temperature of 20°C to 45°C, or 20°C to 40°C, or 22°C to 40°C, or 25°C to 37°C. In some embodiments, the digestion step is incubated at 37°C. One skilled in the art would be able to understand the various parameters and select appropriate conditions for digestion using neutrophil elastase.

[0075] Divide the sample into aliquots In embodiments, before a sample containing a polypeptide to be analyzed is digested with neutrophil elastase, the sample is divided into aliquots. In some cases, the step of dividing the sample into aliquots is performed by simply taking half the volume of the sample before digestion with neutrophil elastase. In some cases, the aliquots can be equalized for polypeptide concentration using, for example, ultraviolet spectrophotometry.

[0076] Selecting a filter When selecting the appropriate MWCO for a particular application, many factors are considered, including the concentration, composition, molecular shape of the sample, and operating conditions such as temperature, pressure, and cross-flow velocity. Other variables related to molecular passage flow are also taken into consideration. For example, linear molecules, high transmembrane pressure (TMP), and low sample concentration can increase molecular passage, while low temperature and membrane fouling can decrease molecular passage. Validation methods for MWCO are not necessarily comparable, as they vary from manufacturer to manufacturer. It is generally recommended in the art to select a MWCO that is at least two times smaller than the molecular weight of the solute being retained.

[0077] For the methods disclosed herein, filter geometry is also a consideration: for volumes between 1 and 2 mL, filter units in which the filter is tilted when the filter unit is perpendicular to the substrate are less desirable than filter units in which the filter is horizontal when the filter unit is perpendicular to the substrate.

[0078] Suitable materials for the MWCO membrane are hydrophilic, including polyethersulfone, polyvinylidene difluoride, and regenerated cellulose.

[0079] Suitable cut-off filters are commercially available, for example, from Pall Corporation (Port Washington, NY) or Millipore, Inc. (Burlington, MA, e.g., Microcon® filters). For the methods disclosed herein, filters from these particular manufacturers have MWCO specifications that are appropriate. Comparable filters from other manufacturers can also be used. The MWCO specifications provided by manufacturers can be trusted because they fairly take into account the differences between MWCO ratings between manufacturers, and the methods disclosed herein take advantage, in part, of the surprising observation that low molecular weight molecules are retained by filters even when these molecules have molecular weights that are equal to or less than (and often substantially lower than) the rated MWCO of the filter (see, e.g., Example 11).

[0080] Filtering the sample using a MWCO filter can use any suitable filtering method using any suitable filter device. Filtering can occur by gravity, capillary force, or conventional centrifugation, including ultracentrifugation.

[0081] Steps to analyze the sample After the polypeptide is digested with neutrophil elastase, the resulting polypeptide fragments can be analyzed by any suitable method. The procedural discussion is not intended to limit in any way the methods that can be used to analyze the prepared polypeptide.

[0082] In general, suitable analytical methods may be chromatography, electrophoresis and spectrometry, some of which may be combined.

[0083] The skilled person has access to handbooks, including, for example, (Gunzler and Williams, 2001), which facilitate the selection of suitable analytical methods as well as, for example, suitable conditions for carrying out these methods.

[0084] Chromatography is a method of separating polypeptide fragments in a mobile phase, which is processed through a structure that holds the stationary phase. Because the polypeptide fragments differ in size and composition, each fragment has a unique partition coefficient. Because of the different partition coefficients, the polypeptides are differentially retained on the stationary phase. Examples of such methods known in the art include gas chromatography, liquid chromatography, high performance liquid chromatography, ultra high performance liquid chromatography, size exclusion chromatography, ion exchange chromatography, affinity chromatography, expanded adsorption fluidized bed chromatography, reversed phase chromatography, and hydrophobic interaction chromatography.

[0085] A summary of some of the known chromatographic methods is given in Table E.

[0086] [Table 7]

[0087] [Table 8]

[0088] The prepared polypeptides can be further analyzed using electrophoretic techniques - gel electrophoresis, free-flow electrophoresis, isoelectric focusing, isotachophoresis, affinity electrophoresis, immunoelectrophoresis, reverse electrophoresis, capillary electrophoresis and capillary zone electrophoresis. The skilled person can obtain overviews and handbooks such as (Kurien and Scofield, 2012; Lord, 2004).

[0089] Electrophoresis can be used to analyze charged molecules, such as polypeptides that are not at their isoelectric point, that are transported through a solvent by an electric field. Polypeptides move at a velocity proportional to their charge density. The mobility of a polypeptide through an electric field depends on: the strength of the electric field, the net charge on the polypeptide, the size and shape of the polypeptide, the ionic strength, and the properties of the matrix through which the polypeptide moves (e.g., viscosity, pore size). Polyacrylamide and agarose are two common support matrices. These matrices function as porous media and behave like molecular sieves. Polyacrylamide forms a support with smaller pore sizes, which is particularly useful in the methods disclosed herein and is ideal for separating the majority of polypeptide fragments.

[0090] Table F provides examples of electrophoretic techniques for polypeptides.

[0091] [Table 9]

[0092] [Table 10]

[0093] [Table 11]

[0094] The prepared polypeptides can be further analyzed using spectrophotometry-mass spectrometry (Rubakhin and Sweedler, 2010), ultraviolet spectrometry, visible spectrometry, fluorescence spectrometry and ultraviolet-visible spectrometry (Nowicka-Jankowska, 1986).

[0095] Table G provides examples of electrophoretic techniques for polypeptides.

[0096] [Table 12]

[0097] The enabling principle of mass spectrometry (MS) consists of ionizing chemical compounds to produce charged molecules or molecular fragments, and then measuring their mass-to-charge ratio. In an exemplary MS procedure, a sample is loaded into an MS instrument and vaporized, the components of the sample are ionized by one of a variety of methods (e.g., by bombarding them with an electron beam) that result in the formation of positively charged particles, the positive ions are then accelerated by a magnetic field, and a calculation is made of the mass-to-charge ratio (m / z) of the particles based on details of the motion of the ions as they pass through the electromagnetic field, and detection of the ions is performed which are classified according to their m / z ratio.

[0098] An illustrative MS instrument has three modules: an ion source, which converts gas phase sample molecules into ions (or, in the case of electrospray ionization, moves ions present in solution into the gas phase); a mass analyzer, which sorts the ions by their mass-to-charge ratio by applying an electromagnetic field; and a detector, which measures the numerical value of an indicator quantity and thus provides data for calculating the abundance of each ion present.

[0099] MS techniques have both qualitative and quantitative uses, including identifying unknown compounds, determining the isotopic composition of elements within a molecule, and determining the structure of a compound by observing its fragmentation. Included are Gas Chromatography-Mass Spectrometry (GC / MS or GC-MS), Liquid Chromatography-Mass Spectrometry (LC / MS or LC-MS), and Ion Mobility Spectrometry / Mass Spectrometry (IMS / MS or IMMS).

[0100] The analytical methods (chromatography, electrophoresis and spectrometry) can be combined, such as liquid chromatography-mass spectrometry, capillary zone electrophoresis coupled to mass spectrometry and ion mobility spectrometry-mass spectrometry.

[0101] automation Various steps of the methods disclosed herein can be performed using liquid handling robots. Such robots dispense reagents, samples, or other liquids into designated containers. The robots are controlled either by software integrated directly into the robot itself or by a connected computer. By automating liquid handling, the methods disclosed herein can be performed with high throughput, fewer errors, and reduced hands-on analyst time.

[0102] Liquid handling robots can be configured with a variety of laboratory tools such as centrifuges, PCR machines, colony pickers, shaking devices, heating instruments, etc. Such customization allows these machines to be adapted to specific methods.

[0103] In some cases, such robots replace the use of pipettes and / or syringes by using acoustics to move liquids (acoustic liquid processing).

[0104] Currently, Agilent Technologies (Santa Clara, CA), Beckman Coulter, Inc. (Indianapolis, IN), Eppendorf North America (Hauppauge, NY), Hamilton Robotics (Reno, NV), Hudson Robotics, Inc. (Springfield, NJ), and Tecan AG (Mannedorf, Switzerland) are some of the manufacturers of such robots.

[0105] Therapeutic Polypeptides Polypeptides, including those that bind to one or more of the following, can be prepared and analyzed in the methods disclosed herein. These include CD proteins, including CD3, CD4, CD8, CD19, CD20, CD22, CD30 and CD34, including those that disrupt receptor binding; HER receptor family proteins, including HER2, HER3, HER4 and EGF receptor; cell adhesion molecules, such as LFA-I, MoI, pl50, 95, VLA-4, ICAM-I, VCAM and αv / β3 integrin. Growth factors such as vascular endothelial growth factor ("VEGF"), growth hormone, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, growth hormone releasing factor, parathyroid hormone, Müllerian inhibitory substance, human macrophage inflammatory protein (MIP-1α), erythropoietin (EPO), nerve growth factors such as NGF-β, platelet derived growth factors (PDGF), fibroblast growth factors including aFGF and bFGF, epidermal growth factor (EGF), transforming growth factors (TGFs) including, in particular, TGF-α and TGF-β, such as TGF-β1, TGF-β2, TGF-β3, TGF-β4 or TGF-β5, insulin-like growth factor I and insulin-like growth factor II (IGF-I and IGF-II), des(1-3)-IGF-I (brain IGF-I) and bone morphogenetic factors. Insulin and insulin-related proteins including insulin, insulin A chain, insulin B chain, proinsulin, and insulin-like growth factor binding proteins. (vii) other blood and serum proteins, including, but not limited to, albumin, IgE and blood group antigens, such as colony stimulating factors and their receptors, including, among others, M-CSF, GM-CSF and G-CSF and their receptors, such as the CSF-1 receptor (c-fms).Receptors and receptor-associated proteins, including, for example, the flk2 / flt3 receptor, the obesity (OB) receptor, the LDL receptor, the growth hormone receptor, the thrombopoietin receptor ("TPO-R", "c-mpl"), the glucagon receptor, the interleukin receptor, the interferon receptor, the T cell receptor, the stem cell factor receptor such as c-Kit, and other receptors. Receptor ligands, including, for example, OX40L, which is the ligand for the OX40 receptor. Neurotrophic factors, including bone-derived neurotrophic factor (BDNF) and neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6). Interferons and interferon receptors, including, for example, interferon-α, -β, and -γ, and their receptors. Interleukins and interleukin receptors, including IL-1-IL-33 and IL-1-IL-33 receptors, including, for example, the IL-8 receptor, among others. Viral antigens, including AIDS enveloped virus antigens. Lipoproteins, calcitonin, glucagon, atrial natriuretic factor, pulmonary surfactant, tumor necrosis factor-α and -β, enkephalinase, RANTES (regulated on activation normally T-cell expressed and secreted), mouse gonadotropin-related peptide, DNAse, inhibin and activin, integrins, protein A or D, rheumatoid factors, immunotoxins, bone morphogenetic proteins (BMPs), superoxide dismutase, surface membrane proteins, decay accelerating factor (DAF), AIDS envelope, transport proteins, homing receptors, addressins, regulatory proteins, immunoadhesins, antibodies. Myostatin, TALL-I, amyloid proteins, including but not limited to amyloid beta protein, thymic stromal lymphopoietin ("TSLP"), RANK ligand ("OPGL"), TALL proteins, including c-kit, TNF receptors, including TNF receptor type 1, TRAIL-R2, angiopoietins, as well as biologically active fragments or analogs or variants of any of the foregoing.

[0106] Exemplary polypeptides and antibodies include Activase® (alteplase); alirocumab, Aranesp® (darbepoetin-α), Epogen® (epoetin α or erythropoietin); Avonex® (interferon beta-1a); Bexxar® (tositumomab); Betaseron® (interferon beta); bococizumab (anti-PCSK9 monoclonal antibody designated L1L3, see U.S. Pat. No. 8,080,243); Campath® (alemtuzumab); Dynepo® (epoetin delta); Velcade® (bortezomib); MLN0002 (anti-α4β7 mAb); MLN1202 (anti-CCR2 chemokine receptor mAb); Enbrel® (etanercept); Eprex® (epoetin alfa); Erbitux® (cetuximab); evolocumab; Genotropin® (somatropin); Herceptin® (trastuzumab); Humatrope® (somatropin [rDNA derived] injection); Humira® (adalimumab); Infergen® (interferon alfa con-1); Natrecor® (nesiritide); Kineret® (anakinra), Leukine® (sargamostim); LymphoC ide® (epratuzumab); Benlysta™ (belimumab); Metalyse® (tenecteplase); Mircera® (methoxypolyethylene glycol epoetin beta); Mylotarg® (gemtuzumab ozogamicin); Raptiva® (efalizumab); Cimzia® (certolizumab pegol); Soliris™ (eculizumab); pexelizumab (anti-C5 complement); MEDI-524 (Numax®); Lucentis® (ranibizumab); edrecolomab (Panorex®); Trabio® (lerdelimumab); TheraCim hR3 (nimotuzumab); Omnitarg (pertuzumab, 2C4); Osidem® (IDM-1);OvaRex® (B43.13); Nuvion® (vigilizumab); cantuzumab mertansine (huC242-DMl); NeoRecormon® (epoetin beta); Neumega® (oprelvekin); Neulasta® (PEGylated filgastrim, PEGylated G-CSF, PEGylated hu-Met-G-CSF); Neupogen® (filgrastim); Orthoclone OKT3® (muromonab-CD3), Procrit® (epoetin alfa); Remicade® (infliximab), Reopro® (abciximab), Actemra® (anti-IL6 receptor mAb), Avastin® (bevacizumab), HuMax-CD4 (zanolimumab), Rituxan® (rituximab); Tarceva® (erlotinib); Roferon-A® (interferon alfa-2a); Simulect® (basiliskine) cimabu); Stelara™ (ustekinumab); Prexige® (lumiracoxib); Synagis® (palivizumab); 146B7-CHO (anti-IL15 antibody, see U.S. Pat. No. 7,153,507), Tysabri® (natalizumab); Valortim® (MDX-1303, an anti-B. anthracis protective antigen mAb); ABthrax™; Vectibix® (panitumumab); Xolair® (omalizumab), ETI211 (anti-MRSA mAb), IL-1 Trap (the Fc portion of human IgG1 and the extracellular domains of both IL-I receptor components (type 1 receptor and receptor accessory protein)), VEGF Trap (the Ig domain of VEGFR1 fused to IgG1 Fc), Zenapax® (daclizumab); Zenapax® (daclizumab), Zevalin® (ibritumomab tiuxetan), Zetia (ezetimibe), atacicept (TACI-Ig), anti-α4β7 mAb (vedolizumab); galiximab (anti-CD80 mAb), anti-CD23 mAb (lumiliximab);BR2-Fc (huBR3 / huFc fusion protein, soluble BAFF antagonist); Simponi™ (golimumab); mapatuzumab (human anti-TRAIL receptor-1 mAb); ocrelizumab (anti-CD20 human mAb); HuMax-EGFR (zalutumumab); M200 (volociximab, anti-α5β1 integrin mAb); MDX-010 (ipilimumab, anti-CTLA-4 mAb and VEGFR-1 (IMC-18F1); anti-BR3 mAb; anti-C. difficile toxin A and toxin BC mAbs MDX-066 (CDA-1) and MDX-1388); anti-CD22 dsFv-PE38 conjugates (CAT-3888 and CAT-8015); anti-CD25 mAb (HuMax-TAC); anti-TSLP antibody; anti-TSLP receptor antibody (U.S. Pat. No. 8,101,182); anti-TSLP antibody designated A5 (U.S. Pat. No. 7,982,016); (anti-CD3 mAb (NI-0401)); adecatumumab (MT201, anti-EpCAM-CD326 mAb); MDX-060, SGN-30, SGN-35 (anti-CD30 mAb); MDX-1333 (anti-IFNAR); HuMax CD38 (anti-CD38 mAb); anti-CD40L mAb; anti-Cripto mAb; anti-CTGF idiopathic pulmonary fibrosis stage 1 fibrogen (FG-3019); anti-CTLA4 mAb; anti-eotaxin 1 mAb (CAT-213); anti-FGF8 mAb; anti-ganglioside GD2 mAbs; anti-sclerostin antibodies (see U.S. Pat. Nos. 8,715,663 or 7,592,429), anti-sclerostin antibodies designated Ab-5 (see U.S. Pat. Nos. 8,715,663 or 7,592,429); anti-ganglioside GM2 mAb; anti-GDF-8 human mAb (MYO-029); anti-GM-CSF receptor mAb (CAM-3001); anti-HepC mAb (HuMax HepC); MEDI-545, MDX-1103 (anti-IFNα mAb); anti-IGF1R mAb; anti-IGF-1R mAb (HuMax-Inflam); anti-IL12 / IL23p40 mAb (briakinumab); anti-IL-23p19 mAb (LY2525623); anti-IL13 mAb (CAT-354); anti-IL-17 mAb (AIN457); anti-IL2Ra mAb (HuMax-TAC);Anti-IL5 receptor mAb; anti-integrin receptor mAb (MDX-O18, CNTO95); anti-IPIO ulcerative colitis mAb (MDX-1100); anti-LLY antibody; BMS-66513; anti-mannose receptor / hCGβ mAb (MDX-1307); anti-mesothelin dsFv-PE38 conjugate (CAT-5001); anti-PDlmAb (MDX-1 106 (ONO-4538)); anti-PDGFRα antibody (IMC-3G3); anti-TGFβ mAb (GC-1008); anti-TRAIL receptor-2 human mAb (HGS-ETR2); anti-TWEAK mAb; anti-VEGFR / Flt-1 mAb; anti-ZP3 mAb (HuMax-ZP3); NVS antibody #1; NVS antibody #2 and amyloid β monoclonal antibody.

[0107] Examples of suitable antibodies for the present methods and pharmaceutical formulations include those shown in Table H. Other examples of suitable antibodies include infliximab, bevacizumab, cetuximab, ranibizumab, palivizumab, abagovomab, abciximab, actoxumab, adalimumab, afelimomab, afutuzumab, alacizumab, alacizumab pegol, ald518, alemtuzumab, alirocumab, altumomab, amatuximab, anatumomab mafenatox, anrukinzumab, apolizumab, arcitumomab, acelizumab, altinumab, atlizumab, atolimumab, tocilizumab, bapineuzumab, basilixima, and the like. , bavituximab, bectumomab, belimumab, benralizumab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bivatuzumab, bivatuzumab mertansine, blinatumomab, brosozumab, brentuximab vedotin, briakinumab, brodalumab, canakinumab, cantuzumab mertansine, caplacizumab, capromab pendetide, carlumab, catumaxomab, cc49, cedelizumab, certolizumab pegol, cetuximab, sitatuzumab bogatox, cixutumumab, clazakisumab , clenoliximab, clivatuzumab tetraxetan, conatumumab, crenezumab, cr6261, dacetuzumab, daclizumab, darotuzumab, daratumumab, demcizumab, denosumab, detumomab, dorlimomab aritox, drozitumab, durigotumab, dupilumab, ecromeximab, eculizumab, edovacomab, edrecolomab, efalizumab, efungumab, elotuzumab, ersilimomab, enabatuzumab, enlimomab pegol, enokizumab, enoticumab, encituximab, epitumomab situxe Tan, epratuzumab, erenumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evolocumab, exibirimab, fanolesomab, faralimomab, farletuzumab, fasinumab, fbta05, felvizumab, fezakinumab, ficlatuzumab, figitumumab, framvotumab, fontolizumab, foralumab, foravirumab, fresolimumab, fullanumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, gevokizumab,Girentuximab, Glembatumumab vedotin, Golimumab, Gomiliximab, GS6624, Ibalizumab, Ibritumomab tiuxetan, Icrucumab, Igovomab, Imciromab, Imgatuzumab, Incracumab, Indatuximab ravtansine, Infliximab, Intetumumab, Inolimomab, Inotuzumab ozogamicin, Ipilimumab, Iratumumab, Itolizumab, Ixekizumab, Keliximab, Labetuzumab, Lebrikizumab, Remaresomab, Lerdelimumab, Lexatumumab, Ribivirumab, Ligelizumab, Lintuzumab, Ribivirumab, Rigelizumab, Lintuzumab, Ribivirumab, Rigelizumab, Lintuzumab, Ribivirumab, Rigelizumab, Lintuzumab, Rigeliz ... Rilumab, lorvotuzumab mertansine, lucatumumab, rumiliximab, mapatuzumab, maslimomab, mavrilimumab, matuzumab, mepolizumab, metelimuab, milatuzumab, minletumomab, mitumomab, mogamulizumab, morolimumab, motavizumab, moxetumomab pasudotox, muromonab-cd3, nacolomab butafenatox, namilumab, naptumomab estafenatox, narutumab, natalizumab, nebacumab, necitumumab, nerelimomab, nesbacumab, nimotuzumab, nivolumab, nofetumomab merpentane, okara Tuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab, onartuzumab, oportuzumab monatox, oregovomab, olticumab, otelixizumab, oxelumab, ozanezumab, ozoralizumab, pasivaximab, palivizumab, panitumumab, panobacumab, palsatuzumab, pascolizumab, pateclizumab, patritumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pintumomab, plakmab, ponezumab, priliximab, pritumumab, PR O140, Quilizumab, Racotumomab, Ladolezumab, Rafivirumab, Ramucirumab, Ranibizumab, Raxibacumab, Regavirumab, Reslizumab, Rilotumumab, Rituximab, Lobatumumab, Loredumab, Romosozumab, Rontalizumab, Rovelizumab, Ruplizumab, Samarizumab, Sarilumab, Satumomabpendetide, Secukinumab, Sevirumab, Sibrotuzumab, Sifalimumab, Siltuximab, Simtuzumab, Siplizumab, Sirukumab, Solanezumab, Solitomab, Sonepcizumab, Sontuzumab, Stamulumab, Sulesomab,Suvisumab, Tabalumab, Tacatuzumab Tetraxetan, Tadocizumab, Talizumab, Tanezumab, Taplitumomab Paptox, Tefibazumab, Terimomab Alitox, Tenatumomab, Tefibazumab, Teneliximab, Teplizumab, Teprotumumab, Tezepelumab, TGN1412, Tremelimumab, Ticilimumab, Tildrakizumab, Tigatuzumab, TNX-650, Tocilizumab, Toralizumab, Tositumomab, Tralokinumab, Trastuzumab, TRBS07, tregalizumab, tucotuzumab celmoleukin, tuvilumab, ublituximab, urelumab, urtoxazumab, ustekinumab, bapaliximab, batelizumab, vedolizumab, veltuzumab, beparimomab, besenkumab, visilizumab, volociximab, borsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, diralimumab, and zolimomab alitox.

[0108] Antibodies further include adalimumab, bevacizumab, blinatumomab, cetuximab, conatumumab, denosumab, eculizumab, erenumab, evolocumab, infliximab, natalizumab, panitumumab, rilotumumab, rituximab, romosozumab, tezepelumab, and trastuzumab, and an antibody selected from Table H.

[0109] [Table 13]

[0110] [Table 14]

[0111] In some embodiments, the therapeutic polypeptide is a BiTE® molecule. BiTE® molecules are engineered bispecific monoclonal antibodies that direct the cytotoxic activity of T cells against cancer cells. They are a fusion of two single chain variable fragments (scFv) of different antibodies or amino acid sequences from four different genes on a single peptide chain of about 55 kilodaltons. One of the scFvs binds to T cells via the CD3 receptor, and the other binds to tumor cells via a tumor-specific molecule. Blinatumomab (BLINCYTO®) is an example of a BiTE® molecule that is specific for CD19. Modified BiTE® molecules, such as those modified to extend their half-life, can also be used in the methods disclosed herein. EXAMPLES

[0112] The Examples section below is presented by way of example only and is not meant to limit the disclosure or claims herein in any way.

[0113] Preparation of Reagents for Examples 1 to 4 Denaturation buffer (7 M guanidine HCl, 100 mM tris, 20 mM methionine, pH 8.3) was prepared by adding 10 mL of 1 M (hydroxymethyl)aminomethane hydrochloride (tris), pH 7.8 (Teknova, Hollister, CA, P / N T1078) to 87.5 mL of 8 M guanidine HCl (Pierce, Rockford, IL, P / N 24115), followed by the addition of 299 mg of L-methionine (J.T. Baker, P / N 2085-05). The pH of this solution was adjusted to pH 8.3 with 6 N hydrochloride (HCl) (Sigma. St. Louis. MO, P / N 84429). The volume was adjusted to 100 mL with HPLC grade water. Reducing solution (500 mM DTT) was prepared by dissolving 7.7 mg pre-weighed dithiothreitol (DTT) (Pierce, Rockford, IL, P / N 20291) in 100 µL of denaturing buffer. Alkylation solution (500 mM NaIAA) was prepared by dissolving 15-65 mg sodium iodoacetate (NaIAA) (Sigma, St. Louis, MO, P / N I-9148) in a sufficient volume of denaturing buffer to produce 500 mM NaIAA. Digestion buffer (100 mM tris, 20 mM methionine, pH 7.8) was prepared by dissolving 299 mg L-methionine in 10 mL of 1 M tris, pH 7.8 and adding 100 mL of HPLC grade water. The pH of this solution was adjusted to pH 7.8 with HCl and the volume was adjusted to 100 mL with HPLC grade water. Enzyme solutions (1 mg / mL trypsin, 1 mg / mL HNE) were prepared by adding 100 μL of digestion buffer to 100 μg of trypsin (Roche, Basel, Switzerland, P / N 03708969001) or 100 μg of HNE (Elastin Products Company, Owensville, MO, P / N SE563). Digestion quenching solution (10% TFA) was prepared by adding 1.0 mL of 100% trifluoroacetic acid (TFA) (Pierce, Rockford, IL, P / N 28904) to 9.0 mL of HPLC grade water.

[0114] Example 2 - Trypsin and HNE Separate Digests Samples were denatured and reduced by adding 57.1 μL of denaturation buffer and 2 μL of reducing solution to 42.9 μL of sample (1–2 mg / mL in formulation buffer) followed by incubation at 37°C for 35 min. After reduction, 9 μL of alkylation solution was added to each sample followed by incubation at room temperature in the dark for 20 min. Alkylation was quenched by the addition of 7 μL of reducing solution. Samples were desalted with Zeba Spin desalting columns (Thermo Scientific, Waltham, MA, P / N 89883) using an Eppendorf 5430 centrifuge (Hamburg, Germany) with a spin at 1,500 × g according to the manufacturer's instructions. Sample concentrations were measured after desalting using either an Implen NanoPhotometer Pearl (Munchen, Germany) or a Thermo Scientific Nanodrop 2000c (Waltham, MA). Enzyme solution (either trypsin or HNE) was added to each sample at an enzyme:substrate ratio of 1:20. Samples were incubated in a water bath for 35 minutes at 37° C. Digestion was quenched by the addition of 6 μL of Digestion Quenching Solution.

[0115] Example 3 - Sequential trypsin and HNE digests Samples were denatured and reduced by adding 57.1 μL of denaturation buffer and 2 μL of reducing solution to 42.9 μL of sample (1–2 mg / mL in formulation buffer) followed by incubation at 37°C for 35 min. After reduction, 9 μL of alkylation solution was added to each sample followed by incubation at room temperature in the dark for 20 min. Alkylation was quenched by the addition of 7 μL of reducing solution. Samples were desalted with Zeba Spin desalting columns (Thermo Scientific, Waltham, MA, P / N 89883) using an Eppendorf 5430 centrifuge (Hamburg, Germany) with a spin at 1,500 × g according to the manufacturer's instructions. Sample concentrations were measured after desalting using either an Implen NanoPhotometer Pearl (Munchen, Germany) or a Thermo Scientific Nanodrop 2000c (Waltham, MA). Trypsin solution was added to each sample at an enzyme:substrate ratio of 1:20. Samples were incubated in a water bath at 37°C for 35 minutes. HNE solution was added to each sample at an enzyme:substrate ratio of 1:20. Samples were incubated in a water bath at 37°C for 30 minutes. Digestion was quenched by the addition of 6 μL of Digestion Quenching Solution.

[0116] Example 4 - Mixture of tryptic digest and tryptic-HNE sequential digest Samples were denatured and reduced by adding 57.1 μL of denaturation buffer and 2 μL of reducing solution to 42.9 μL of sample (1 mg / mL in formulation buffer) followed by incubation at 37° C. for 35 minutes. After reduction, 9 μL of alkylation solution was added to each sample followed by incubation at room temperature in the dark for 20 minutes. Alkylation was quenched by the addition of 7 μL of reducing solution. Samples were desalted using Zeba desalting columns using an Eppendorf 5430 centrifuge at 1,500×g spinning according to the manufacturer's instructions. Sample concentrations were measured after desalting using either a NanoPhotometer Pearl or a Nanodrop 2000c. Trypsin solution was added to each sample at a 1:20 enzyme:substrate ratio. Samples were incubated in a 37° C. water bath for 35 minutes. After trypsin digestion, each sample was split into two equal aliquots (approximately 55 μL each). 3 μL of Digestion Quenching Solution was added to the first aliquot, which was then set aside. To the second aliquot, HNE solution was added at an enzyme:substrate ratio of 1:20. This aliquot was incubated in a water bath at 37° C. for 30 minutes, after which 3 μL of Digestion Quenching Solution was added. The two aliquots were then combined in a 1:1 ratio and then mixed gently.

[0117] Preparation of Reagents for Examples 5 - 6 and 7 Denaturation buffer (6 M guanidine HCl, 200 mM tris, 20 mM methionine, pH 8.3) was prepared by adding 20 mL of 1 M (hydroxymethyl)aminomethane hydrochloride (tris), pH 8.3 (Teknova, St. Louis, MO, P / N T1083) to 87.5 mL of 8 M guanidine HCl (Pierce, Rockford, IL, P / N 24115), followed by adding 299 mg of L-methionine (JT Baker, P / N 2085-05). The pH of this solution was adjusted to pH 8.3 using either 1 N hydrochloric acid (HCl) (Ricca, Arlington, TX, P / N R3700100-120A) or 1 N sodium hydroxide (NaOH) (Merck, Kenilworth, NJ, P / N 1.09137.100). The volume was adjusted to 100 mL with HPLC grade water. The reducing solution (500 mM DTT) was prepared by dissolving 7.7 mg of pre-weighed dithiothreitol (DTT) (Pierce, Rockford, IL, P / N 20291) in 100 μL of denaturing buffer. Alkylation solution (500 mM NaIAA) was prepared by dissolving 15-65 mg of sodium iodoacetate (NaIAA) (Sigma, St. Louis, MO, P / N I-9148) in a sufficient volume of denaturation buffer to produce 500 mM NaIAA. Digestion buffer (50 mM tris, 20 mM methionine, pH 7.8) was prepared by dissolving 299 mg of L-methionine in 10 mL of 1 M tris, pH 7.8, and adding 100 mL of HPLC grade water. The pH of this solution was adjusted to pH 7.8 with either 1 N hydrochloride (HCl) (Ricca, Arlington, TX, P / N R3700100-120A) or 1 N sodium hydroxide (NaOH) (Merck, Kenilworth, NJ, P / N 1.09137.100) and the volume was adjusted to 100 mL with HPLC grade water.Enzyme solutions (1 mg / mL trypsin, 1 mg / mL HNE) were prepared by adding 100 μL of digestion buffer to 100 μg trypsin (Roche, Basel, Switzerland, P / N 03708969001) or 100 μg HNE (Elastin Products Company, Owensville, MO, P / N SE563). Digestion quenching solution (8 M guanidine HCl, 250 mM acetate, pH 4.7) was prepared by dissolving 76.4 g guanidine HCl (Sigma, St. Louis, MO, P / N 50933) and 1.0 g sodium acetate (Sigma, P / N 32319) in 95 mL of HPLC grade water. 716 μL of glacial acetic acid (Sigma, St. Louis, MO, P / N 320099) was then added and the pH was adjusted to pH 4.7 with either HCl or NaOH. The volume was then adjusted to 100 mL with HPLC grade water.

[0118] Example 6 - MWCO spin filter assisted sequential digestion 100 μg of sample in formulation buffer was added to a 30 kDa molecular weight cutoff spinner consisting of a membrane unit placed in a centrifuge tube for filtrate collection (Millipore, Billerica, MA, P / N MRCF0R030 or Pall, Port Washington, NY, P / N OD030C34). The device was spun at 14,000×g for 15 minutes using an Eppendorf 5430 centrifuge. The filtrate was discarded. 200 μL of denaturing buffer was added to the sample and the filter device was spun at 14,000×g for 15 minutes and the filtrate was discarded; this was repeated two more times. For each sample, 3 μL of reducing solution was added to 37 μL of denaturing buffer and 40 μL of this solution was added to the filter device. Samples were denatured and reduced by incubating in a 37° C. water bath for 45 minutes. The samples were then spun at 14,000×g for 15 minutes and the filtrate was discarded. For each sample, 7 μL of alkylation solution was added to 33 μL of denaturation buffer and 40 μL of this solution was added to the filter device. The samples were alkylated by incubating at room temperature in the dark for 20 minutes. The samples were then spun at 14,000×g for 15 minutes and the filtrate was discarded. For each sample, 4 μL of denaturation solution was added to 36 μL of denaturation buffer and 40 μL of this solution was added to the filter device to quench the alkylation. The samples were then spun at 14,000×g for 15 minutes and the filtrate was discarded. 200 μL of digestion buffer was added to the samples and the filter device was spun at 14,000×g for 15 minutes and the filtrate was discarded; this was repeated two more times to remove the denaturant, reducing agent and alkylating agent. For each sample, 5 μL of trypsin solution was added to 35 μL of digestion buffer and 40 μL of this solution was added to the filter device (1:20 enzyme:substrate ratio). Samples were incubated in a 37°C water bath for 60 minutes. The filter device was transferred to a new collection tube (Collection Tube 2). The original collection tube (Collection Tube 1) was removed. The filter device was centrifuged at 14,000 x g for 15 minutes. The filtrate containing the tryptic peptides was retained in Collection Tube 2.20 μL of digestion buffer was added to the filter device and the filter device (in collection tube 2) was spun at 14,000×g for 15 minutes and the filtrate was retained in collection tube 2; this was repeated one more time. The filter device was transferred back to collection tube 1 and collection tube 2 was removed. For each sample, 5 μL of HNE solution was added to 35 μL of digestion buffer and 40 μL of this solution was added to the filter device (now in collection tube 1, 1:20 enzyme:substrate ratio based on starting material). The samples were incubated in a 37° C. water bath for 30 minutes. The filter device was transferred to collection tube 2. Collection tube 1 was discarded. The filter device was centrifuged at 14,000×g for 15 minutes. The filtrate containing the peptides resulting from the HNE digestion was retained in collection tube 2 (together with the tryptic peptides from the previous step). 20 μL of Digestion Buffer was added to the filter device and the filter device (in collection tube 2) was spun at 14,000×g for 15 minutes, retaining the filtrate in collection tube 2; this was repeated one additional time. The digestion was quenched by adding 160 μL of Digestion Quenching Buffer to collection tube 2.

[0119] Example 7 - MWCO spin filter assisted continuous digestion, shortened plate 200 μL of denaturing buffer was added to a 30 kDa molecular weight cutoff spinner consisting of a membrane unit placed in a centrifuge tube for filtrate collection (Millipore, Billerica, MA, P / N MRCF0R030 or Pall, Port Washington, NY, P / N OD030C34). The device was spun at 14,000×g for 10 minutes using an Eppendorf 5430 centrifuge. The filtrate was discarded. 100 μg of sample in formulation buffer was added to the filter device and spun at 14,000×g for 10 minutes. The filtrate was discarded. For each sample, 3 μL of reducing solution was added to 37 μL of denaturing buffer and 40 μL of this solution was added to the filter device. Samples were denatured and reduced by incubation in a 37° C. water bath for 30 minutes. For each sample, 7 μL of alkylation solution was added to 33 μL of denaturation buffer, and 40 μL of this solution was added to the filter device. Samples were alkylated by incubating at room temperature in the dark for 20 minutes. For each sample, 4 μL of denaturation solution was added to 36 μL of denaturation buffer, and 40 μL of this solution was added to the filter device to quench the alkylation. Samples were then spun at 14,000×g for 15 minutes, and the filtrate was discarded. 200 μL of digestion buffer was added to the sample, and the filter device was spun at 14,000×g for 15 minutes, and the filtrate was discarded; this was repeated two more times to remove the denaturant, reducing agent, and alkylating agent. For each sample, 5 μL of trypsin solution was added to 35 μL of digestion buffer, and 40 μL of this solution was added to the filter device (1:20 enzyme:substrate ratio). Samples were incubated in a 37° C. water bath for 60 minutes. The filter device was transferred to a new collection tube (Collection Tube 2). The original collection tube (Collection Tube 1) was removed. The filter device was centrifuged at 14,000 x g for 10 minutes. The filtrate containing the tryptic peptides was retained in Collection Tube 2.20 μL of digestion buffer was added to the filter device and the filter device (in collection tube 2) was spun at 14,000×g for 10 minutes and the filtrate was retained in collection tube 2; this was repeated one more time. The filter device was transferred back to collection tube 1 and collection tube 2 was removed. For each sample, 5 μL of HNE solution was added to 35 μL of digestion buffer and 40 μL of this solution was added to the filter device (now in collection tube 1, 1:20 enzyme:substrate ratio based on starting material). The samples were incubated in a 37° C. water bath for 30 minutes. The filter device was transferred to collection tube 2. Collection tube 1 was discarded. The filter device was centrifuged at 14,000×g for 10 minutes. The filtrate containing the peptides resulting from the HNE digestion was retained in collection tube 2 (together with the tryptic peptides from the previous step). 20 μL of Digestion Buffer was added to the filter device and the filter device (in collection tube 2) was spun at 14,000×g for 10 minutes, with the filtrate retained in collection tube 2; this was repeated one additional time. The digestion was quenched by adding 160 μL of Digestion Quenching Buffer to collection tube 2. A comparison highlighting the differences between protocols 4 and 5 is summarized in Table 9.

[0120] Example 8 - Ultra Performance Liquid Chromatography (UPLC) Conditions For all samples, mobile phase A consisted of 0.1% formic acid in water and mobile phase B consisted of 0.1% formic acid in acetonitrile. For the initial experiments using protocols 1 and 2, peptides were separated using a CSH C18 1.7 μm, 2.1×150 mm UPLC column (Waters, Milford, MA, P / N 186005298). After crosslinking data was acquired, the remaining experiments using the methods from Examples 2-4 were carried out using a BEH C18 1.7 μm, 2.1×150 mm UPLC column (Waters, Milford, MA, P / N 186003556). UPLC separations were performed using either a Thermo Scientific U-3000 system (Waltham, MA), a Waters Acquity H-Class system (Milford, MA), and / or an Agilent 1290 system (Santa Clara, CA) utilizing the gradients (depending on the experiment) outlined in Tables 1-3. Approximately 3-4 μg of sample was loaded onto the column, based on the starting material.

[0121] Example 9 - Mass Spectroscopy Conditions The resulting peptides from the digestion were analyzed using a Thermo Scientific Q Exactive (Waltham, MA), Thermo Scientific Q Exactive Plus (Waltham, MA) or Thermo Scientific Q Exactive BioPharma (Waltham, MA). As multiple instruments were used, data collection parameters varied slightly depending on the instrument. The instruments were operated in a data-dependent manner (top 4-8) over a scan range of 200-2,000 m / z. The AGC target was set to 1E6 for MS1 scans and 5E5 for tandem mass spectrometry (MSMS) scans. MS1 scans were collected at either 35,000 or 140,000 resolution, and MS2 scans were collected at 17,500 resolution. An isolation window of 2-4 m / z was specified for the MSMS scans. Unused charge states and charge states greater than 8 were excluded from the MSMS. Dynamic exclusion was set for 10 seconds. A lock mass of m / z 391.28430 is now possible.

[0122] Example 10 - Data Analysis MS data were searched using MassAnalyzer (multiple versions of MassAnalyzer were used since data were collected over several months). Carboxymethylation was identified as a static modification. Depending on the experiment, truncations were identified as either non-specific or C-terminal to the amino acid KRVITAL. For the full search, the signal-to-noise ratio was set to 20, a mass accuracy of 15 ppm was specified, and confidence was set to 0.95. For sequence coverage maps, the minimum peak area was set to 1% of the base peak, the relative peak area threshold was set to 17%, the minimum confidence was set to 0.95, and the highest peptide mass was set to 15,000.

[0123] Example 11 - Results An initial experiment investigating the effectiveness of HNE for digestion of BiTE® molecules was a direct comparison of trypsin and HNE digestion for BiTE®-3 using Protocol 1 and Gradient 1. Trypsin digestion did not produce peptides that could be used to characterize attributes in any of the CDRs. Digestion with HNE, on the other hand, gave rise to several peptides that corresponded to the linker region. Many of these peptides had low signal intensity (<10 6 ), and this lack of specificity (14 peptides were identified) could potentially invalidate the quantification of attributes in this region. However, the specificity of HNE was thought to be related to the length of the substrate digested (Stein et al., 1987). The properties of this enzyme were exploited by first digesting BiTE®-3 with trypsin to generate potential substrates with shorter amino acid lengths, followed by digestion with HNE (Protocol 2). This sequential digestion resulted in high signal intensities (>1.5 × 10 7 ) was generated.

[0124] After demonstrating the feasibility of sample preparation utilizing trypsin-HNE digestion to monitor attributes in the α-CD3 CDR of interest, additional molecules (BiTE®-1, half-life extended BiTE®; BiTE®-2, canonical BiTE®; and CDH19, half-life extended BiTE®; BiTE®-3, in addition to the half-life extended BiTE®) that had been subjected to photodegradation (12 million lux hours cool white light, t = 3 days) and forced deamidation (pH 8.5, 50, t = 3 days) were prepared using protocol 2 and gradient 1. For the unstressed samples, sequence coverage for α-CD3 CDR1 was highly reproducible (Figure 4). One or two peptides corresponding to potential asparagine deamidation sites within this CDR consistently showed high signal intensity (>10 7) was identified. Similarly, for all molecules except CDH19, a single peptide corresponding to a potential tryptophan oxidation site was identified with high signal intensity. Forced deamidation conditions produced peaks corresponding to deamidation in the α-CD3 CDR1 for all four molecules that were not observed in the unstressed samples (Figure 3 shows BiTE®-1 as a representative sample). MSMS (Figure 4) confirmed that the first two eluting peaks correspond to the -GNS-motif, whereas the last eluting peak corresponds to deamidation of the -GNF-motif. Comparison of the modification percentages for all four molecules (Table 11.1) highlights both potential deamidation sites that were susceptible to modification in this CDR and that had to be monitored. Oxidation of CDR tryptophans was also observed after photodegradation at a significantly lower percentage (Table 11.2). No increase in deamidation was observed for the second CDR immediately adjacent to the large linker peptide (α-CD3 CDR2). These initial results confirmed not only that there are unstable sites within the α-CD3 CDR that cannot be monitored using trypsin digestion, but also that these modifications can be identified and monitored using sequential trypsin-HNE digestions.

[0125] [Table 15]

[0126] [Table 16]

[0127] Although trypsin-HNE digestion produced high quality peptides for the two α-CD3 CDRs of interest, quantification of sequence coverage and modifications for the remaining molecule still required the tryptic peptides utilized by conventional MAMs.

[0128] A single analysis was performed on a 1:1 mixture of trypsin-digested samples combined with an aliquot of a sample digested with trypsin followed by HNE (Figure 5). An additional strength of this approach of analyzing a mixture of two digests lies in the fact that data for two peptide maps are obtained in essentially one injection; not only does this allow greater sequence coverage to be identified, but in the case of BiTE®-2, peptides containing a crucial attribute resulting from trypsin-HNE digestion (CDR aspartic acid isomerization) produced better signal quality and reproducibility than the corresponding tryptic peptides. Furthermore, the UPLC gradient utilized for the separation was switched to gradient 2 and the column was switched from a Waters CSH C18 column to a Waters BEH C18 column. The robustness of this approach was evaluated by analyzing unstressed, photodegraded, pH-jumped and pyrolyzed samples corresponding to two samples (BiTE®-2 and BiTE®-3). Trypsin digested samples, trypsin-HNE sequential digested samples, and a 1:1 mixture of trypsin digested and trypsin-HNE digested samples were prepared by two analysts and analyzed using two UPLC systems (Thermo U-3000 and Waters Acquity H-Class) connected to two different mass spectrometers (Thermo QExactive and Thermo QExactive Plus) (Figure 8). All samples were prepared in duplicate, so a total of four injections were obtained for each condition.

[0129] Table 11.3 summarizes the quantitative results of deamidation from α-CD3 CDR1 from both BiTE®-2 and BiTE®-3 as representative peptides with asparagine deamidation. For the data presented in this table, photolysis was performed for 2 days using 192,000 lux hours; pH jump decay was performed at pH 8.4 at 37° C. for 7 days; and pyrolysis was performed at 40° C. for 4 weeks. These results indicate that the step of mixing the trypsin digest with the trypsin-HNE digest had only a minor effect on the quantitative results. Decreasing the gradient nevertheless resulted in clear separation of the three deamidated species (-GNS-unmodified, separated by about 1.5 min from peak 1, -GNS-peak 1, separated by about 0.5 min from peak 2, -GNS-peak 2, separated by about 1.3 min from -GNF- (data not shown). Furthermore, the precision of the deamidation levels for both molecules was comparable whether the trypsin-HNE digest was run separately or mixed with the trypsin digest. Because deamidation and aspartate isomerization were typically the most challenging modifications to chromatographically separate from the corresponding unmodified peptides, Table 11.4 includes the D of BiTE®-3 as a representative peptide with aspartate isomerization. 510 Quantitative results for isomerization are summarized. For the data presented in this table, photolysis was performed for 2 days using 192,000 lux hours; pH-jump degradation was performed for 7 days at pH 8.4, 37° C.; and thermolysis was performed for 4 weeks at 40° C. Despite containing threonine and leucine residues, a portion of the sequence of BiTE®-3 is slightly cleaved during sequential digestion with HNE, so intact tryptic peptides were used to determine the D, regardless of whether a tryptic digest, a tryptic-HNE sequential digest, or a 1:1 mixture of the two digests was analyzed. 510 It was possible to quantify the isomerization. 510Since isomerization levels were consistent regardless of digestion conditions, this again reinforced the observation that mixing these digests has little effect on attribute quantification. These results demonstrate that mixing two parallel digests is a reasonable approach for BiTE® molecules (as well as other polypeptides), and that doing so has little effect on modification quantification, regardless of whether tryptic or tryptic-HNE peptides are used.

[0130] [Table 17]

[0131] [Table 18]

[0132] MAM utilizing the method described above (Protocol 3, Gradient 2) was evaluated for suitability for BiTE®-2 (ATM-000401) and BiTE®-3 (ATM-000391). However, during the analysis, the new peak detection procedure (sieve) incorporated into the method identified numerous new peaks. This resulted in a system suitability failure for the sequence. After detailed evaluation, numerous peaks corresponded to failed tryptic cleavage. However, some of these peaks were also observed in the final system suitability injection (but not in the initial pre-run injection). These observations suggested that the effectiveness of trypsin was somehow hindered, but furthermore, they suggested that one of the enzymes was still active despite acidification of the sample after digestion. However, quantification of the attributes was not affected by the novel peaks (only the system suitability check). Troubleshooting indicated that the desalting column utilized in Protocols 1-3 did not completely remove guanidine from the pre-digested sample. Because trypsin effectiveness can be inhibited by low levels of guanidine, it was likely that variable removal of guanidine by the desalting column resulted in inconsistent tryptic digestion. Furthermore, because acid quenching of tryptic digestion has been reported, it was speculated that oxidation would not sufficiently quench the HNE digestion. Enzyme activity decreased due to acidification, but would result in the observation of new peaks if multiple injections were performed from a single vial after a period of time; this, in turn, would result in failure of the system to be qualified.

[0133] To address these issues, a thorough exploration of multiple preparation methods was performed. These included evaluating multiple desalting columns (Zeba, Biospin, NAP-5), a preparation method that does not require guanidine (Rapigest) for denaturation, and a method based on MWCO filters available from multiple manufacturers (Pall and Microcon®, Millipore, Waltham, MA) with multiple molecular weight cutoffs (30 kDa, 5 kDa as specified by the manufacturer). Additionally, the digestion quenching buffer was updated to contain a higher concentration of guanidine to inactivate both enzymes. Protocols 1-3 In previous studies, acid quenching typically resulted in a final sample pH of 2. The updated digestion quenching buffer was low enough to inhibit trypsin and HNS activity, but high enough to minimize artifactual modifications (e.g., deamidation and isomerization) that may result from prolonged exposure to low pH. Because this quenching buffer contained guanidine, the initial wash steps in the UPLC gradient were extended; all experiments utilizing a guanidine quenching buffer required the use of gradient 3 to fully desalt samples prior to MS analysis.

[0134] The conclusion of this research study is that the MWCO filter-based method (Protocol 4 above, previously reported Filter-Aided Sample Preparation (FASP) method (Wisniewski et al., 2009)) outperformed other preparation methods based on several metrics (e.g., repeatability, recovery, quantification, sequence coverage, concentration range, etc.). A schematic of Protocol 4 is presented in Figure 7. In this method, molecules are captured on top of a 30 kDa MWCO spin filter and all of the denaturation, reduction, alkylation, desalting and digestion occurs on the filter. After digestion, the resulting peptides are collected via centrifugation. In the sequential digestion outlined in Protocol 4, after trypsin digestion, tryptic peptides are collected and the species that do not pass through the filter are then subjected to digestion with HNE. One surprising observation of this method was that the 8 kDa linker peptide of interest was retained by the 30 kDa filter, despite recommendations from the manufacturers, Microcon® and Pall, respectively, to "use a membrane with a MWCO at least 2-fold smaller than the molecular weight of the protein solute you intend to concentrate" and "select a MWCO 3-6-fold smaller than the molecular weight of the solute to be retained." Surprisingly, the 8 kDa peptide was retained by a MWCO nearly 4-fold larger than the peptide itself, even though the manufacturers recommended using a MWCO 2-6-fold smaller than the species to be retained.

[0135] The retention of the 8 kDa peptide by the 30 kDa MWCO filter with membranes made of different materials (Microcon® membrane is regenerated cellulose, Pall membrane is modified polyethersulfone) highlights that this is not a manufacturer specific artifact. Not only did this novel use of MWCO filters enable sequential digestion, but a direct comparison of this method utilizing a 30 kDa MWCO filter versus a 5 kDa MWCO filter demonstrated that the 30 kDa filter method was significantly superior in terms of trypsin digestion efficiency, repeatability, precision of attribute quantification, and recovery compared to the 5 kDa filter method. The main advantage of this method is that the enzyme specificity conferred by trypsin digestion is preserved for the bulk of the molecule, and only the large peptides that are difficult to characterize by MS are further digested into smaller peptides due to their retention on the filter. This is illustrated in FIG. 8, which highlights the trypsin specificity of peptides identified for BiTE®-1, BiTE®-2, and BiTE®-3 (triplicate analyses of each molecule) using filters manufactured by both Microcon® and Pall. In these results, over 95% of the identified peptides typically corresponded to trypsin digests, while the remaining identified peptides primarily corresponded to the large 8 kDa linker peptide of interest (FIG. 9). Quantification of attributes for BiTE®-2 using filters from both manufacturers was comparable to the results for the parallel digest (protocol 3) (Table 11.5). Similar results were observed for BiTE®-1 and BiTE®-3. This method more completely solved the novel peak problem compared to the other methods discussed above by removing guanidine, and the enzymes were further inactivated either by updated quenching buffer or by retention on MWCO filters. In Figure 10, a BiTE®-3 sample prepared using Protocol 3 was reinjected after about 4 days. Several peaks were present only in the initial injection, but they were not present in the second injection.In contrast, samples prepared using Protocol 4 were consistent even after 4 days had elapsed between injections (Figure 11). These results suggest that the MWCO filter-based preparation method outlined in Protocol 4 is robust, producing quantitative results consistent with those previously reported, and eliminating many of the underlying causes of the novel peaks observed using Protocol 3.

[0136] [Table 19]

[0137] One of the major concerns regarding Protocol 4 was the length of time required to prepare samples; sequential digestion utilizing Protocol 4 would require more than 8 hours. In addition to analyst fatigue and other factors, the amount of time required to prepare samples using this method would introduce artifactual modifications resulting from extended exposure to ambient laboratory conditions. To address these concerns, a shortened filter-based preparation method (Protocol 5) was developed. Table 11.6 highlights the differences between Protocols 4 and 5. The bulk of the time savings introduced by Protocol 5 was made possible by eliminating unnecessary centrifugation steps, reducing centrifugation times, and reducing incubation times. These reductions theoretically yield a time savings of roughly 2 hours, but in practice, the time savings were typically more than 3 hours. While still slightly longer than solution-based preparation methods such as Protocol 3, these modifications reduced sample preparation times to a point comparable to solution-based methods.

[0138] [Table 20]

[0139] In FIG. 12, an overlay is presented comparing BiTE®-2 samples prepared using protocols 4 and 5 (each preparation was performed in triplicate). The only notable peak difference observed was a shoulder at about 24 minutes and at about 47 minutes that was present in all samples prepared using protocol 4, but not in the samples prepared using protocol 5. These peaks were attributed to non-specific carboxymethylation resulting from over-alkylation. Similarly, the alkylation levels for all cysteine ​​residues were compared (Table 11.7), since the denaturation / reduction incubation time was decreased and the rotation step between reduction and alkylation was eliminated. For the data presented in this table, the percentage of alkylation was determined by searching the data using MassAnalyzer, identifying carboxymethylation as a variable modification (rather than a static modification as typically defined). The alkylation levels for all BiTE®-1 samples were greater than 99.7%, regardless of whether they were prepared using protocols 4 and 5. Similar results were observed for BiTE®-2 and BiTE®-3, suggesting the time reduction introduced by Protocol 5 did not show any negative impact on alkylation levels. Attribute quantification was comparable when samples were prepared using both Protocols 4 and 5, which in turn was consistent with historically observed modification levels (Table 11.8). For the data in this table, each preparation was performed in triplicate. D 54 +D 57 +D 62 Isomerization of I for MDR-001581 51 -R 65 Quantification was performed using C 44 -E 65was used for quantitation in the filter-based experiments due to the more efficient trypsin digestion. Because these initial results for Protocol 5 were promising, a limited robustness evaluation was performed (2 analysts, BiTE®-2 samples prepared in triplicate). Novel peak detection passed for all samples (e.g., no novel peaks were detected) and attribute quantitation for all six injections was consistent with MDR-001581 (Table 11.9). Based on these results, along with comparison of other metrics such as trypsin specificity, total area identified, recovery of the 8 kDa linker peptide, and percent of identified area corresponding to the enzyme used for digestion, Protocol 5 introduces significant time savings in preparation time without a significant negative impact on data quality.

[0140] [Table 21]

[0141] [Table 22]

[0142] References All references are incorporated herein by reference in their entirety. Crimmins,DL,SMMische,and NDDenslow.2001.Chemical Cleavage of Proteins in Solution.In Current Protocols in Protein Science.John Wiley&Sons,Inc. Doucet,A.,and C.M.Overall.2011.Broad coverage identification of multiple proteolytic cleavage site sequences in complex high molecular weight proteins using quantitative proteomics as a complement to edman sequencing.Molecular&cellular proteomics:MCP.10:M110 003533. Gunzler,H.,and A.Williams.2001.Handbook of analytical techniques.Wiley-VCH,Weinheim,Germany.1198 pp. Janoff,A.,and J.Scherer.1968.Mediators of inflammation in leukocyte lysosomes.IX.Elastinolytic activity in granules of human polymorphonuclear leukocytes.J Exp Med.128:1137-1155. Kurien,B.T.,and R.H.Scofield.2012.Protein electrophoresis:methods and protocols.Humana Press;Springer,New York.xiv,648 p.pp. Li,A.,R.C.Sowder,L.E.Henderson,S.P.Moore,D.J.Garfinkel,and R.J.Fisher.2001.Chemical Cleavage at Aspartyl Residues for Protein Identification.Analytical Chemistry.73:5395-5402. Lord,G.A.2004.Capillary electrophoresis of proteins and peptides,Edited by M.A.Strege and A.L.Lagu(Methods in Molecular Biology,Volume 276,Series Editor J.M.Walker).Humana Press,Totowa,New Jersey,2004,332 pp,US$125.00.Biomedical Chromatography.18:875-875. Nowicka-Jankowska,T.1986.Analytical visible and ultraviolet spectrometry.Elsevier; Distributors for the United States and Canada,Elsevier Science Pub.Co.,Amsterdam;New York New York,NY,USA.xvi,690 p.pp. Pontius,J.,L.Wagner,and G.Schuler.2003.UniGene:a unified view of teh transcriptome.In The NCBI Handbook.National Center for Biotechnology Information,Bethesda(MD). Rawlings,N.D.,M.Waller,A.J.Barrett,and A.Bateman.2014.MEROPS:the database of proteolytic enzymes,their substrates and inhibitors.Nucleic acids research.42:D503-509. Rohani,R.,M.Hyland,and D.Patterson.2011.A refined one-filtration method for aqueous based nanofiltration and ultrafiltration membrane molecular weight cut-off determination using polyethylene glycols.Journal of Membrane Science.382:278-290. Rubakhin,S.S.,and J.V.Sweedler.2010.Mass spectrometry imaging:principles and protocols.Humana Press,New York.xiv,487 p.pp. Sinha,S.,W.Watorek,S.Karr,J.Giles,W.Bode,and J.Travis.1987.Primary structure of human neutrophil elastase.Proc Natl Acad Sci USA.84:2228-2232. Stein,R.L.,A.M.Strimpler,H.Hori,and J.C.Powers.1987.Catalysis by human leukocyte elastase:mechanistic insights into specificity requirements.Biochemistry.26:1301-1305. Tanabe,K.,A.Taniguchi,T.Matsumoto,K.Oisaki,Y.Sohma,and M.Kanai.2014.Asparagine-selective cleavage of peptide bonds through hypervalent iodine-mediated Hofmann rearrangement in neutral aqueous solution.Chemical Science.5:2747-2753. Tanford,C.1968.Protein Denaturation.In Advances in Protein Chemistry.Vol.23.C.B.Anfinsen,M.L.Anson,J.T.Edsall,and F.M.Richards,editors.Academic Press.121-282. The UniProt,C.2017.UniProt:the universal protein knowledgebase.Nucleic acids research.45:D158-D169. Unspecified.2007.Table 2.List of proteases commonly used for fragmenting proteins.Cold Spring Harbor Protocols.2007:pdb.tab2ip13. Wisniewski,J.R.,A.Zougman,N.Nagaraj,and M.Mann.2009.Universal sample preparation method for proteome analysis.Nature methods.6:359-362.

Claims

1. A method for preparing a polypeptide for multi-attribute method (MAM) analysis, wherein the polypeptide is a trypsin cleavage resistant polypeptide, the method comprising: a. cleaving said polypeptide in a sample in a first digestion with trypsin, said cleaving generating at least two fragments of said polypeptide; b. cleaving said at least two fragments of said polypeptide with neutrophil elastase; and c. subjecting said sample to MAM analysis after cleaving said at least two fragments of said polypeptide with said neutrophil elastase. The method includes:

2. A method for preparing a polypeptide for multi-attribute method (MAM) analysis, wherein the polypeptide is a trypsin cleavage resistant polypeptide, the method comprising: a. cleaving said polypeptide in a sample in a first digestion with trypsin, said cleaving generating at least two fragments of said polypeptide; b. dividing the sample containing the at least two fragments of the polypeptide into a first aliquot and a second aliquot; c. digesting the at least two fragments of the first aliquot with neutrophil elastase; and d. performing MAM analysis of the first aliquot and the second aliquot. The method includes:

3. 3. The method of claim 2, wherein the first and second aliquots are combined after digestion of the at least two fragments of the polypeptide with the neutrophil elastase.

4. 3. The method of claim 1 or 2, further comprising the step of applying a filter having a molecular weight cut-off to the sample prior to the first digestion, wherein the first digestion digests the sample on the filter.

5. The method of claim 1 or 2, wherein the polypeptide is denatured prior to cleaving the polypeptide in the sample in the first digest.

6. The method of claim 1 or 2, wherein the polypeptide is alkylated prior to cleaving the polypeptide in the sample in the first digest.

7. a. Prior to cleaving the polypeptides in the sample in the first digest, the polypeptides are denatured and either alkylated or reduced; or b. Prior to cleaving the polypeptides in the sample in the first digest, the polypeptides are denatured, reduced and alkylated; The method according to claim 1 or 2.

8. The method of claim 1 or 2, wherein the neutrophil elastase is human neutrophil elastase (EC 3.4.21.37).

9. 3. The method of claim 1 or 2, wherein the analyzing step comprises at least one technique selected from the group consisting of chromatography, electrophoresis, spectrometry, and combinations thereof.

10. 3. The method of claim 1 or 2, wherein the polypeptide is a therapeutic polypeptide, and optionally the therapeutic polypeptide is selected from the group consisting of an antibody or an antigen-binding fragment thereof, and a derivative of an antibody or antibody fragment.

11. 11. The method of claim 10, wherein the therapeutic polypeptide is a bispecific T cell engager molecule.

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