Method for preparing trypsin-resistant polypeptides for mass spectrometry analysis

Abstract

Methods for preparing polypeptides for analysis are provided. [Solution] A method for preparing a polypeptide for analysis, comprising: a. a step of cleaving the polypeptide in a sample in a first digestion, wherein the cleaving step produces at least two fragments of the polypeptide; b. a step of cleaving the at least two fragments of the polypeptide with neutrophil elastase; and c. a step of analyzing the sample after the at least two fragments of the polypeptide have been cleaved with neutrophil elastase.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 642,444, filed on 13 March 2018, the entire contents of which are incorporated herein by reference.

[0002] Sequence List This application is filed together with an electronic sequence listing. The sequence listing, provided as a file titled "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. More specifically, the subject matter presented herein relates to the preparation of samples for detecting polypeptides contained therein, such as therapeutic polypeptides. The method disclosed herein is a sequential digestion in polypeptide preparation, wherein the final digestion involves the use of neutrophil elastase. [Background technology]

[0004] Multi-attribute mapping (MAM) relies on the enzymatic digestion of molecules, followed by characterization and quantification of key attributes by mass spectrometry. Modifications in complementarity-determining regions (CDRs) are of particular concern because they can affect the potency and / or safety of the molecule. Trypsins that cleave at the C-terminus of lysine and arginine residues are typically used as optimal enzymes for peptide mapping and MAM for a number of reasons, including high digestion specificity and frequency of lysine and arginine residues; trypsin digestion more typically yields peptides with a basic residue at the C-terminus that have an optimal length for analysis by mass spectrometry. 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 adjacent to the long-chain 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 applicable to trypsin digestion (Figure 1). Therefore, the attribute of interest within the CDR domain cannot be monitored by MAM due to the large size of this peptide (approximately 8 kDa), the difficulty in separating modified versions of the peptide by chromatography, poor peptide recovery and / or ionization, and the general challenges associated with interpreting mass spectroscopy for peptides of this size. Furthermore, this linker region does not contain residues sensitive to commonly used secondary proteases such as Asp-N, Lys-C, and Glu-C. While chymotrypsin cleaves at tryptophan residues, the use of this enzyme was not pursued due to both its low specificity and the potential for oxidation at tryptophan residues to affect the quantification of this attribute. Similarly, numerous BiTE® molecules further possess shorter linker peptides (approximately 5–7 kDa) adjacent to the target-specific CDR, which are equally difficult to monitor using trypsin digestion. For example, the potential CDR aspartate isomerization site is located within a 5.7 kDa linker peptide.In summary, these attributes can have a significant impact on targets and / or CD3 binding that require MAM-based monitoring. [Overview of the Initiative] [Means for solving the problem]

[0005] In a first aspect, the Specified provides a method for preparing a polypeptide for analysis, comprising the steps of: cleaving a polypeptide in a sample in a first digestion, wherein the cleaving step generates at least two fragments of the polypeptide, the at least two fragments of the polypeptide subsequently digested by neutrophil elastase; and analyzing the sample after digesting the at least two fragments of the polypeptide with neutrophil elastase.

[0006] In a second embodiment, the Spectrum provides a method for preparing a polypeptide for analysis, comprising the steps of: cleaving a polypeptide in a sample in a first digestion, wherein the cleaving step generates 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 at least two fragments of the first aliquot with neutrophil elastase; and analyzing the first and second aliquots. In some partial embodiments, the first and second aliquots are combined after digestion of at least two polypeptide fragments with neutrophil elastase; in such cases, the first and second aliquots may contain approximately equal amounts of polypeptide.

[0007] In both the first and second embodiments, the step of cleaving the polypeptide in the first digestion includes proteolytic cleavage or chemical cleavage. In those embodiments including proteolytic cleavage, such cleavage is carried out by a protease, where the protease has different activity from neutrophil elastase. In some embodiments, 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-parts of these first and second embodiments, the protease is selected from the group consisting of trypsin, Asp-N and Glu-C and combinations thereof; in yet other sub-parts, the protease is trypsin. However, when chemical cleavage is used to fragment polypeptides in the sample in the first and second embodiments, such cleavage is carried out by a chemical substance selected from the group consisting of cyanogen bromide, 2-nitro-5-thiocyanobenzoic acid, hydroxylamine and BNPS-skatole and combinations thereof.

[0008] Furthermore, polypeptides may be denatured, and / or reduced, and / or alkylated before cleaving polypeptides in the sample during the first digestion. Neutrophil elastase may be, for example, human neutrophil elastase (EC3.4.21.37). When analyzing a sample, the analysis may include at least one technique selected from the group consisting of chromatography, electrophoresis, spectroscopy, or spectroscopic measurement and combinations thereof. For example, if the technique for analyzing a sample involves chromatography, the chromatographic technique can be selected from the group consisting of gas chromatography, liquid chromatography, high-performance liquid chromatography, ultrahigh-performance liquid chromatography, size exclusion chromatography, ion exchange chromatography, affinity chromatography, extended adsorption fluidized bed chromatography, reversed-phase chromatography, hydrophobic interaction chromatography, and combinations thereof; if the technique is electrophoresis, the electrophoretic technique can be selected from the group consisting of gel electrophoresis, free-flow electrophoresis, electrofocus, isostatic electrophoresis, affinity electrophoresis, immunoelectrophoresis, reverse electrophoresis, and capillary electrophoresis, capillary zone electrophoresis, and combinations thereof; and if the technique is spectroscopy (or spectroscopic measurement), the spectroscopic (or spectroscopic measurement) technique can be selected from mass spectrometry, ultraviolet spectroscopy, visible spectroscopy, fluorescence spectroscopy, and ultraviolet-visible spectroscopy, and combinations thereof. In some specific sub-embodiments, the technique includes liquid chromatography-mass spectroscopy; in other cases, the technique includes capillary zone electrophoresis linked to mass spectrometry.

[0009] In a third aspect, disclosed herein is a method for preparing a polypeptide for analysis, comprising the steps of: denaturing, reducing, and alkylating the polypeptide; digesting the polypeptide with trypsin to produce trypsin-cleaved polypeptide fragments; splitting the trypsin-cleaved polypeptide fragments into a first aliquot and a second aliquot; digesting the first aliquot of the trypsin-cleaved polypeptide fragments with neutrophil elastase; combining the first and second aliquots in a ratio of about 1:1; and analyzing the combined aliquots. Neutrophil elastase may be, for example, human neutrophil elastase (EC3.4.21.37). When analyzing a sample, the analysis may 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 a sample involves chromatography, the chromatographic technique can be selected from gas chromatography, liquid chromatography, high-performance liquid chromatography, ultrahigh-performance liquid chromatography, size exclusion chromatography, ion exchange chromatography, affinity chromatography, extended adsorption fluidized bed chromatography, reversed-phase chromatography, hydrophobic interaction chromatography, and combinations thereof; if the technique is electrophoresis, the electrophoretic technique can be selected from the group consisting of gel electrophoresis, free-flow electrophoresis, electrofocus, isostatic electrophoresis, affinity electrophoresis, immunoelectrophoresis, reverse electrophoresis, and capillary electrophoresis, capillary zone electrophoresis, and combinations thereof; and if the technique is spectroscopy, the spectroscopic (or spectroscopic measurement) technique can be selected from mass spectrometry, ultraviolet spectroscopy, visible spectroscopy, fluorescence spectroscopy, and ultraviolet-visible spectroscopy, and combinations thereof. In some specific sub-embodied aspects, the technique includes liquid chromatography-mass spectroscopy; in other cases, the technique includes capillary zone electrophoresis linked to mass spectrometry.

[0010] In a fourth aspect, the foregoing discloses a method for preparing a polypeptide for analysis, comprising the steps of: preparing a sample containing 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; where the second protease is neutrophil elastase; and the first protease is a method different from that of the second protease. In this aspect, the first protease may include a protease selected from the group consisting of trypsin, Asp-N, and Glu-C; furthermore, the polypeptide may be denatured and / or reduced and / or alkylated before digestion with the first polypeptide. The neutrophil elastase may be, for example, human neutrophil elastase (EC3.4.21.37). When analyzing the sample, the analysis may 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 a sample involves chromatography, the chromatographic technique can be selected from gas chromatography, liquid chromatography, high-performance liquid chromatography, ultrahigh-performance liquid chromatography, size exclusion chromatography, ion exchange chromatography, affinity chromatography, extended adsorption fluidized bed chromatography, reversed-phase chromatography, hydrophobic interaction chromatography, and combinations thereof; if the technique is electrophoresis, the electrophoretic technique can be selected from the group consisting of gel electrophoresis, free-flow electrophoresis, electrofocus, isostatic electrophoresis, affinity electrophoresis, immunoelectrophoresis, reverse electrophoresis, and capillary electrophoresis, capillary zone electrophoresis, and combinations thereof; and if the technique is spectroscopy or spectroscopic measurement, the spectroscopic (or spectroscopic measurement) technique can be selected from mass spectrometry, ultraviolet spectroscopy, visible spectroscopy, fluorescence spectroscopy, and ultraviolet-visible spectroscopy, and combinations thereof.In some specific sub-parts, the technique includes liquid chromatography-mass spectroscopy; in other cases, the technique includes capillary zone electrophoresis linked to mass spectroscopy. The filter may have, for example, a molecular weight cutoff of 30 kDa.

[0011] Furthermore, in a fifth aspect, the foregoing discloses a method for preparing a polypeptide for analysis by liquid chromatography, capillary zone electrophoresis or mass spectrometry, comprising the steps of: preparing a sample containing the polypeptide; applying the sample to a 30 kDa MWCO filter; capturing the sample on the filter to produce a concentrate (retentate); denaturing the sample concentrate on the filter; reducing the sample concentrate 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 the active protease in the filtrate with guanidine; and analyzing the filtrate by liquid chromatography, capillary zone electrophoresis or mass spectrometry; where the second protease is neutrophil elastase, and the first protease is a method different from that of the second protease. In such a configuration, the neutrophil elastase may be human neutrophil elastase (EC3.4.21.37).

[0012] In these five embodiments, the method may be carried out by automation, at least in part. In some partial embodiments, the addition and replacement of the solution are handled by an automated liquid processing device or a robot.

[0013] Furthermore, in these five embodiments, the methods disclosed herein may be used to prepare and analyze therapeutic polypeptides. Therapeutic polypeptides may be selected from the group consisting of antibodies or their antigen-binding fragments, derivatives of antibodies or antibody fragments, and fusion polypeptides. Examples of antibodies and antibody constructs include infliximab, bevacizumab, cetuximab, ranibizumab, palivizumab, avagovomab, absiximab, actoxumab, adalimumab, aferimomab, aftuzumab, aracizumab, aracizumab pegol, ald518, alemtuzumab, alirocumab, artumomab, amatsuximab, anatumomab mafenatox, anlukinzumab, apolizumab, artimomab, aselizumab, artinumab, atorizumab, atrolimumab, and Silizumab, bapineuzumab, basiliximab, bavituximab, vectumomab, belimumab, benralizumab, vertilimumab, besilesomab, bevacizumab, bezlotoxumab, bisilomab, vibatuzumab, vibatuzumab meltansine, blinatumomab, brosozumab, brentuximab vedotin, briakinumab, brodalumab, canakinumab, cantuzumab meltansine, caplacizumab, capromab pendetide, carlumab, catsumakisomab, CC49, sedelizumab, sertoli Zumab pegol, cetuximab, sitatuzumab bogatox, sixtumumab, crazakizumab, clenoliximab, cribatuzumab tetraxetan, conatumumab, crenezumab, cr6261, dasetuzumab, dacrizumab, darotuzumab, daratumumab, demcizumab, denosumab, detumomab, dorurimomab aritox, dorozizumab, duligotuzumab, dupilumab, eclomeximab, eculizumab, edovacomab, edrecolomab, efalizumab, efungumab, elotuzumab Elsilimo-b, Enabatuzumab, Enlimo-b pegol, Enokizumab, Enoticumab, Encituximab, Epitumomab citucetan, Epratuzumab, Erenumab, Erlizumab, Erzumakisomab, Etalacizumab, Etrolizumab, Evolocumab, Exhibivirumab, Fanoresomab, Faralimo-b, Farletuzumab, Facinumab, FBTA05, Felbizumab, Fezakinumab, Ficlatuzumab, Figitumumab, Flambotumab, Fontrizumab, Foralumab,Folavirumab, Fresolimmab, Fluranumab, Futuximab, Galiximab, Ganitumab, Gantenerumab, Gabirimomab, Gemtuzumab Ozogamicin, Gevokizumab, Gilentuximab, Grembatumumab Vedotin, Golimumab, Gomiliximab, GS6624, Ibalizumab, Ibritumomab Chiuxetan, Icurumab, Igobomab, Imsilomab, Imugatuzumab, Incrakumab, Indatuximab Tansine, Infliximab, Intetumumab, Inorimomab, Inotuzumab Ozogamicin, Ipilimumab, Iratumumab, Itri Zumab, ixekizumab, keriximab, rabetsuzumab, lebrikizumab, remalesomab, reldelimumab, lexatumumab, rivivirumab, rigerizumab, lintuzumab, lirirumab, rorbotuzumab meltansine, lucatumumab, lumiliximab, mapatuzumab, masurimomab, mapurilimumab, matsuzumab, mepolizumab, meterimumab, miratuzumab, minretumomab, mitumomab, mogamulizumab, morolimmab, motabizumab, moxetumomab pasdotox, muromonab-cd3, nacolomabutafenatox, namilumab, naptumomab esta Fenatox, nalnatumab, natalizumab, nevacumab, necitumumab, nererimomab, nesbakumab, nimotuzumab, nivolumab, nofetumomab, merpentan, okalatuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab, onarutuzumab, oporutuzumab, monatox, olegobomab, orticumab, otelixizumab, oxerumab, ozanezumab, ozoralizumab, padibaximab, palivizumab, panitumumab, panobakumab, pulsatuzumab, pascolizumab, patechrizumab, pat Ritumab, pemtumomab, perakizumab, pertuzumab, pexerizumab, pizilizumab, pintumomab, pracumubab, ponezumab, priliximab, pritumumab, PRO140, quilizumab, lacosumomab, radrezumab, rafibirumab, ramucirumab, ranibizumab, laxibakumab, regavirumab, reslizumab, rilotumumab, rituximab, lobatumumab, loredumab, romosozumab, lontarizumab, loberizumab, luprizumab, samarizumab, sarilumab, saturomab pendecide, secukinumab, sevilumab, sibrotuzumabSifalimumab, siltuximab, simtuzumab, ciprizumab, silumab, solanezumab, solitomab, sonepcizumab, sontuzumab, stamlumab, thresomab, suvizumab, tabarmab, takatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, tapritumomab paptox, tefibazumab, terimomab aritox, tenatumomab, tefibazumab, teneriximab, teprizumab, teprotumumab, tezeperumab, TGN1412, tremelimumab, tisilimmab, tildrakizumab, tigatuzumab, TNX-6 50. Examples include tocilizumab, tralizumab, tocitumomab, tralokinumab, trastuzumab, TRBS07, toregalizumab, tucotzumab cermoloukin, tubirumab, ubrituximab, urerumab, urtoxazumab, ustekinumab, bapariximab, baterizumab, vedolizumab, bertuzumab, bepalimomab, besenkumab, vizilizumab, borosiximab, borsetuzumab mafodotin, botumumab, zaltumumab, zanolimumab, zatuximab, diralimumab, zolimomab aritox, and their antibodies shown in Table H. In other embodiments, therapeutic polypeptides include glycoproteins, CD polypeptides, HER receptor polypeptides, cell adhesion polypeptides, growth factor polypeptides, insulin polypeptides, insulin-related polypeptides, coagulation polypeptides, coagulation-related polypeptides, albumin, IgE, blood group antigens, colony-stimulating factors, receptors, neurotrophic factors, interferons, interleukins, viral antigens, lipoproteins, calcitonin, glucagon, atrial natriuretic factor, pulmonary surfactants, tumor necrosis factor α and β, enkephalinases, and mouse gonadotropin-related peptides. Tide, DNAse, inhibin, activating, integrin, protein A, protein D, rheumatoid factor, immunotoxin, bone morphogenetic protein, superoxide dismutase, surface membrane polypeptide, disintegration promoter, AIDS envelope, transport polypeptide, homing receptor, adresin, regulatory polypeptide, immunoadhesin, myostatin, TALL polypeptide, amyloid polypeptide, thymic interstitial lymphopoietin, RANK ligand, c-kit polypeptide, TNF receptor and angiopoietin, and their bioactive fragments.It is a polypeptide selected from a group consisting of analogs or their variants.

[0014] In some cases, the therapeutic polypeptide is BiTE® (bispecific T cell engager). [Brief explanation of the drawing]

[0015] [Figure 1] This figure shows the conserved α-CD3 domains across multiple BiTE® molecules (canonical and half-life extended). Trypsin digestion produces an 8 kDa peptide containing two complementarity-determining regions (CDRs) along with multiple hotspots. [Figure 2] This figure shows the enzyme specificity for human neutrophil elastase. P1 corresponds to the C-terminus of a single amino acid, and P1' corresponds to the N-terminus of the following residue. The relative font size corresponds to the possibility of cleavage at that particular residue (adjusted from the MEROPS database; (Rawlings et al., 2014)). [Figure 3] This figure shows the extracted ion chromatograms (EICs) of unstressed and forced-deamidated (pH 8.5, 50°C, t=3 days) samples of BiTE®-1, indicating the deamidated species in both the unmodified and forced-deamidated samples. [Figure 4] This figure shows the tandem mass spectrometry (MSMS) results for the three deamidated species observed in Figure 3. The diagnostic b3 ion shifts by +0.9838 Da at peak 3 compared to peaks 1 and 2. This mass shift corresponds to deamidation. [Figure 5] This diagram illustrates the differences between a single digestion of the BiTE(registered trademark) molecule, a conventional MAM approach, and a MAM approach that incorporates a 1:1 mixture of samples digested with trypsin and samples digested with neutrophil elastase after being digested with trypsin. [Figure 6]A diagram showing the experimental design used to evaluate the effectiveness and robustness of Protocol 3 (for details, see the examples below). [Figure 7] A diagram schematically showing the oversample preparation outlined in Protocol 4 (for example, for further details, see the examples below). [Figure 8] A diagram showing the trypsin specificity of Protocol 4 for BiTE®-1, BiTE®-2, and BiTE®-3. Specificity was first calculated by the MassAnalyzer by identifying strict trypsin cleavage in the search parameters. The data were then re-searched using cleavage at the C-terminus of the residues KRVITAL (low specificity). Trypsin specificity was the total area of peptides identified using the strict trypsin search divided by the total area of peptides identified using the KRVITAL cleavage search. This value was expressed as a percentage. [Figure 9] A diagram showing the percentage of the total area corresponding to the 8 kDa linker peptide for BiTE®-1, BiTE®-2, and BiTE®-3. The data were investigated by the MassAnalyzer using cleavage at the C-terminus of the 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 cleavage search. This value was expressed as a percentage. The percentage of the total area corresponding to the 8 kDa linker peptide is higher for BiTE®-2, which does not contain the Fc domain added for half-life extension as a canonical BiTE® molecule. [Figure 10] A diagram showing an overlay of the total ion chromatograms (TIC) of repeated injections of a BiTE®-3 sample prepared using Protocol 3. The injection solutions were prepared from the same vial and separated over approximately 4 days. [Figure 11]Figure showing the TIC overlay of repeated injections of BiTE®-3 samples prepared using Protocol 4. The injection solutions were prepared from the same vial and separated over approximately 4 days. [Figure 12] Figure showing the TIC overlay comparing three BiTE®-2 samples each prepared using Protocols 4 and 5. The two peaks (arrows) observed in four samples for all three protocols correspond to non-specific alkylation.

Mode for Carrying Out the Invention

[0016] To address the problem of cleavage-resistant polypeptides for MAM analysis, the cleavage site and specificity for a number of commercially available enzymes were tested. Human neutrophil elastase (HNE; EC 3.4.21.37, also known as elastase-2, human leukocyte elastase (HLE), myeloid serine protease, mejurasin, and PMN elastase) has been 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 non-specifically, which is a disadvantage where the frequency decreases as the substrate length decreases (Stein et al., 1987). Assuming the amino acid sequence of the target polypeptide, digestion with HNE could function as a means to monitor the desired region within the polypeptide, aside from the tendency of HNE to cleave the polypeptide non-specifically.

[0017] For example, the inventors observed that when bispecific T cell engager (BiTE®) molecules are digested solely by HNE, the resulting peptides are extremely numerous and of low abundance (due to nonspecific cleavage). For instance, Figure 1 shows the conserved α-CD3 domains (canonical and half-life extended) across numerous BiTE® molecules. Trypsin digestion produces an 8 kDa peptide containing two complementarity-determining regions (CDRs) along with numerous hotspots that are difficult to identify and characterize using conventional mass spectrometry (MS) experiments. This peptide also lacks residues that are susceptible to cleavage by other widely used enzymes, such as Glu-C and Asp-N.

[0018] The inventors also unexpectedly observed that, following trypsin digestion on the filter, both the conserved 8 kDa linker peptide and the molecularly specific linker peptide of the BiTE® molecule were retained by a 30 kDa molecular weight cutoff (MWCO) filter, regardless of whether the filter membrane was composed of regenerated cellulose or polyethersulfone. The step of enhancing the specificity of HNE by first digesting with a non-HNE protease and the step of utilizing the unexpected retention of the linker peptide resulting from first protease digestion by a 30 kDa MWCO (or other cutoff) filter enabled the development of a novel robust method for characterizing the BiTE® molecule (and other polypeptides) and quantifying key attributes.

[0019] A novel sample preparation method is disclosed, which involves using a first digestion step followed by digestion by HNE.

[0020] definition Both the general explanation above and the detailed explanation below are illustrative and descriptive, and not limiting. The use of the singular form includes the plural form unless otherwise specified. The use of "or" means "and / or" unless otherwise specified. The use of the term "contains" and other forms such as "contains" and "includes" is not limiting. The terms "element" or "component" include both elements and components containing one unit and elements and components containing two or more subunits unless otherwise specified. The use of the term "part" can include a part or all of a part. For example, when a numerical range such as 1 to 5 is mentioned, all intervening values ​​such as 1, 2, 3, 4 and 5 and their fractions such as 1.5, 2.2, 3.4 and 4.1 are explicitly included.

[0021] When "approximately" or "~" modifies a quantity (e.g., "approximately" 3 mM), it means that there may be fluctuations around the modified quantity. These fluctuations can be caused by various means, such as typical measurement and processing procedures, careless errors, and the purity of the components.

[0022] The terms “includes” and “contains” are intended to mean that the Method includes the enumerated elements but does not exclude other unenumerated elements. The terms “essentially consist of” and “essentially consist of,” when used in the Method disclosed herein, include the enumerated elements but exclude unenumerated elements that alter the fundamental nature of the Method, but do not exclude other unenumerated elements. The terms “consist of” and “comprising” when used to define a Method exclude substantial Method steps. Embodiments defined by each of these evolving terms are included within the scope of this disclosure.

[0023] "Interconnected" means related, both directly and indirectly. For example, one 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] The terms "protein," "peptide," and "polypeptide" are used synonymously because they all refer to a chain of amino acids in which each amino acid is connected to its neighbor by peptide bonds.

[0025] "Denaturation," "denaturing," and "to denaturate" refer to the process by which polypeptides lose, at least partially, the quaternary, tertiary, and secondary structures found within them in their natural state through the application of external stress or reagents (i.e., denaturants).

[0026] "Modifying agent" means any substance, composition, energy, or state that can modify a polypeptide. Examples of modifying agents include strong acids or strong bases, inorganic salts, organic solvents, radiation, chaotropic agents, heat, or combinations thereof.

[0027] "Denatured polypeptide," "denatured protein," etc., refers to polypeptides whose secondary, tertiary, and / or quaternary structures have been altered from their natural polypeptides. Polypeptides can be completely denatured or partially denatured. "Undenatured polypeptide" or "undenatured protein" (and similar terms) refers to polypeptides that maintain their secondary, tertiary, and, where applicable, quaternary structures. "Natural polypeptide" or "natural protein" (and similar terms) refers to naturally occurring polypeptides that have any major reference sequence listed in the UniProt Knowledgebase database (The UniProt, 2017) or the UniGene database (Pontius et al., 2003).

[0028] A “reduced polypeptide” or “reduced protein” (and similar terms) refers to a polypeptide in which at least one of its interchain or intrachain disulfide bonds is broken. Such bonds can be formed between reducing thiol groups, such as those available on cysteine ​​residues.

[0029] "Alkylated polypeptide" or "alkylated protein" (and similar) refers to a polypeptide to which an alkyl group has been transferred. In practice, polypeptides are often alkylated at a thiol group (for example, so that it is available at a cysteine ​​residue) to prevent the formation of a reduced thiol, or the reformation of disulfide bonds or crosslinks after reduction.

[0030] In the context of polypeptides, "digestion" refers to the fragmentation of the polypeptide into two or more fragments, which is mediated by another substance, chemical, or enzyme.

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

[0032] "Protease," "peptidase," "protein-degrading cleaving enzyme," "endoproteinase," and "proteinase" all refer to enzymes, specifically polypeptides or fragments thereof that catalyze the hydrolysis of peptide bonds. Proteases include amino acid sequence variants of known proteases that catalyze peptide bond hydrolysis, even when such catalytic activity is reduced within the variant.

[0033] Enzyme-catalyzed reactions are classified according to the Enzyme Committee numbering system. Proteases catalyze peptide bond hydrolysis, and they are classified as follows: aminopeptidases (EC3.4.11), dipeptidases (EC3.4.13), dipeptidylpeptidases and tripeptidylpeptidases (EC3.4.14), peptidyldipeptidases (EC3.4.15), serine carboxypeptidases (EC3.4.16), metallocarboxypeptidases (EC3.4.17), and systolic proteases (EC3.4.18). These are classified into the EC3.4 class, which includes in-type carboxypeptidases, ω-peptidases (EC3.4.19), serine proteases (EC3.4.21), cysteine ​​proteases (EC3.4.22), aspartate endopeptidases (EC3.4.23), metallopeptidases (EC3.4.24), threonine endopeptidases (EC3.4.25), and endopeptidases with unknown catalytic mechanisms (EC3.4.99). Specific examples of proteases include trypsin (EC3.4.21.4), endoproteinase Asp-N (EC3.4.24.33), and endoproteinase Glu-C (EC3.4.21.19).

[0034] Elastases are proteases that hydrolyze elastin, the elastic fiber polypeptide of the extracellular matrix (except for those classified as EC3.4.21.70). There are eight human genes that encode elastases (Table A):

[0035] [Table 1]

[0036] [Table 2]

[0037] Neutrophil elastase (sometimes called elastase-2, leukocyte elastase, bone marrow serine protease, meduracin, or PMN elastase) is an elastase secreted by neutrophils and macrophages during inflammation that breaks down bacteria. Human neutrophil elastase (HNE) was first reported in 1968 (Janoff and Scherer, 1968). HNE has been shown to differ from pancreatic elastase in that soy trypsin inhibitors and salivary kallikline inhibitors inhibit HNE but not pancreatic elastase activity. HNE maintains its activity at physiological salt concentrations, while pancreatic elastase activity decreases. HNE is at least 10 times more resistant to serum elastase inhibitors than pancreatic elastase (as defined by Janoff and Scherer, 1968). Finally, HNE is also more resistant to low pH than pancreatic elastase (Janoff and Scherer, 1968).

[0038] Sinha et al. (Sinha et al., 1987) were the first to report the amino acid sequence of HNE, demonstrating that this polypeptide has only 43% sequence homology to porcine pancreatic elastase. The UniProt database for UniProtKB - P08246 (ELNE_HUMAN) (The UniProt, 2017) states that ELANE encodes HNE as a 267-amino acid precursor polypeptide consisting of a signal peptide (amino acid residues 1-27), a propeptide domain (residues 28-29), and a mature polypeptide (residues 30-267). The mature polypeptide contains a peptidase S1 domain (residues 30-247). Disulfide bonds can be found between residues 55 and 71, 151 and 208, 181 and 187, and 198-223. Asparagine residues at positions 88, 124, and 174 can be glycosylated (N-linked). The sequences for HNE are shown in Table B. In Table B, the signal peptide is written in bold, with the propeptide domain underlined once. The mature polypeptide is written in italics, with the S1 peptidase domain underlined twice and written in both italics and bold.

[0039] [Table 3]

[0040] HNE can hydrolyze almost all polypeptide members of the extracellular matrix, including several types of collagen, fibronectin, proteoglycans, heparin, and cross-linked fibrin.

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

[0042] In situations where polypeptide fragments are generated, "chemical cleavage" refers to the fragmentation of polypeptides by chemical substances. These "chemical substances" are non-proteinogenic substances or compounds. These chemical substances can be organic or inorganic.

[0043] "Molecular weight cutoff" or "MWCO" means that the MWCO of a membrane is an expression of the membrane selectivity for solute molecules of various molecular weights (MW), where the MW value (denoted in Daltons (Da)) is derived from the solute molecules that yield a 90% rejection rate when a range of different MW solutes are filtered in the target solvent (which is water for most liquid-based pressure-driven membrane applications), where the rejection rate is given by equation (1):

number

[0044] MWCO (Membrane Mass Concentration) is determined experimentally using dextran, polyethylene glycol, and proteins of various molecular weights to evaluate the MWCO of a membrane or filter. The rejection rate depends on numerous solutes and process parameters such as solute type, concentration, hydrodynamics, pressure, temperature, and pH. MWCO measurements are typically performed in separate experiments using different solutes, each with a predetermined MW.

[0045] For low MW ranges (approximately less than 10 kDa), polyethylene glycol (PEG), n-alkanes, and oligostyrenes can be used in solute inhibition assays. For high MW ranges (approximately greater than 10 kDa), dextran and sugars are often used (Rohani et al., 2011).

[0046] method Disclosed herein is a method relating to the steps of digesting a polypeptide in a sample using a first protease and a second protease, wherein the first protease produces at least two fragments of the polypeptide, and these at least two fragments of the polypeptide are subsequently digested by the second protease; and analyzing the sample after digestion with the second protease, wherein the second protease is neutrophil elastase; and the first protease is different from the second protease. Other steps in this method may include the steps of digesting the polypeptide in the sample with the first protease and then splitting the sample into two aliquots, and then digesting the polypeptide contained 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. The analysis may include chromatography, electrophoresis, spectroscopy, and combinations thereof.

[0047] In some embodiments, the method includes the steps of applying a sample to a filter having an 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 may further incorporate a filtration step. In some embodiments, the MWCO filter retains a significant proportion of polypeptides and polypeptide fragments, even when the size of these polypeptides or polypeptide fragments has a MW that is significantly smaller than the MWCO of the filter.

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

[0049] Polypeptides can be modified using various art-accredited techniques and modifying agents. In some embodiments, multiple modifying agents are used together, either simultaneously or sequentially. For example, SDS and heating can be combined to modify polypeptides.

[0050] Protein denaturation can be carried out by any means that disrupts the quaternary, tertiary, or secondary polypeptide structure. For example, 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 be manipulated to further accelerate denaturation. These components are often used together to effectively unravel the polypeptide chain.

[0051] Examples of additional 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 into hydrophilic groups: anionic, cationic, nonionic, and amphoteric. Anionic and cationic detergents are more likely to denature, and examples of these 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, amphoteric 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 intramolecular salt (SB3-8), and d(decyldimethylammonio)propanesulfonate intramolecular salt (SB3-10). Anionic detergents are preferred, and SDS is particularly preferred.

[0053] The denaturing agent may be heat (for most polypeptides), for example, a high temperature of 30°C or higher. The denaturing agent may include stirring. In some embodiments, essentially or substantially low-salt or salt-free agents may denature the polypeptide.

[0054] The denaturing agent may be a solvent such as ethanol or other alcohols.

[0055] The step of denaturing polypeptides has been extensively tested and reported; for example, see (Tanford, 1968) for details. Those skilled in the art will understand the methods of denaturing polypeptides, given the properties of the polypeptides and the numerous denaturing agents to be selected.

[0056] polypeptide reduction A reduced polypeptide is a polypeptide that has been exposed to reducing conditions sufficient to reduce reducible residues in its polypeptide structure, such as cysteine. If a reduced polypeptide contains a thiol group or a sulfur-containing residue, the thiol group within the reduced polypeptide is reduced. A reduced polypeptide containing a cysteine ​​residue has a sulfur atom of the reduced cysteine ​​residue, which may be denoted as "-SH". A reduced polypeptide can also be a disulfide bond-containing polypeptide. A disulfide bond-containing polypeptide can become a reduced polypeptide by being exposed to reducing conditions that induce the rupture of one or more disulfide bonds (disulfide crosslinks) within the disulfide bond-containing polypeptide.

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

[0058] [Table 4]

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

[0060] Those skilled in the art will understand how to reduce a polypeptide, given the properties of the polypeptide and the reducing reagent to be selected.

[0061] Alkylate the polypeptides contained in the sample. The "step of inactivating reactive thiol groups" means blocking free thiol groups within the polypeptide to prevent undesirable thiol-disulfide exchange reactions. Alkylating agents are substances that induce hydrogen substitution by alkyl groups.

[0062] The frequent alkylation of free cysteine ​​following their reduction prevents the formation and reformation of disulfide bonds that could otherwise be formed between the free thiols of cysteine ​​residues. Commonly used alkylating agents include n-ethyl maleimide (NEM), iodoacetamide (IAA), and iodoacetic acid. Other suitable alkylating agents include dithiobis(2-nitro)benzoic acid; acrylamide; 4-vinylpyridine; nitrogen mustards, e.g., chlorambucyl and cyclophosphamide; cisplatin; nitrosourea, e.g., carmustine, lomustine, and semustine; alkyl sulfonates, e.g., busulfan; ethyleneimine, e.g., thiotepa; and triazines, e.g., decarbazine. Those skilled in the art are familiar with reagents that can be used to protect sulfhydryl groups and methods for using such reagents.

[0063] polypeptide digestion The methods disclosed herein include the step of cleaving an analyte polypeptide in a sample in a first digestion, wherein the cleavage step generates at least two fragments of the polypeptide. Any method for fragmenting a polypeptide can be used on the premise that at least two fragments of the polypeptide are generated; furthermore, complete degradation of the polypeptide to, for example, a single amino acid is undesirable.

[0064] Such digestion steps involve the use of a protease as a convenient means of cleavage (wherein the protease is not neutrophil elastase). Any protease can be used as long as it cleaves the polypeptide into at least two fragments. The rationale for this first digestion before digestion with neutrophil elastase is that it provides the polypeptide fragments to neutrophil elastase (such as human neutrophil elastase), which has increased specificity compared to when presented with intact polypeptide.

[0065] In some embodiments, a mixture of two or more proteases may be used. In other embodiments, the first digestion may consist of a number of sequential digestions. For example, the first digestion is carried out using a first protease, and then in a subsequent reaction, the second digestion is carried out using a second protease (where none of the proteases are neutrophil elastases). The second (or subsequent) digestions are completed to improve the specificity of the neutrophil elastase used in the step of further cleaving the polypeptide with the neutrophil elastase.

[0066] Examples of useful proteases (but not all) 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 peptide bond selectivity and EC number, are shown in Table D, taken from (Unspecified, 2007). The sources listed for each protease are for illustrative purposes only; many of these proteases are commercially available.

[0068] In some embodiments, protein:protease 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 approximately 100 ng / mL to 1 mg / mL, or approximately 100 ng / mL to 500 μg / mL, or approximately 100 ng / mL to 100 μg / mL, or approximately 1 μg / mL to 1 mg / mL, or approximately 1 μg / mL to 500 μg / mL, or approximately 1 μg / mL to 100 μg / mL, or approximately 10 μg / mg to 1 mg / mL, or approximately 10 μg / mg to 500 μg / mL, or approximately 10 μg / mg to 100 μg / mL. In some embodiments, the digestion step is 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 to 8 hours, or 1 to 6 hours, or 1 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. In some embodiments, the digestion step is incubated at 37°C. As those skilled in the art will know, in vitro protease digestion is well understood in the art, and appropriate conditions (buffer, incubation time, amount of protease, volume, etc.) can be selected.

[0069] [Table 5]

[0070] [Table 6]

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

[0072] In some embodiments, the first digestion may include a series of digestions, where at least one of these digestions includes a step using a chemical such as CNBr, NTCB, hydroxylamine, formic acid, and BNPS-skatole. For example, the first digestion may be carried out using a chemical that cleaves the polypeptide into at least two fragments, and then in a subsequent reaction, the second digestion may be carried out using a protease (where the protease is not a 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 a neutrophil elastase.

[0073] In the methods disclosed herein, subsequent digestion is carried out using 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 approximately 100 ng / mL to 1 mg / mL, or approximately 100 ng / mL to 500 μg / mL, or approximately 100 ng / mL to 100 μg / mL, or approximately 1 μg / mL to 1 mg / mL, or approximately 1 μg / mL to 500 μg / mL, or approximately 1 μg / mL to 100 μg / mL, or approximately 10 μg / mg to 1 mg / mL, or approximately 10 μg / mg to 500 μg / mL, or approximately 10 μg / mg to 100 μg / mL. In some embodiments, the digestion step is 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 to 8 hours, or 1 to 6 hours, or 1 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. Those skilled in the art will be able to understand the various parameters and select appropriate conditions for digestion using neutrophil elastase.

[0075] Divide the sample into aliquots. In some embodiments, the sample containing the polypeptide to be analyzed is divided into aliquots before digestion with neutrophil elastase. In some cases, the step of dividing the sample into aliquots is carried out 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, for example, using ultraviolet spectrophotometry.

[0076] Filter Selection When selecting an appropriate MWCO for a specific application, numerous factors are considered, including sample concentration, composition, molecular shape, and operating conditions such as temperature, pressure, and cross-flow rate. Other variables related to molecular channel flow are also taken into account. For example, linear molecules, high transmembrane pressure (TMP), and low sample concentration may increase molecular channels, while low temperature and membrane contamination may decrease them. Verification methods for MWCOs are not always comparable, as they vary from manufacturer to manufacturer. In this field, it is generally recommended to select an MWCO with a molecular weight at least twice that of the retained solute.

[0077] The shape of the filter is also considered in the methods disclosed herein. For volumes of 1-2 mL, a filter unit in which the filter is inclined when the filter unit is perpendicular to the substrate is less desirable than a filter unit in which the filter is horizontal when the filter unit is perpendicular to the substrate.

[0078] Suitable materials for MWCO membranes are hydrophilic. Suitable hydrophilic materials include polyethersulfone, polyvinylidene difluoride, and regenerated cellulose.

[0079] Suitable cutoff 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 specific manufacturers have appropriate MWCO specifications. Comparable filters from other manufacturers may also be used. The MWCO specifications provided by manufacturers are reliable because they fairly take into account the differences in MWCO ratings between manufacturers, and the methods disclosed herein make good use of the remarkable observation that some low molecular weight molecules are retained by the filter even when these molecules have molecular weights below (and often substantially below) the filter's rated MWCO (see, for example, Example 11).

[0080] The step of filtering the sample using an MWCO filter can be performed using any suitable filtering device and any suitable filtering method. The filtering step can be performed by gravity, capillary force, or general centrifugation, including ultracentrifugation.

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

[0082] Generally, suitable analytical methods may include chromatography, electrophoresis, and spectroscopic analysis. Some of these methods can be combined.

[0083] Those skilled in the art can utilize, for example, handbooks that facilitate the selection of appropriate analytical methods and the appropriate conditions for carrying out those methods, including, for example (Gunzler and Williams, 2001).

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

[0085] A summary of some known chromatography methods is shown in Table E.

[0086] [Table 7]

[0087] [Table 8]

[0088] The prepared polypeptides can also be further analyzed using electrophoresis—gel electrophoresis, free-flow electrophoresis, isoelectric focusing, isokinetic electrophoresis, affinity electrophoresis, immunoelectrophoresis, inverse electrophoresis, capillary electrophoresis, and capillary zone electrophoresis. Those skilled in the art 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 transported through a solvent by an electric field and do not exist at their isoelectric point. Polypeptides move at a rate proportional to their charge density. The mobility of polypeptides 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 a smaller pore size and is particularly useful in the methods disclosed herein, and is ideal for separating the majority of polypeptide fragments.

[0090] Table F shows examples of polypeptide electrophoresis techniques.

[0091] [Table 9]

[0092] [Table 10]

[0093] [Table 11]

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

[0095] Table G shows examples of polypeptide electrophoresis techniques.

[0096] [Table 12]

[0097] The principle that enables mass spectrometry (MS) consists of the steps of ionizing a chemical compound to generate charged molecules or molecular fragments, and then measuring the mass-to-charge ratio. In an exemplary MS procedure, the sample is loaded into an MS instrument and vaporized, the components of the sample are ionized by one of various methods that cause the formation of positively charged particles (e.g., by bombarding them with an electron beam), the positive ions are then accelerated by a magnetic field, and as they pass through the electromagnetic field, calculations are performed on the mass-to-charge ratio (m / z) of the particles based on the details of the ion motion, and the ions are detected and classified according to their m / z ratios.

[0098] An example MS instrument has three modules: an ion source that 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 spectrometer that classifies the ions by their mass-to-charge ratio by applying an electromagnetic field; and a detector that measures numerical values ​​of indicator quantities, thus providing 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. These include gas chromatography-mass spectroscopy (GC / MS or GC-MS), liquid chromatography-mass spectroscopy (LC / MS or LC-MS), and ion mobility spectroscopy / mass spectroscopy (IMS / MS or IMMS).

[0100] Analytical methods (chromatography, electrophoresis, and spectroscopic analysis) can be combined. For example, combinations such as liquid chromatography-mass spectroscopy, capillary zone electrophoresis linked to mass spectroscopy, and ion mobility spectroscopy-mass spectroscopy.

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

[0102] Liquid processing robots can be configured using various laboratory equipment such as centrifuges, PCR machines, colony pickers, shakers, and heating devices. 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 containing binding to one or more of the following can be prepared and analyzed by the methods disclosed herein: These include CD proteins, including CD3, CD4, CD8, CD19, CD20, CD22, CD30, and CD34, including those that interfere with receptor binding; HER receptor family proteins, including HER2, HER3, HER4, and EGF receptors; cell adhesion molecules, such as LFA-I, MoI, pl50, 95, VLA-4, ICAM-I, VCAM, and αv / β3 integrins. 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 duct inhibitors, human macrophage inflammatory protein (MIP-1α), erythropoietin (EPO), nerve growth factors such as NGF-β, platelet-derived growth factor (PDGF), fibroblast growth factors including aFGF and bFGF, epidermal growth factor (EGF), especially transforming growth factors (TGF) including 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(l-3)-IGF-I (brain IGF-I), and bone induction factors. Insulin and insulin-related proteins, including insulin A chain, insulin B chain, proinsulin, and insulin-like growth factor-binding proteins. In particular, coagulation and coagulation-related proteins such as factor VIII, tissue factor, von Willebrand factor, protein C, α-1-antitrypsin, plasminogen activators such as urokinase and tissue plasminogen activator ("t-PA"), bombazine, thrombin and thrombopoietin; (vii) other blood proteins and serum proteins including but not limited to albumin, IgE and blood group antigens; colony-stimulating factors and their receptors, including, for example, M-CSF, GM-CSF and G-CSF and their receptors such as the CSF-1 receptor (c-fms).Receptors and receptor-related proteins, including, for example, the flk2 / flt3 receptor, obesity (OB) receptor, LDL receptor, growth hormone receptor, thrombopoietin receptor ("TPO-R", "c-mpl"), glucagon receptor, interleukin receptor, interferon receptor, T cell receptor, stem cell factor receptors 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 relaxin A chain, relaxin B chain, and prorelaxin; for example, interferon-α, -β, and -γ and their receptors. Interleukins and interleukin receptors, including IL-1 to IL-33 and IL-1 to IL-33 receptors, for example, the IL-8 receptor in particular. Viral antigens, including AIDS enveloped virus antigens. Lipoproteins, calcitonin, glucagon, atrial natriuretic factor, pulmonary surfactants, tumor necrosis factor-α and -β, enkephalinase, RANTES (regulated on activation normally T-cell expressed and secreted), mouse gonadotropin-related peptides, DNAse, inhibin and activin. Integrins, protein A or D, rheumatoid factor, immunotoxins, bone morphogenetic proteins (BMPs), superoxide dismutase, surface membrane proteins, denaturation-promoting factors (DAFs), AIDS envelope, transport proteins, homing receptors, adresin, regulatory proteins, immunoadhesins, antibodies. Myostatin, TALL-I, amyloid proteins, for example, but not limited to, amyloid-beta protein, thymic interstitial lymphocyte generating factor ("TSLP"), RANK ligand ("OPGL"), TALL proteins containing c-kit, TNF receptors including TNF receptor type 1, TRAIL-R2, angiopoietin, and any of the aforementioned bioactive fragments, analogs, or variants.

[0106] Exemplary polypeptides and antibodies include Activase® (alteplase); alirocumab, Aranesp® (darbepoetin-α), Epogen® (epoetin-α or erythropoietin); Avonex® (interferon β-1a); Bexxar® (tositumomab); Betaseron® (interferon β); vococizumab (anti-PCSK9 monoclonal antibody designated as L1L3, see U.S. Patent No. 8080243); Campath® (aremutuzumab); Dynepo® (epoetin-δ); Velcade® (bortezomib); MLN0002 (anti-α4β7) mAb);MLN1202 (anti-CCR2 chemokine receptor mAb);Enbrel(registered trademark) (etanercept);Eprex(registered trademark) (epoetin α);Erbitux(registered trademark) (cetuximab);Evolocumab;Genotropin(registered trademark) (somatropin);Herceptin(registered trademark) (trastuzumab);Humatrope(registered trademark) (somatropin [rDNA-derived] injection);Humira(registered trademark) (adalimumab);Infergen(registered trademark) (interferon αcon-1);Natrecor(registered trademark) (nesiritide);Kineret(registered trademark) (anakinra), Leukine(registered trademark) (sargamostim);LymphoC ide(registered trademark) (epratuzumab); Benlysta(trademark) (belimumab); Metalyse(registered trademark) (tenecteplase); Mircera(registered trademark) (methoxypolyethylene glycol epoetin β); Mylotarg(registered trademark) (gemtuzumab ozogamicin); Raptiva(registered trademark) (efalizumab); Cimzia(registered trademark) (certolizumab pegol); Soliris(trademark) (eculizumab); pexerizumab (anti-C5 complement); MEDI-524 (Numax(registered trademark)); Lucentis(registered trademark) (ranibizumab); edrecolomab (Panorex(registered trademark)); Trabio(registered trademark) (reldelimumab); TheraCim hR3 (nimotuzumab); Omnitarg (pertuzumab, 2C4); Osidem(registered trademark) (IDM-1);OvaRex(registered trademark) (B43.13); Nuvion(registered trademark) (vizilizumab); Cantuzumab meltansine (huC242-DMl); NeoRecormon(registered trademark) (epoetin β); Neumega(registered trademark) (oprelbequin); Neulasta(registered trademark) (PEGylated filgastrim, PEGylated G-CSF, PEGylated hu-Met-G-CSF); Neupogen(registered trademark) (filgrastim); Orthoclone OKT3(registered trademark) (muromonab-CD3), Procrit(registered trademark) (epoetin α); Remicade(registered trademark) (infliximab), Reopro(registered trademark) (absiximab), Actemra(registered trademark) (anti-IL6 receptor mAb), Avastin(registered trademark) (bevacizumab), HuMax-CD4(zanorimumab), Rituxan(registered trademark) (rituximab); Tarceva(registered trademark) (erlotinib); Roferon-A(registered trademark) (interferon α-2a); Simulect(registered trademark) (basilicum Simab; Stellara (trademark) (ustekinumab); Prexige (registered trademark) (lumiracoxib); Synagis (registered trademark) (palivizumab); 146B7-CHO (anti-IL15 antibody, see U.S. Patent No. 7153507); Tysabri (registered trademark) (natalizumab); Valortim (registered trademark) (MDX-1303, anti-anthrax (B. anthracis) protective antigen mAb); ABthrax (trademark); Vectibix (registered trademark) (panitumumab); Xolair (registered trademark) (omalizumab); ETI211 (anti-MRSA) mAb), IL-1 Trap (Fc portion of human IgG1 and extracellular domains of both IL-I receptor components (type 1 receptor and receptor co-protein)), VEGF Trap (Ig domain of VEGFR1 fused with IgG1 Fc), Zenapax® (daclizumab); Zenapax® (daclizumab), Zevalin® (ibritumomab tiuxetan), Zetia (ezetimab), Atasicept (TACI-Ig), anti-α4β7 mAb (vedolizumab); galiximab (anti-CD80 mAb), anti-CD23 mAb (lumiliximab);BR2-Fc (huBR3 / huFc fusion protein, soluble BAFF antagonist); Simponi (trademark) (golimumab); Mapatuzumab (human anti-TRAIL receptor-1 mAb); Ocrelizumab (anti-CD20 human mAb); HuMax-EGFR (saltumumab); M200 (boroxiximab, 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 conjugate (CAT-3888 and CAT-8015); anti-CD25 mAb (HuMax-TAC); anti-TSLP antibody; anti-TSLP receptor antibody (US Patent No. 8101182); anti-TSLP antibody designated as A5 (US Patent No. 7982016); (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 mAb; Anti-sclerostin antibody (see U.S. Patent No. 8715663 or No. 7592429), anti-sclerostin antibody designated as Ab-5 (U.S. Patent No. 8715663 or No. 7592429); 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);Examples include anti-IL5 receptor mAbs; anti-integrin receptor mAbs (MDX-Ol8, 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 antibodies suitable for this method and pharmaceutical formulations are listed in Table H. Other examples of suitable antibodies include infliximab, bevacizumab, cetuximab, ranibizumab, palivizumab, avagovomab, absiximab, actoxumab, adalimumab, aferimomab, aftuzumab, aracizumab, aracizumab pegol, ald518, alemtuzumab, alirocumab, artumomab, amatsuximab, anatumomab mafenatox, anlukinzumab, apolizumab, artitumomab, aselizumab, artinumab, atolizumab, atromumab, tocilizumab, bapineuzumab, and basilixima. B, bavituximab, vectumomab, belimumab, benralizumab, vertilimumab, besilesomab, bevacizumab, bezlotoxumab, bisilomab, vibatuzumab, vibatuzumab meltansine, blinatumomab, brosozumab, brentuximab vedotin, briakinumab, brodalumab, canakinumab, cantuzumab meltansine, caplacizumab, capromab pendetide, carlumab, katumakisomab, CC49, sedelizumab, certolizumab pegol, cetuximab, sitatuzumab bogatox, sixtumumab, crazakizumab Crenoliximab, cribatuzumab tetraxetan, conatumumab, crenezumab, cr6261, dasetuzumab, dacrizumab, darotuzumab, daratumumab, demcizumab, denosumab, detumomab, dorulimomab aritox, dorozizumab, duligotuzumab, dupilumab, eclomeximab, eculizumab, edovacomab, edrecolomab, efalizumab, efungumab, elotuzumab, elcilimomab, enabatuzumab, enlimomab pegol, enokizumab, enochikumab, encituximab, epitumomab cituximab Tan, epratuzumab, erenumab, erlizumab, erzmakisomab, etalacizumab, etrolizumab, evolocumab, excivivirumab, fanolesomab, falarimomab, falletuzumab, facinumab, fbta05, felbizumab, fezakinumab, ficratuzumab, figitumumab, frambotumab, fontrizumab, foralumab, folavirumab, fresolimmab, fluranumab, futuximab, galiximab, ganitumab, gantenerumab, gabirimomab, gemtuzumab ozogamicin, gevokizumab,Gilentuximab, Grembatumumab Vedotin, Golimumab, Gomiliximab, GS6624, Ibalizumab, Ibritumomab Chiuxetan, Icurcumab, Igobomab, Imsilomab, Imugatuzumab, Incrakumab, Indatuximab Tansine, Infliximab, Intetumumab, Inorimomab, Inotuzumab Ozogamicin, Ipilimumab, Iratumumab, Itorizumab, Ixekizumab, Keriximab, Labetuzumab, Lebrikizumab, Remalesomab, Reldelimumab, Lexatumumab, Rivivirumab, Rigerizumab, Lintuzumab, Li Rilumab, lorbotuzumab meltansine, lucatumumab, lumiliximab, mapatuzumab, masulimomab, mapulimumab, matsuzumab, mepolizumab, meterimumab, milatuzumab, minretumomab, mitumomab, mogamulizumab, morolimumab, motabizumab, moxetumomab pasdotox, muromonab-cd3, nacolomab butafenatox, namilumab, naptumomab estafenatox, nalnatumomab, natalizumab, nevacumab, necitumumab, nererimomab, nesbakumab, nimotuzumab, nivolumab, nofetumomab merpentan, okara Tuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab, onaltuzumab, oporutuzumab monatox, olegobomab, orticumab, otelixizumab, oxerumab, ozanezumab, ozoralizumab, padibaximab, palivizumab, panitumumab, panobacumab, pulsatuzumab, pascolizumab, pateclizumab, patrizumab, pemtumomab, perakizumab, pertuzumab, pexerizumab, pizilizumab, pintumomab, prakmab, ponezumab, priliximab, pritumumab, PR O140, quilizumab, lacosumomab, radrezumab, rafivirumab, ramucirumab, ranibizumab, laxibakumab, regavirumab, reslizumab, rilotumumab, rituximab, lobatumumab, loredumab, romosozumab, lontalizumab, loberizumab, luprizumab, samarizumab, sarilumab, satumomab pendecide, secukinumab, sevilumab, cibrotuzumab, cifarimumab, siltuximab, simtuzumab, ciprizumab, silumab, solanezumab, soritomab, sonepcizumab, sontuzumab, stamlumab, thresomab,Suvizumab, Tabalumab, Takatuzumab Tetraxetan, Tadocizumab, Talizumab, Tanezumab, Tapritumomab Paptox, Tefibazumab, Terimomab Aritox, Tenatumomab, Tefibazumab, Teneriximab, Teprizumab, Teprotumumab, Tezeperumab, TGN1412, Tremerimumab, Ticilimumab, Childraquizumab, Tigatuzumab, TNX-650, Tocilizumab, Tralizumab, Tositumomab, Tralokinumab Examples include trastuzumab, TRBS07, tregalizumab, tucothuzumab cermoloykin, tuvirumab, ubrituximab, urerumab, urtoxazumab, ustekinumab, bapariximab, baterizumab, vedolizumab, bertuzumab, bepalimomab, besenkumab, vizilizumab, borosiximab, borsetuzumab mafodotin, botumumab, zaltumumab, zanorimumab, zatuximab, diralimumab, and zolimomab aritox.

[0108] Other antibodies include adalimumab, bevacizumab, blinatumomab, cetuximab, conatumumab, denosumab, eculizumab, erenumab, evolocumab, infliximab, natalizumab, panitumumab, rilotumumab, rituximab, romosozumab, tezeperumab, and trastuzumab, as well as antibodies selected from Table H.

[0109] [Table 13]

[0110] [Table 14]

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

[0112] The section of examples below is presented for illustrative purposes only and is not intended to limit the disclosures or claims of this specification.

[0113] Preparation of reagents for Examples 1-2 to 4 A denaturation buffer (7M guanidine HCl, 100mM tris, 20mM 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 8M guanidine HCl (Pierce, Rockford, IL, P / N 24115), followed by the addition of 299 mg of L-methionine (JTBaker, P / N 2085-05). The pH of this solution was adjusted to pH 8.3 using 6N hydrochloride (HCl) (Sigma, St. Louis, MO, P / N 84429). The volume was adjusted to 100 mL using 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. The alkylating solution (500 mM NaIAA) was prepared by dissolving 15–65 mg of sodium iodide acetate (NaIAA) (Sigma, St. Louis, MO, P / N I-9148) in a volume of denaturing buffer sufficient to produce 500 mM NaIAA. The digestion buffer (100 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 using HCl, and the volume was adjusted to 100 mL using HPLC-grade water. The enzyme solution (1 mg / mL trypsin, 1 mg / mL HNE) was prepared by adding 100 μg 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). The 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 - Individual digests of trypsin and HNE Samples were denatured and reduced by adding 57.1 μL of denaturing buffer and 2 μL of reducing solution to 42.9 μL of sample (1–2 mg / mL in the 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 adding 7 μL of reducing solution. Samples were desalted using a Zeba Spin desalting column (Thermo Scientific, Waltham, MA, P / N 89883) with rotation at 1,500 × g using an Eppendorf 5430 centrifuge (Hamburg, Germany) 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). An enzyme solution (either trypsin or HNE) was added to each sample in an enzyme:substrate ratio of 1:20. The samples were incubated in a water bath at 37°C for 35 minutes. Digestion was quenched by adding 6 μL of digestion quenching solution.

[0115] Example 3 - Continuous digest of trypsin and HNE Samples were denatured and reduced by adding 57.1 μL of denaturing buffer and 2 μL of reducing solution to 42.9 μL of sample (1–2 mg / mL in the 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 adding 7 μL of reducing solution. Samples were desalted using a Zeba Spin desalting column (Thermo Scientific, Waltham, MA, P / N 89883) with rotation at 1,500 × g using an Eppendorf 5430 centrifuge (Hamburg, Germany) 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 in an enzyme:substrate ratio of 1:20. The samples were incubated in a water bath at 37°C for 35 minutes. HNE solution was added to each sample in an enzyme:substrate ratio of 1:20. The samples were incubated in a water bath at 37°C for 30 minutes. Digestion was quenched by adding 6 μL of digestion quenching solution.

[0116] Example 4 - Mixture of trypsin digest and trypsin-HNE serial digest The samples were denatured and reduced by adding 57.1 μL of denaturing buffer and 2 μL of reducing solution to 42.9 μL of sample (1 mg / mL in the formulation buffer), followed by incubation at 37°C for 35 minutes. After reduction, 9 μL of alkylating solution was added to each sample, followed by incubation at room temperature in the dark for 20 minutes. Alkylation was quenched by adding 7 μL of reducing solution. The samples were desalted using a Zeba desalting column with an Eppendorf 5430 centrifuge at 1,500 × g rotation according to the manufacturer's instructions. The sample concentration was measured after desalting using either a NanoPhotometer Pearl or Nanodrop 2000c. Trypsin solution was added to each sample in an enzyme:substrate ratio of 1:20. The samples were incubated in a 37°C water bath for 35 minutes. After trypsin digestion, each sample was divided into two equal aliquots (approximately 55 μL each). 3 μL of digestion quenching solution was added to the first aliquot and then set aside. HNE solution was added to the second aliquot in 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 slowly mixed.

[0117] Preparation of reagents for Examples 5-6 and 7 A denaturation buffer (6M guanidine HCl, 200mM tris, 20mM 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 8M guanidine HCl (Pierce, Rockford, IL, P / N 24115), followed by the addition of 299 mg of L-methionine (JTBaker, P / N 2085-05). The pH of this solution was adjusted to pH 8.3 using either 1N hydrochloric acid (HCl) (Ricca, Arlington, TX, P / N R3700100-120A) or 1N sodium hydroxide (NaOH) (Merck, Kenilworth, NJ, P / N 1.09137.100). The volume was adjusted to 100 mL using 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 denaturation buffer. Alkylation solution (500 mM NaIAA) was prepared by dissolving 15–65 mg of sodium iodide acetate (NaIAA) (Sigma, St. Louis, MO, P / N I-9148) in a sufficient volume of denaturing 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 7.8 using either 1N hydrochloride (HCl) (Ricca, Arlington, TX, P / N R3700100-120A) or 1N sodium hydroxide (NaOH) (Merck, Kenilworth, NJ, P / N 1.09137.100), and the volume was adjusted to 100 mL using HPLC-grade water.Enzyme solutions (1 mg / mL trypsin, 1 mg / mL HNE) were prepared by adding 100 μg 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 solutions (8 M guanidine HCl, 250 mM guanidine acetate, pH 4.7) were prepared by dissolving 76.4 g of guanidine HCl (Sigma, St. Louis, MO, P / N 50933) and 1.0 g of sodium acetate (Sigma, P / N 32319) in 95 mL of HPLC-grade water. Next, 716 μL of glacial acetic acid (Sigma, St. Louis, MO, P / N 320099) was added, and the pH was adjusted to pH 4.7 using either HCl or NaOH. The volume was then adjusted to 100 mL using HPLC-grade water.

[0118] Example 6 - MWCO spin filter-assisted continuous digestion 100 μg of the sample in the formulation buffer was added to a 30 kDa molecular weight cutoff rotating apparatus 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). This apparatus was rotated at 14,000 × g for 15 minutes using an Eppendorf 5430 centrifuge. The filtrate was discarded. 200 μL of denaturation buffer was added to the sample, and the filter apparatus was rotated 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 denaturation buffer, and 40 μL of this solution was added to the filter apparatus. The samples were denatured and reduced by incubation in a 37°C water bath for 45 minutes. Next, the samples were rotated at 14,000 × g for 15 minutes, and the filtrate was discarded. For each sample, 7 μL of alkylating solution was added to 33 μL of denaturing buffer, and 40 μL of this solution was added to the filter apparatus. The samples were alkylated by incubation at room temperature in the dark for 20 minutes. Next, the samples were rotated at 14,000 × g for 15 minutes, and the filtrate was discarded. For each sample, 4 μL of denaturing solution was added to 36 μL of denaturing buffer, and 40 μL of this solution was added to the filter apparatus to quench the alkylation. Next, the samples were rotated at 14,000 × g for 15 minutes, and the filtrate was discarded. 200 μL of digesting buffer was added to the samples, and the filter apparatus was rotated at 14,000 × g for 15 minutes, and the filtrate was discarded; this was repeated two more times to remove the denaturing agent, 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 apparatus (enzyme:substrate ratio of 1:20). The samples were incubated in a 37°C water bath for 60 minutes. The filter apparatus was transferred to a new collection tube (collection tube 2). The first collection tube (collection tube 1) was removed. The filter apparatus was centrifuged at 14,000 × g for 15 minutes. The filtrate containing the trypsin peptide was retained in collection tube 2.20 μL of digestion buffer was added to the filter apparatus, and the filter apparatus (in collection tube 2) was rotated at 14,000 × g for 15 minutes, with the filtrate retained in collection tube 2; this was repeated once more. The filter apparatus was returned 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 apparatus (this time in collection tube 1, with an enzyme:substrate ratio of 1:20 based on the starting material). The samples were incubated in a 37°C water bath for 30 minutes. The filter apparatus was transferred to collection tube 2. Collection tube 1 was discarded. The filter apparatus was centrifuged at 14,000 × g for 15 minutes. The filtrate containing the peptide resulting from HNE digestion was retained in collection tube 2 (along with the trypsin peptide from the previous step). 20 μL of digestion buffer was added to the filter device, and the filter device (in collection tube 2) was rotated at 14,000 × g for 15 minutes, with the filtrate retained in collection tube 2; this was repeated once more. 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 denaturation buffer was added to a 30 kDa molecular weight cutoff rotating apparatus 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). This apparatus was rotated at 14,000 × g for 10 minutes using an Eppendorf 5430 centrifuge. The filtrate was discarded. 100 μg of the sample in the formulation buffer was added to the filter apparatus and rotated 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 denaturation buffer, and 40 μL of this solution was added to the filter apparatus. The 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 apparatus. The samples were alkylated by incubation 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 apparatus to quench the alkylation. The samples were then rotated 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 apparatus was rotated 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 apparatus (enzyme:substrate ratio of 1:20). The samples were incubated in a 37°C water bath for 60 minutes. The filtered material was transferred to a new collection tube (collection tube 2). The first collection tube (collection tube 1) was removed. The filtered material was centrifuged at 14,000 × g for 10 minutes. The filtrate containing the trypsin peptide was retained in collection tube 2.20 μL of digestion buffer was added to the filter apparatus, and the filter apparatus (in collection tube 2) was rotated at 14,000 × g for 10 minutes, with the filtrate retained in collection tube 2; this was repeated once more. The filter apparatus was returned 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 apparatus (this time in collection tube 1, with an enzyme:substrate ratio of 1:20 based on the starting material). The samples were incubated in a 37°C water bath for 30 minutes. The filter apparatus was transferred to collection tube 2. Collection tube 1 was discarded. The filter apparatus was centrifuged at 14,000 × g for 10 minutes. The filtrate containing the peptide resulting from HNE digestion was retained in collection tube 2 (along with the trypsin peptide from the previous step). 20 μL of digestion buffer was added to the filter device, and the filter device (in collection tube 2) was rotated at 14,000 × g for 10 minutes, with the filtrate retained in collection tube 2; this was repeated once more. 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 - Conditions for ultra-high-performance liquid chromatography (UPLC) For all samples, mobile phase A consisted of a 0.1% formic acid aqueous solution, 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 obtaining crosslinking data, the remaining experiments using the methods from Examples 2-4 were performed using a BEH C18 1.7 μm, 2.1 × 150 mm UPLC column (Waters, Milford, MA, P / N 186003556). UPLC separation was performed using either a Thermo Scientific U-3000 system (Waltham, MA), a Waters Acquity H-Class system (Milford, MA), or an Agilent 1290 system (Santa Clara, CA), utilizing the gradients outlined in Tables 1-3 (depending on the experiment). Approximately 3-4 μg of sample was loaded onto the column based on the starting material.

[0121] Example 9 - Conditions for mass spectrometry Peptides resulting from digestion were analyzed using Thermo Scientific Q Exactive (Waltham, MA), Thermo Scientific Q Exactive Plus (Waltham, MA), or Thermo Scientific Q Exactive BioPharma (Waltham, MA). Due to the use of multiple instruments, data acquisition parameters varied slightly depending on the instrument. 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 to 5E5 for tandem mass spectrometry (MSMS) scans. MS1 scans were acquired at either 35,000 or 140,000 resolution, and MS2 scans were acquired at 17,500 resolution. An isolation window of 2–4 m / z was identified for MSMS scans. Unused charge states and charge states greater than 8 were excluded from MSMS. Dynamic exclusion was set to 10 seconds. Lock mass at m / z 391.28430 is now possible.

[0122] Example 10 - Data Analysis MS data were searched using MassAnalyzer (multiple versions of MassAnalyzer were used as data were collected over several months). Carboxymethylation was identified as a static modification. Experimentally, cleavage was identified as either nonspecific or at the C-terminus of the amino acid KRVITAL. For the full search, the signal-to-noise ratio was set to 20, the mass precision to 15 ppm was set to 15 ppm, and the confidence level to 0.95. For the sequence coverage map, the minimum peak area was set to 1% of the base peak, the relative peak area threshold to 17%, the minimum confidence level to 0.95, and the maximum peptide mass to 15,000.

[0123] Example 11 - Results Initial experiments investigating the effectiveness of HNE for the digestion of the BiTE® molecule involved a direct comparison of trypsin digestion and HNE digestion of BiTE®-3 using Protocol 1 and Gradient 1. Trypsin digestion did not produce any peptides that could be used to characterize attributes in any CDR. On the other hand, digestion with HNE produced several peptides corresponding to the linker region. Many of these peptides had low signal intensity (<10). 6 It was identified as ), and this lack of specificity (14 peptides were identified) could potentially render the quantification of attributes in this region invalid. However, the specificity of HNE was thought to be related to the length of the substrate being digested (Stein et al., 1987). The properties of this enzyme were utilized by first digesting BiTE(registered trademark)-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 intensity (>1.5 × 10⁻⁶). 7 A single peptide possessing the following characteristics was produced.

[0124] After demonstrating the feasibility of sample preparation using trypsin-HNE digestion to monitor attributes in the α-CD3 CDR of interest, additional molecules that had undergone photodecay (12 million lux hours of cool white light, t=3 days) and forced deamidation (pH 8.5, 50, t=3 days) were prepared using Protocol 2 and Gradient 1, in addition to unstressed samples (BiTE®-1, half-life extended BiTE®; BiTE®-2, canonical BiTE®; and CDH19, half-life extended BiTE®; BiTE®-3, half-life extended BiTE®). For 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, single peptides corresponding to potential tryptophan oxidation sites were identified with high signal intensity. Forced deamidation conditions produced peaks corresponding to deamidation at α-CD3 CDR1 for all four molecules, which were not observed in unstressed samples (Figure 3 shows BiTE®-1 as a representative sample). MSMS (Figure 4) confirmed that the two peaks eluting the first two correspond to the -GNS- motif, while the last eluted peak corresponds to the deamidation of the -GNF- motif. A comparison of modification percentages for all four molecules (Table 11.1) highlights both the potentially deamidation sites in this CDR that were susceptible to modification and that needed to be monitored. Oxidation of CDR tryptophan was also observed after photodecay 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 the existence of unstable sites within α-CD3 CDRs that cannot be monitored using trypsin digestion, but also that these modifications can be identified and monitored using trypsin-HNE serial digestion.

[0125] [Table 15]

[0126] [Table 16]

[0127] Digestion of trypsin-HNE produced high-quality peptides for the two α-CD3 CDRs of interest, but sequence coverage and modification quantification for the remaining molecules still required trypsin peptides utilized by conventional MAM.

[0128] Single analysis was performed on a 1:1 mixture of trypsin-digested samples combined with aliquots of the sample digested with trypsin and then HNE (Figure 5). An additional strength of this approach, which analyzes a mixture of the two digests, lies in the fact that data for two peptide maps are obtained essentially in a single injection; this not only allows for the identification of greater sequence coverage, but in the case of BiTE®-2, the peptide containing the crucial attribute (CDR aspartate isomerization) resulting from trypsin-HNE digestion produced better signal quality and reproducibility than the corresponding trypsin peptide. Furthermore, the UPLC gradient used for 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 samples, photo-decayed samples, pH jump samples, and pyrolysis samples corresponding to the two samples (BiTE®-2 and BiTE®-3). Trypsin digestion samples, trypsin-HNE sequential digestion samples, and 1:1 mixtures of trypsin digestion and trypsin-HNE digestion 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). Since two samples were prepared for each condition, a total of four injections were obtained.

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

[0130] [Table 17]

[0131] [Table 18]

[0132] We evaluated whether the MAM using the above-described method (Protocol 3, Gradient 2) was suitable for BiTE®-2 (ATM-000401) and BiTE®-3 (ATM-000391). However, during the analysis, a new peak detection procedure (sieving) incorporated into the method identified numerous new peaks. This resulted in a failure of the sequence's system compatibility. After detailed evaluation, numerous peaks corresponded to failed trypsin cleavage. However, some of these peaks were also observed in the final system compatibility injection (but not in the initial pre-injection). These findings suggested that the effectiveness of trypsin was somehow impaired, and further suggested that one of the enzymes remained active despite the acidification of the post-digestion sample. However, attribute quantification was not affected by the new peaks (only the system compatibility check). Troubleshooting indicated that the desalting column used in Protocols 1-3 did not completely remove guanidine from the pre-digestion sample. Since the efficacy of trypsin can be inhibited by low levels of guanidine, it was highly likely that the removal of guanidine variability by desalting columns resulted in inconsistent trypsin digestion. Furthermore, since acidic quenching of trypsin digestion has been reported, it was presumed that oxidation would not sufficiently quench HNE digestion. Enzyme activity decreased due to acidification, but if multiple injections were performed from a single vial after a certain period of time, it would result in the observation of novel peaks; this, in turn, would lead to a failure in assessing the suitability of the system.

[0133] To address these issues, a thorough exploration of multiple preparation methods was conducted. These included steps to evaluate multiple desalting columns (Zeba, Biospin, NAP-5), preparation methods that do not require guanidine (Rapigest) for denaturation, and methods based on MWCO filters available from multiple manufacturers (Pall and Microcon® (Millipore (Waltham, MA))), as well as multiple molecular weight cutoffs (30 kDa, 5 kDa as specified by the manufacturers). Furthermore, the digestion quenching buffer was updated to include a high concentration of guanidine to inactivate both enzymes. Protocols 1-3 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 artificial modifications (e.g., deamidation and isomerization) that could result from prolonged exposure to low pH. Because this quenching buffer contained guanidine, the initial washing step in the UPLC gradient was extended; all experiments utilizing guanidine quenching buffer required the use of gradient 3 to completely desalt the sample before MS analysis.

[0134] The conclusion of this study is that the MWCO filter-based method (Protocol 4 above, the previously reported filter-assisted sample preparation (FASP) method (Wisniewski et al., 2009)) was superior to other preparation methods based on several numerical indicators (e.g., repeatability, recovery rate, quantification, sequence coverage, concentration range, etc.). A schematic diagram of Protocol 4 is shown in Figure 7. In this method, molecules are captured on the top of a 30 kDa MWCO spin filter, and all processes of denaturation, reduction, alkylation, desalting, and digestion occur on the filter. After digestion, the resulting peptides are collected by centrifugation. In the sequential digestion outlined in Protocol 4, after trypsin digestion, trypsin peptides are collected, and species that do not pass through the filter are then subjected to HNE digestion. One of the surprising observations of this method was that the 8 kDa linker peptide of interest was retained by a 30 kDa filter, despite recommendations from manufacturers Microcon® and Pall, respectively, to "use a MWCO membrane at least twice as small as the molecular weight of the protein solute to be concentrated" and "select an MWCO 3 to 6 times smaller than the molecular weight of the solute to be retained." Surprisingly, the 8 kDa peptide was retained by an MWCO nearly four times larger than the peptide itself, even though the manufacturers recommended using an MWCO 2 to 6 times smaller than the species to be retained.

[0135] The retention of 8kDa peptides by 30kDa MWCO filters with membranes made of different materials (Microcon® membrane is regenerated cellulose, and Pall membrane is modified polyethersulfone) emphasizes that this is not a manufacturer-specific artifact. This novel use of MWCO filters alone enabled continuous digestion; a direct comparison of this method using 30kDa MWCO filters over 5kDa MWCO filters demonstrated that the 30kDa filtering method is significantly superior to the 5kDa filtering method in terms of trypsin digestion efficiency, repeatability, accuracy of attribute quantification, and recovery. The main advantage of this method is that the enzyme specificity given by trypsin digestion is conserved for the bulk of the molecule, and only large peptides that are difficult to characterize by MS are further digested into smaller peptides so that they are retained on the filter. This is illustrated in Figure 8, which highlights the trypsin specificity of peptides identified for BiTE®-1, BiTE®-2, and BiTE®-3 (three analyses for each molecule) using filters manufactured by both Microcon® and Pall. In these results, over 95% of the identified peptides corresponded typically to trypsin digestion, while the remaining identified peptides mainly corresponded to the large 8 kDa linker peptide of interest (Figure 9). Quantification of attributes for BiTE®-2 using filters from both manufacturers was comparable to the results for parallel digestion (Protocol 3) (Table 11.5). Similar results were observed for BiTE®-1 and BiTE®-3. This method more completely solves the problem of novel peaks by removing guanidine compared to other methods discussed above, and these enzymes were further inactivated either by renewed quenching buffer or by retention on MWCO filters. In Figure 10, BiTE®-3 samples prepared using Protocol 3 were reinjected approximately 4 days later. 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 remained consistent even four days after injection (Figure 11). These results suggest that the MWCO filter-based preparation method outlined in Protocol 4 is robust, produces quantitative results consistent with previously reported findings, and eliminates many of the underlying causes of the novel peaks observed using Protocol 3.

[0136] [Table 19]

[0137] One of the main concerns with Protocol 4 was the length of time required to prepare the sample; continuous digestion using Protocol 4 would take more than 8 hours. In addition to analyst fatigue and other factors, the amount of time required to prepare the sample using this method would introduce artifact 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 time savings introduced by Protocol 5 was made possible by eliminating unnecessary centrifugation steps, reducing centrifugation time, and reducing incubation time. These reductions theoretically resulted in roughly 2 hours of time savings, 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 time to a point comparable to solution-based methods.

[0138] [Table 20]

[0139] Figure 12 presents an overlay comparing BiTE®-2 samples prepared using protocols 4 and 5 (each preparation was performed three times). The only notable peak differences observed were shoulders on peaks at approximately 24 minutes and approximately 47 minutes that were present in all samples prepared using protocol 4 but not present in any of the samples prepared using protocol 5. The cause of these peaks was attributed to non-specific carboxymethylation resulting from overalkylation. Similarly, the denaturation / reduction incubation time was decreased and the rotation step between reduction and alkylation was eliminated to compare the alkylation levels for all cysteine residues (Table 11.7). For the data presented in this table, the percentage of alkylation was determined by steps of searching the data using MassAnalyzer and identifying carboxymethylation as a variable modification (rather than the typically defined static modification). The alkylation levels for all BiTE®-1 samples were greater than 99.7%, regardless of whether protocol 4 or 5 was used for preparation. Similar results observed for BiTE®-2 and BiTE®-3, which suggest a reduction in time introduced by protocol 5, showed no negative impact on the alkylation levels. The quantification of attributes was comparable when samples were prepared using both protocols 4 and 5, which also agreed with the historically observed modification levels in sequence (Table 11.8). For the data in this table, each preparation was performed three times. D 54 +D 57 +D 62 Isomerization of was quantified using I for MDR-001581 51 -R 65 was used. C 44 -E 65It was used for quantification in filter-based experiments for more efficient trypsin digestion. Since these initial results for Protocol 5 were promising, a limited robustness assessment was performed (2 analysts, 3 BiTE®-2 samples prepared each). Novel peak detection passed for all samples (e.g., no novel peaks were detected), and attribute quantification for all 6 injections was consistent with MDR-001581 (Table 11.9). Based on these results, along with comparisons of other evaluation metrics such as trypsin specificity, total area identified, recovery rate of 8kDa linker peptides, and percentage 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 to them 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 polypeptides of bispecific T cell engager molecules for analysis, a. A step of preparing a sample containing a polypeptide of a bispecific T cell engager molecule, b. The step of applying the sample to a filter having a molecular weight cutoff of 30 kDa, c. The step of digesting the polypeptide in the sample on the filter with a first protease containing trypsin, d. The step of digesting the polypeptide in the sample on the filter with a second protease containing neutrophil elastase, e. The step of analyzing the aforementioned sample It includes a through e in that order. The first protease is different from the second protease. method.

2. The method according to claim 1, wherein the polypeptide is denatured on the filter before being digested by the first protease.

3. The method according to claim 1, wherein the polypeptide is alkylated on the filter before being digested by the first protease.

4. The method according to claim 1, wherein, before the polypeptide is digested by the first protease, the polypeptide is denatured on the filter and subjected to either reduction or alkylation.

5. The method according to claim 1, wherein the polypeptide is denatured, reduced, and alkylated on the filter before being digested with the first protease.

6. The method according to claim 1, wherein the neutrophil elastase is human neutrophil elastase (EC3.4.21.37).

7. The method according to claim 1, wherein the step of analyzing the sample comprises at least one technique selected from the group consisting of chromatography, electrophoresis, spectroscopy, and combinations thereof.

8. The technique for analyzing the aforementioned sample is, a. Chromatography selected from the group consisting of gas chromatography, liquid chromatography, high-performance liquid chromatography, ultrahigh-performance liquid chromatography, size exclusion chromatography, ion exchange chromatography, affinity chromatography, extended adsorption fluidized bed chromatography, reversed-phase chromatography, hydrophobic interaction chromatography, and combinations thereof; b. Electrophoresis selected from the group consisting of gel electrophoresis, free-flow electrophoresis, isoelectric focusing, isokinetic electrophoresis, affinity electrophoresis, immunoelectrophoresis, inverse electrophoresis, capillary electrophoresis, and combinations thereof; or c. Spectroscopic methods or spectroscopic measurements selected from the group consisting of mass spectrometry, ultraviolet spectroscopy, visible spectroscopy, fluorescence spectroscopy, ultraviolet-visible spectroscopy, and combinations thereof. The method according to claim 7, including the method described in claim 7.

9. The method according to claim 8, wherein the technique includes liquid chromatography-mass spectroscopy.

10. The method according to claim 8, wherein the technique includes capillary zone electrophoresis linked to mass spectrometry.

11. A method for preparing polypeptides of bispecific T cell engager molecules for analysis by liquid chromatography, capillary zone electrophoresis, or mass spectrometry, a. A step of preparing a sample containing a polypeptide of a bispecific T cell engager molecule, b. The step of applying the sample to a 30 kDa MWCO filter, c. A step of filtering the sample by passing it through the filter in order to produce a concentrated solution, d. A step of denaturing the polypeptide in the sample concentrate on the filter, e. A step of reducing the polypeptide in the sample concentrate on the filter, f. A step of alkylating the polypeptide in the sample on the filter, g. A step of digesting the polypeptide in the sample on the filter with a first protease containing trypsin, h. The step of filtering the sample by passing it through the same filter and holding the filtrate, i. A step of digesting the polypeptide in the sample on the filter with a second protease, j. A step of filtering the sample through the same filter into the filtrate of step (h), k. A step of quenching the active protease in the filtrate with guanidine, l. The step of analyzing the filtrate by at least one technique selected from the group consisting of liquid chromatography, capillary zone electrophoresis, or mass spectrometry. It includes a to l in order, Here, i. The second protease mentioned above is neutrophil elastase, ii. The first protease is different from the second protease. method.

12. The method according to claim 11, wherein the neutrophil elastase is human neutrophil elastase (EC3.4.21.37).

13. The method according to claim 11, wherein the technique includes capillary zone electrophoresis linked to mass spectrometry.

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