Methods for middle down antibody characterization

Abstract

To provide novel methods for antibody characterization sequencing.SOLUTION: Provided is a method for cleaving an antibody, comprising: mixing the antibody with cathepsin L, cathepsin D, or a combination of cathepsin L and cathepsin D to obtain one or more antibody fragments, the antibody comprising a light chain comprising a light chain variable region (VL) and a light chain constant region (CL) and a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region (CH), and the cathepsin L and / or D cleaving the antibody between the VL and CL regions and / or between the VH and CH regions to create VL and / or VH antibody fragments and CL and / or CH antibody fragments; and optionally isolating one or more of the antibody fragments after the cleavage.SELECTED DRAWING: Figure 8

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 053,899, filed July 20, 2020, the entire contents of which are incorporated herein by reference.

[0002] Field The present disclosure relates to novel methods for antibody characterization sequencing, such as middle-down antibody characterization and sequencing, for example, for sequencing de novo antibodies, identifying known antibodies in a sample, or validating the sequence of an antibody in a sample. In some embodiments, the methods include exposing an antibody to cathepsin D, cathepsin L, and / or cathepsin D and cathepsin L, followed by mass spectrometry and sequence identification and deconvolution. [Background technology]

[0003] background Approaches currently used to identify protein sequences, such as antibody sequences, in samples include bottom-up, middle-down, and top-down analysis. Bottom-up analysis, used in the majority of cases, often involves the use of several different proteases (e.g., 4-5 proteases) to generate relatively short overlapping peptides (e.g., 9-30 amino acids long or about 1-5 kDa) for liquid chromatography (LC) and mass spectrometry (MS). When such protocols are performed to determine the sequence of an unknown antibody (i.e., de novo sequencing), for example, a large number of mass spectra must be generated and then assembled using specialized sequencing programs that use information about extracted mass shifts between product ion peaks. One limitation of this approach is that the accuracy of the sequence analysis may depend on obtaining very high quality MS (e.g., tandem MS (MS / MS) fragmentation efficiency and low mass error) data and detecting peptides that span the entire protein. Additionally, bottom-up analysis may pose software challenges as it may require correctly piecing together sequence information from a large number of small peptides into a complete protein sequence. A middle-down approach using fewer peptides or protein fragments may help to alleviate these software challenges.

[0004] The middle-down approach can be used, for example, for antibodies where the antibody is first cleaved near or within the hinge region between the CH1 and CH2 regions of the heavy chain to generate, for example, F(ab')2 fragments, F(ab') fragments, Fc fragments, Fd fragments, and VL-CL fragments. The most commonly used enzyme is the cysteine ​​proteinase (IdeS) from Streptococcus pyogenes, which cleaves after the hinge region to generate F(ab')2 fragments and Fc / 2 fragments. After reduction of the disulfide bonds, light chain (LC) fragments, Fd' fragments, and Fc fragments can result, each of which is approximately 25 kDa in size and approximately 200 amino acids in length.

[0005] These relatively long fragments are difficult to completely fragment when analyzed using common MS instruments and may therefore not be suitable for de novo sequencing. For example, currently common MS instruments include time-of-flight with collision-induced dissociation or Orbitrap™ instruments with high-energy collision dissociation (HCD), which lack electron-induced dissociation (a highly efficient and orthogonal fragmentation technique) and lack ultraviolet photodissociation (suitable for very large polypeptides). Thus, the middle-down approach, which provides only limited coverage, may need to be complemented with bottom-up sequencing methods for de novo antibody sequencing. Thus, there is a need for alternative middle-down methods that generate protein fragments that are more suitable for commonly used industry-standard LC-MS / MS CID and HCD instruments and therefore more practical. Summary of the Invention

[0006] overview The disclosure includes, inter alia, a method for cleaving an antibody, comprising mixing the antibody with cathepsin L, cathepsin D, or a combination of cathepsin L and cathepsin D to obtain one or more antibody fragments, the antibody comprising a light chain comprising a light chain variable region (VL) and a light chain constant region (CL), and a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region (CH), and cathepsin L and / or cathepsin D cleaves the antibody between the VL and CL regions and / or between the VH and CH regions to generate VL and / or VH antibody fragments and CL and / or CH antibody fragments. In some embodiments, the method also comprises isolating one or more of the antibody fragments after cleavage. In some embodiments, the antibody fragments are not isolated after cleavage. In some embodiments, after cleavage, the one or more antibody fragments are analyzed by mass spectrometry.

[0007] For example, the disclosure also encompasses a method for analyzing the sequence of an antibody, comprising: (a) cleaving an antibody with cathepsin L, cathepsin D, or a combination of cathepsin L and cathepsin D to obtain one or more antibody fragments, wherein the antibody comprises a light chain comprising a light chain variable region (VL) and a light chain constant region (CL), and a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region (CH), and cathepsin L and / or cathepsin D cleaves the antibody between the VL and CL regions and / or between the VH and CH regions to generate VL and / or VH antibody fragments, and CL and / or CH antibody fragments, to obtain one or more antibody fragments; (b) optionally isolating one or more of the antibody fragments after cleavage; and (c) performing mass spectrometry (MS) of the one or more antibody fragments.

[0008] In any of the above methods, in some embodiments, the antibody is an IgG antibody, such as a human IgG1, IgG2, IgG2A, IgG2B, or IgG4 antibody. In some embodiments, the cleavage generates a VL fragment and / or a VH fragment. In some such embodiments, the MS analysis is performed on the VL fragment and / or the VH fragment. In some methods herein, the heavy chain constant region comprises at least a CH1 region, and optionally further comprises a hinge, a CH2 region, and / or a CH3 region. In some methods herein, the antibody heavy chain constant region comprises at least a CH1 region, a hinge, and a CH2 region, and cathepsin L and / or cathepsin D further cleaves the antibody between the CH1 region and the hinge.

[0009] In some embodiments, the antibody is cleaved by cathepsin L. In some embodiments, the antibody is cleaved by cathepsin D. In some embodiments, the antibody is cleaved by a combination of cathepsin L and cathepsin D. In some cases, an additional enzyme, such as IdeS or another protease, is also used. In other cases, the enzyme used for cleavage consists of cathepsin L, consists of cathepsin D, or consists of a combination of cathepsin L and cathepsin D. In some cases, the method includes incubating the antibody with both cathepsin L and cathepsin D simultaneously.

[0010] In some embodiments, cleavage is performed to achieve at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% cleavage between the VL and CL regions and between the VH and CH regions. In some cases, cleavage is performed at or below the hinge to achieve at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% cleavage. In some cases, the antibody is an IgG antibody and is cleaved by a combination of cathepsin L and cathepsin D, and cleavage results in a VL fragment, a VH fragment, a CL fragment, a CH1 fragment, a CH2 fragment, a CH3 fragment, a CH2-CH3 fragment, a CL+CH1 (binding) fragment, a F(ab') fragment, and a F(ab')2 fragment. In some embodiments, cleavage results in a fragment comprising each of the light chain CDR1, light chain CDR2, and light chain CDR3, e.g., a VL fragment, an F(ab') fragment, or an F(ab')2 fragment, and / or a fragment comprising each of the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, e.g., a VH, F(ab') fragment, or an F(ab')2 fragment.

[0011] In some methods herein, cleavage is carried out by incubating the antibody with cathepsin L, cathepsin D, or a combination of cathepsin D and cathepsin L at pH 2-8 (e.g., pH 2-7, pH 2-6, pH 3-6, pH 3-5, pH 2, pH 2.5, pH 3, pH 3.5, pH 4, pH 4.5, pH 5, pH 5.5, pH 6, pH 7, or pH 8), at a temperature between room temperature and 50° C., in the presence of up to 50% organic solvent (e.g., acetonitrile, methanol, ethanol, or isopropyl alcohol), and the antibody is in a native state. In some methods herein, the cleavage is performed in the presence of one or more organic solvents (e.g., methanol, ethanol, isopropyl alcohol, or acetonitrile) at a concentration of 0-50%, 5-50%, 5-30%, 0-30%, 10-30%, 0-10%, 5-15%, 10-20%, 15-25%, or 20-30%. In some methods herein, including methods involving one or more organic solvents (e.g., methanol, ethanol, isopropyl alcohol, or acetonitrile) at a concentration of 0-50%, 5-50%, 5-30%, 0-30%, 10-30%, 0-10%, 5-15%, 10-20%, 15-25%, or 20-30%, the pH is 3-5, e.g., 3, 3.5, 4, 4.5, or 5. In some such cases, the pH is 3.5-4.5. In some such cases, the pH is 4. In some cleavage reactions, 30% or less of an organic solvent is present. In some such cases, the cleavage is performed in the presence of 10-30% of an organic solvent (e.g., 10-30% acetonitrile, 10-30% methanol, 10-30% ethanol, or 10-30% isopropyl alcohol). In some cleavage reactions, 10% or less of an organic solvent is present. In some cleavage reactions, the temperature is 37°C to 50°C.

[0012] The present disclosure also provides a method for analyzing an antibody sequence, comprising: (a) cleaving an antibody with a combination of cathepsin L and cathepsin D to obtain one or more antibody fragments comprising at least a light chain variable region (VL) fragment and / or a heavy chain variable region (VH) fragment, wherein (i) the antibody is in a native state; (ii) the antibody comprises a light chain comprising a light chain variable region (VL) and a light chain constant region (CL), and a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region (CH); and (iii) the antibody comprises a light chain comprising a light chain variable region (VL) and a light chain constant region (CL), and the light chain variable region (VH) and the heavy chain constant region (CH) are cleaved with cathepsin L and cathepsin D in combination to obtain one or more antibody fragments comprising at least a light chain variable region (VL) and / or a heavy chain variable region (VH) fragment, wherein (i) the antibody is in a native state; and (ii) the antibody comprises a light chain comprising a light chain variable region (VL) and a light chain constant region (CL), and a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region (CH), and wherein cathepsin L and cathepsin D are combined to obtain one or more antibody fragments comprising at least a light chain variable region (VL) and / or a heavy chain variable region (VH) fragment. D cleaving the antibody between the VL and CL regions and / or between the VH and CH regions to produce a VL antibody fragment and / or a VH antibody fragment and a CL antibody fragment and / or a CH antibody fragment; (iii) obtaining one or more antibody fragments, wherein the cleavage is performed at a temperature of 25°C to 50°C and a pH of 3 to 5 in the presence of 30% or less of an organic solvent; (b) optionally isolating one or more of the antibody fragments after cleavage; and (c) performing mass spectrometry (MS) of the one or more antibody fragments.

[0013] In any of the methods herein, after cleavage, the one or more antibody fragments may be subjected to one or more of buffer exchange, chromatography (e.g., liquid chromatography such as high performance liquid chromatography or capillary electrophoresis), filtration (e.g., molecular weight cut-off filtration), reduction of disulfide bonds, exposure to guanidine hydrochloride, or alkylation. For example, one or more of these steps may be performed prior to MS analysis or on a portion of the sample undergoing MS analysis (e.g., reduction or alkylation of a portion of the sample to allow comparison of MS assignments with and without these modifications). In some embodiments, the one or more antibody fragments after cleavage are isolated by chromatography or filtration. In some such cases, the one or more antibody fragments are isolated by liquid chromatography.

[0014] In some embodiments, if MS analysis is performed, the MS comprises LC-MS or LC-MS / MS. In other cases, the one or more antibody fragments are not isolated after cleavage. In some embodiments, if mass analysis is performed, the mass analysis comprises direct infusion mass spectrometry (DIMS), static spray infusion mass spectrometry, or flow injection mass spectrometry.

[0015] In some embodiments, if mass spectrometry is performed, the mass spectrometry data is used to determine the amino acid sequence of at least a 10 amino acid stretch of one antibody fragment. In some embodiments, the amino acid sequence of at least a 15 amino acid stretch of one antibody fragment is determined. In some embodiments, the amino acid sequence of at least a 20 amino acid stretch of one antibody fragment is determined. In some embodiments, the amino acid sequence of at least one antibody CDR region is determined. In some embodiments, the sequences of the VH and / or VL CDR1, CDR2 and CDR3 are determined. In some embodiments, the complete amino acid sequence of at least one antibody fragment, such as a VH fragment and / or a VL fragment, is determined. In some embodiments, the sequence is determined by top-down analysis. In some embodiments, the sequence is further analyzed or confirmed by bottom-up analysis. In some embodiments, the amino acid sequence of the antibody VH and / or VL region is unknown. In some embodiments, the amino acid sequence of the antibody is unknown. Some embodiments also include performing MS on the CL and / or CH regions of the antibody, for example the CL, CH1, CH2 and / or CH3 fragments generated from cleavage. In some cases, any method herein further comprises performing Edman degradation on at least one antibody fragment. In some methods herein, the cleavage is performed at a ratio of cathepsin L and / or cathepsin D to antibody of 1:20 to 1:2000, 1:20 to 1:500, 1:50 to 1:500, 1:100 to 1:500, 1:200 to 1:1000, 1:200 to 1:2000, 1:500 to 1:2000, 1:1000 to 1:2000, or 1:20, 1:50, 1:100, 1:200, 1:300, 1:400, 1:500, or 1:1000. In some methods herein, the antibody remains in a native state after cleavage. In some methods herein, the antibody is not treated with a denaturing agent or an agent that reduces disulfide bonds during or after cleavage. In some methods herein, the antibody retains its disulfide bonds during or after cleavage.Some methods herein include performing mass spectrometry (MS) of one or more antibody fragments after cleavage, where the antibody remains in its native state and is not treated with denaturing agents or agents that reduce disulfide bonds prior to MS analysis.

[0016] The disclosure also includes compositions comprising antibody fragments generated according to the above methods. In some cases, the compositions include IgG antibody fragments generated from cleavage with cathepsin L, cathepsin D, or a combination of cathepsin L and cathepsin D, where the antibody fragments include one or both of a VH fragment and a VL fragment, and at least one, at least two, or at least three of the following fragments: a CL fragment, a CH1 fragment, a CH2 fragment, a CH3 fragment, a CH2-CH3 fragment, a CL+CH1 (combined) fragment, a F(ab') fragment, and a F(ab')2 fragment. In some cases, the VH fragment and / or the VL fragment include a length of 90-150 amino acids, such as 95-140 amino acids, such as 100-140 amino acids, or such as 100-120 amino acids. In some such cases, the VH fragment and / or the VL fragment have a molecular mass of 10-16 kDa, such as 10-13 kDa, such as 10-12 kDa, such as 10-11 kDa, or such as 11-12 kDa.

[0017] The disclosure also includes kits for use in digesting proteins with cathepsin L, cathepsin D, or a combination of cathepsin L and cathepsin D, the kits including (a) cathepsin L and / or cathepsin D, (b) one or more reaction buffers, and optionally (c) instructions for use in digesting proteins. In some cases, the kits provide reagents for use in cleaving antibodies according to the above methods. In some kits, the reaction buffer is pH 2-8, pH 2-7, pH 2-6, pH 2-5, pH 3-6, pH 3-5, pH 3-4, pH 4-5, pH 2, pH 2.5, pH 3, pH 3.5, pH 4, pH 4.5, pH 5, pH 5.5, pH 6, pH 7, or pH 8, and the reaction buffer includes one or more organic solvents. In some embodiments, the reaction buffer comprises one or more organic solvents (e.g., methanol, ethanol, isopropyl alcohol, or acetonitrile) at a concentration of 0-50%, 5-50%, 5-30%, 0-30%, 10-30%, 0-10%, 5-15%, 10-20%, 15-25%, or 20-30%. In some cases, the reaction buffer is pH 3, pH 3.5, pH 4, pH 4.5, or pH 5. In some kits, the reaction buffer is pH 4.

[0018] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the scope of the claims. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate specific embodiments or aspects and, together with the description, serve to explain the principles described herein. [Brief description of the drawings]

[0019] [Figure 1]Figures 1A-1D show the spectrum of rituximab exposed to cathepsin L. Figures 1A-1B show the full spectrum of rituximab exposed to cathepsin L, including the CDR L3 clip at a mass of approximately 10 kDa, the "one arm" clip at approximately 47 kDa, the complement of the one arm clip at approximately 100 kDa (e.g., 100,238 is a complete monoclonal antibody (mAb) with G1F / G1F of 147,400 missing 47,178 along with 18 of water), and the intact mAb at approximately 147 kDa. Figures 1C-1D show the charge deconvolution of the same rituximab exposed to cathepsin L spectra, focusing on the m / z range (3000-4000) and m range (45000-49000) of the one arm clip.

[0020] [Diagram 2] Figures 2A-D show the spectrum of rituximab exposed to cathepsin D. Figures 2A-B show the 1-arm clip. Figures 2C-D show the F(ab')2 clip.

[0021] [Diagram 3] Figures 3A-D show the spectrum of obinutuzumab exposed to cathepsin L. Figures 3A-B show the intact mAb peaks. 148630.4Da is a good match for the mAb with G0 / G0 glycosylation, 148,834.1 is a good match for the extra GlcNAc, and 149038.0 has two extra GlcNAcs. Obinutuzumab has been glycoengineered to have mostly fucosylated glycans. Figures 3C-D show the one-arm clip.

[0022] [Figure 4]Figures 4A-H show the spectrum of obinutuzumab exposed to cathepsin D. Figures 4A-B show the full spectrum. Figures 4C-D show the intact mAb peaks. 148628.3 Da is a good match for the mAb with G0 / G0 glycosylation, 148,832.9 is a good match for the extra GlcNAc, and 149034.7 has two extra GlcNAcs. Figures 4E-F show the intact 1-arm clip. Figures 4G-H show the F(ab')2 clip.

[0023] [Diagram 5] Figures 5A-5D show the spectrum of eculizumab exposed to cathepsin L. Figures 5A-5B show the full spectrum. Figures 5C-5D show the CDR H3 clip.

[0024] [Figure 6] Figures 6A-6D show the spectrum of eculizumab exposed to cathepsin D. Figures 6A-6B show the full spectrum. Figures 6C-6D show the F(ab')2 clip.

[0025] [Figure 7] Figures 7A-B show native mass spectra of rituximab digested by cathepsin D. Figure 7A shows that deconvolution of the full m / z range gives peptides from 9 to 98 kDa, indicating significant amounts of intact Ab (approximately 147 kDa). The peak at 97,684 is generated from cleavage below the hinge of LLGGPSVF.L. Asymmetric clips such as 97,538 formed by F(ab')LLGGPSVF.L + F(ab')LLGGPSV.F are also observed and are shown in the inset. Figure 7B shows that deconvolution over a 47 kDa region reveals the multiplicity of clips observed at the specific site location, F(ab'), shown on the IgG1 crystal structure (PDB1HZH).

[0026] [Figure 8]FIG. 8 shows the preferred cleavage sites of cathepsin L (gray line) and cathepsin D (solid black line) alone and in combination on human IgG1, IgG2-B and IgG1 antibodies. In the IgG1 class, cathepsin L and D caused cleavage at the heavy (HC) and light (LC) chain CDR3, above the hinge (F(ab')), and throughout the heavy chain hinge region (gray and black dashed lines), respectively. Only cathepsin D uniquely cleaved the sequence PSVFL.F to give F(ab')2. (solid black line.) The combination of cathepsin L and cathepsin D caused additional cleavage at the positions indicated by the black line with diamonds at each end. For eculizumab (IgG2-B), cleavage at the hinge was not observed due to the different disulfide pattern. For the IgG1 bispecific, no cleavage was observed at the Fc anti-CD3 arm (hole) compared to that observed at the anti-Her2 (knob) arm.

[0027] [Figure 9] Figures 9A-B show a comparison of the digestion efficiency of cathepsin L and cathepsin D across different treatments. Figure 9A shows all identified polypeptides reported in Tables 3 and 11. Polypeptides were summed and taken relative to the intensity of intact trastuzumab. Figure 9B shows the UV peak areas (corresponding to EIC elution times) of all peptide peaks taken as a ratio to the main Ab peak. Error bars represent the standard deviation of the measurements.

[0028] [Figure 10] Figures 10A-10E show examples of UV and TIC measurements of one-pot cathepsin L and cathepsin D digests. The insets show selected MS spectra averaged over their elution time windows and deconvoluted with intact mass (Protein Metrics). The masses in these figures may vary slightly from those given in the tables disclosed herein, since the exact masses depend on the time, mass, and m / z ranges, as well as other parameters used for charge deconvolution.

[0029] [Figure 11] 11A-11D show spectra of a static injection of a cathepsin pH 4 sample. The sample was buffer exchanged into 50 mM ammonium acetate on an ultra-high mass range (UHMR) mass spectrometer using a Biorad Microspin® column. MS1 spectra were acquired at 17,500 resolution at 200 m / z and charge states were confirmed in SIM mode at 50K or 100K resolution. FIG. 11A shows that the Rf settings were tuned and optimized for high mass (>4000 m / z). FIG. 11B shows that the Rf settings were tuned and optimized for mid-range mass (1500-4000 m / z). FIG. 11C and FIG. 11D show high-resolution mass spectra of the 12.1 kDa and 47 kDa species, respectively.

[0030] [Figure 12] Figure 12 shows Edman degradation assignments of optimized digests of cathepsin L and cathepsin D. Amino acids in "()" are less confident than those identified with good confidence.

[0031] [Figure 13] Figures 13A-C show MS2 spectra of selected cathepsin digestion products: Figure 13A shows the mass spectrum of the 12.1 kDa species, Figure 13B shows the mass spectrum of the 47 kDa species, and Figure 13C shows the mass spectrum of the 98 kDa species.

[0032] [Figure 14] Figures 14A-B show the annotation of selected polypeptides from top to bottom. Coverage of (Figure 14A) 12,121.5 Da and (Figure 14B) 98 kDa products is shown. Light grey y ions correspond to HC sequences ending in G, dark grey to GG, triangles to y ions + unspecified covalent cross-linking modifications. Black represents HC b ions or LC b / y ions. Spectra were collected by nESI injection, deconvoluted using the Xtract algorithm and matched to fragments with a tolerance of 10 ppm using ProSight Lite and in-house programs.

[0033] [Figure 15] Figures 15A-C show the amino acid enriched motifs. Figure 15A shows cathepsin L. Figure 15B shows cathepsin D. Figure 15C shows one-pot cathepsin L and cathepsin D digests. Amino acid selectivity for p4=p4' was assessed in Seq2Logo 2.0

[71] .

[0034] [Figure 16] FIG. 16 shows a molecular model of trastuzumab and the cleavage sites observed after the optimized digestion protocol. The trastuzumab (PDB 6BI2) LC is shown on the right / top (towards the bottom) and the HC AA 1-221 is shown on the left / top (towards the bottom). The HC CH2 and CH3 regions are shown in black and combined, and the structure shown is a single Ab arm (half of an antibody). Trastuzumab F (Ab') was aligned to residues 1-214 of the full-length IgG1 crystal structure (PDB 1HZH) using the in-house program GYST and modeled in PyMol 2.3.5. The CH1, CH2, and hinge regions of the aligned IgG1 (AA228-478) shared 90.9% identity with trastuzumab, with all cleavage sites falling on the same region of homology. Disulfide bonds are not shown and Fc glycans are shown as white sticks, with any cleavage sites shown as sphere models and colored black. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] Further Description of Specific Embodiments definition Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art.

[0036] In this application, the use of "or" means "and / or" unless specifically stated otherwise. In the context of a multiple dependent claim, the use of "or" refers only to any one or more of the preceding independent or dependent claims. Also, terms such as "element" or "component" encompass both elements and components that comprise a single unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.

[0037] As described herein, any concentration range, percentage range, ratio range, or integer range, unless otherwise indicated, should be understood to include every integer value within the recited range, and, where appropriate, fractions thereof (such as tenths and hundredths of integers).

[0038] Units, prefixes, and symbols are shown in the format accepted by the International System of Units (SI). Numerical ranges are inclusive of the numbers that define the range. The headings provided herein are not limitations of the various aspects of the disclosure that may be had by reference to the specification as a whole. Thus, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0039] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0040] As used herein, "sample" refers to any specimen that may contain proteins or antibodies requiring analysis.

[0041] The terms "polypeptide" and "protein" are used interchangeably and refer to a polymer of amino acid residues. Such polymers of amino acid residues may contain natural and / or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. The term also includes polymers of amino acids that have modifications, such as, for example, glycosylation, sialylation, or are conjugated to other molecules.

[0042] As used herein, a "peptide" is a relatively short polymer of amino acids, such as about 4 to 50 amino acids.

[0043] The term "antibody" or "Ab" is used herein in the broadest sense and encompasses various antibody structures, including, but not limited to, monoclonal antibodies (mAbs), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies), so long as they exhibit the desired antigen-binding activity. As used herein, the term refers to a molecule that comprises at least the complementarity determining region (CDR)1, CDR2, and CDR3 of a heavy chain and at least the CDR1, CDR2, and CDR3 of a light chain, and that is capable of binding to an antigen. The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, human antibodies, and antibodies of various species, such as mouse and cynomolgus monkey. The term also encompasses antigen-binding fragments. The term "antigen-binding fragment" includes, but is not limited to, fragments of an antibody that are capable of binding to an antigen, such as Fv, single-chain Fv (scFv), Fab, Fab', and (Fab')2. In contrast, a "full-length antibody" refers to an antibody molecule that comprises all of its normal variable and constant region portions. As with other proteins and peptides, in some embodiments, antibodies can include various types of post-translational modifications, such as glycosylation.

[0044] A "fragment" of a protein, polypeptide or antibody ("antibody fragment") generally refers to a portion or region of a larger molecule, such as an Fc or F(ab')2 fragment of an antibody or peptide, that has been enzymatically cleaved from the protein. For example, protein fragments such as antibody fragments may be generated by enzymatic cleavage in the methods disclosed herein, or may be generated during a mass spectrometry process.

[0045] A protein or antibody in the "native state" herein means one that retains its native folded structure and disulfide bonds and has not been denatured, unfolded, or reduced to remove disulfide bonds. A protein in the native state can be, for example, in the presence of sufficient organic solvent to relax the native fold without denaturing or unfolding the protein.

[0046] The term "heavy chain" or "HC" refers to a polypeptide comprising at least a heavy chain variable region, with or without a leader sequence. In some embodiments, the heavy chain comprises at least a portion of a heavy chain constant region. The term "full-length heavy chain" refers to a polypeptide comprising a full-length heavy chain variable region and a full-length heavy chain constant region, with or without a leader sequence, and with or without a C-terminal lysine (K). The term "mature full-length heavy chain" refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, without a leader sequence, with or without a C-terminal lysine (K). In some embodiments, the heavy chain comprises a post-translational modification, such as a pyroglutamate modification, while in other cases, the heavy chain does not comprise such a modification.

[0047] The term "heavy chain variable region" or "VH" refers to a region of a heavy chain that includes heavy chain complementarity determining region (CDR)1, framework region (FR)2, CDR2, FR3, and CDR3. In some cases, the VH also includes part or all of FR1 before CDR1 and / or part or all of FR4 after CDR3. A "full-length VH" is a VH that includes the complete FR1 and FR4 that precede and follow the CDR1, FR, CDR2, FR3, and CDR3 segments, respectively. As used herein, a first protein segment that is "prior to" or "precedes" a second segment means that the first segment is N-terminal to the second segment. A first segment that is "after" a second segment is C-terminal to the second segment. The VH of a human IgG antibody typically includes amino acids 1-117 of the heavy chain, according to EU numbering.

[0048] The term "light chain" or "LC" refers to a polypeptide comprising at least a light chain variable region, with or without a leader sequence. In some embodiments, a light chain comprises at least a portion of a light chain constant region. The term "full-length light chain" refers to a polypeptide comprising a full-length light chain variable region and a full-length light chain constant region, with or without a leader sequence. The term "mature full-length light chain" refers to a polypeptide comprising a light chain variable region and a light chain constant region, without a leader sequence. In some cases, the light chain may include post-translational modifications, but in other cases it does not.

[0049] The term "light chain variable region" or "VL" refers to a region that comprises light chain CDR1, FR2, HVR2, FR3, and HVR3. In some embodiments, the light chain variable region also comprises at least a portion of FR1 and / or FR4. A "full-length VL" is a VL that comprises the complete FR1 and FR4 that precede and follow, respectively, the CDR1, FR, CDR2, FR3, CDR3 segments. The VL of a human antibody typically comprises amino acids 1-107 of the heavy chain, according to EU numbering.

[0050] As used herein, the term "heavy chain constant region" or "CH" refers to a heavy chain variable region followed by one, two, or three heavy chain constant regions, C H 1. C H 2, and C H The term "hinge" refers to a region encompassing the heavy chain constant regions CH1, CH2, and CH3, as well as any segments connecting those regions. The heavy chain constant region of an IgG antibody typically has three regions called CH1, CH2, and CH3, with a short "hinge" region between CH1 and CH2. The CH2 and CH3 regions collectively form the "Fc" fragment of the antibody. The "hinge" region of an IgG class antibody refers to a short amino acid sequence region between the CH1 and CH2 portions of the heavy chain that is relatively flexible in the antibody's native state. In the EU numbering system, the hinge region of a human IgG antibody is found at about amino acids 216-230 of the heavy chain, the CH1 region is found at about amino acids 118-215, the CH2 is found at about amino acids 231-340, and the CH3 is found at about amino acids 341-447.

[0051] Non-limiting exemplary heavy chain constant regions include gamma, delta, and alpha. Non-limiting exemplary heavy chain constant regions also include epsilon and mu. Additionally, an antibody comprising a gamma constant region is an IgG antibody, an antibody comprising a delta constant region is an IgD antibody, and an antibody comprising an alpha constant region is an IgA antibody. Additionally, an antibody comprising a mu constant region is an IgM antibody, and an antibody comprising an epsilon constant region is an IgE antibody. Certain isotypes can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 antibodies (comprising a gamma 1 constant region), IgG2 antibodies (comprising a gamma 2 constant region), IgG3 antibodies (comprising a gamma 3 constant region), and IgG4 antibodies (comprising a gamma 4 constant region). IgA antibodies include, but are not limited to, IgA1 antibodies (comprising an alpha 1 constant region) and IgA2 antibodies (comprising an alpha 2 constant region). IgM antibodies include, but are not limited to, IgM1 and IgM2.

[0052] In some embodiments, the heavy chain constant region, framework regions, or light chain constant region contain one or more mutations (or substitutions), additions, or deletions that confer desired characteristics to the antibody. For example, a non-limiting exemplary mutation is the deletion of the human IgG4 hinge region (constant domain CCPSCP) that changes the IgG4 motif CPSCP to CPPCP, which is similar to the corresponding motif in IgG1. H 1 and C H 2) which may result in more stable IgG4 antibodies. See, e.g., Angal et al., Mol. Immunol. 30:105-108 (1993); Bloom et al., Prot. Sci. 6:407-415 (1997); Schuurman et al., Mol. Immunol. 38:1-8 (2001).

[0053] As used herein, the term "light chain constant region" or "CL" refers to the region following the VL region and may be abbreviated as "CL". Non-limiting exemplary light chain constant regions include λ and κ. The CL of a human antibody typically comprises amino acids 108-214 of the heavy chain, according to EU numbering.

[0054] An "IgG" or "immunoglobulin G" antibody is one of several mammalian antibody classes (others are, e.g., IgA, IgM, etc.) and contains a gamma constant region. It is a tetrameric protein formed from two heavy chains and two light chains. The light chains typically contain a variable region and a light chain constant region (VL and CL, respectively). The heavy chains typically contain a heavy chain variable region (VL) followed by CH1, hinge, CH2, and CH3 constant regions.

[0055] As used herein, a protease such as cathepsin D or L may cleave an antibody "between" two regions, such as VL and CL or VH and CH (i.e., CH1). In such cases, for example, in the case of cleavage between VL and CL or VH and CH, this means that the cleavage occurs just before CDR3, within CDR3, or within FR4 of VL or VH, but before the 20th amino acid in the CL or CH (i.e., CH1) segment of the antibody. This is shown, for example, in the examples herein. For example, cleavage occurring between the CH1 and CH2 regions may occur before, within, or after the hinge region, resulting in one fragment containing most of the CH1 region and another fragment containing most of the CH2 region.

[0056] As used herein, a protease cleavage site "preceding," "prior to," "before," or "above" a particular amino acid region or position means that cleavage occurs N-terminal to that region or position. A cleavage occurring "following," "after," or "below" a particular position or region means a cleavage C-terminal to that position or region. Similarly, a region or amino acid position that "precedes," "prior to," "before," or "above" another amino acid region or position is N-terminal to the other amino acid region or position. A region or amino acid position that "follows," "after," or "below" another region or position is C-terminal to the other region or position.

[0057] "Cathepsin L" includes lysosomal proteases expressed in eukaryotic cells and can be naturally or recombinantly produced. Cathepsin L can be derived from various eukaryotic organisms, such as humans or other mammals. Cathepsin L can also include engineered variants of natural cathepsin L that retain cathepsin L activity but can improve, for example, activity, yield, shelf life or stability. Cathepsin L is also known as cathepsin L1. In some embodiments, cathepsin L is human cathepsin L, for example recombinant human cathepsin L. Mammalian cathepsin L is expressed from the CTSL1 gene. An exemplary human cathepsin L includes the sequence of SEQ ID NO: 10 and can also be purchased from Sigma (catalog number C6854).

[0058] "Cathepsin D" includes lysosomal proteases expressed in eukaryotic cells and can be naturally or recombinantly produced. Cathepsin D can be derived from various eukaryotic organisms, such as humans or other mammals. Cathepsin D can also include engineered variants of natural cathepsin D that retain cathepsin D activity but can improve, for example, activity, yield, shelf life or stability. In some embodiments, cathepsin D is human cathepsin D, such as recombinant human cathepsin D. Mammalian cathepsin D is expressed from the CTSD gene. An exemplary human cathepsin L includes the sequence of SEQ ID NO: 9 and can also be purchased from Sigma (catalog number C8696).

[0059] "De novo" sequencing of a protein or protein fragment refers to determining the sequence of that protein or fragment, such as a full-length antibody molecule or antigen-binding fragment or other antibody fragment, where the sequence is not known beforehand by other means. In other words, the sequence is determined "from scratch," without reliance on any previously known sequence information.

[0060] "Bottom-up" protein sequencing methods refer to methods in which proteins are digested to form peptides that are typically short in length or size (e.g., about 3-5 kDa or about 5-30 amino acids in length) by an enzyme such as trypsin, the peptides are analyzed by mass spectrometry, and then assembled via software, such as by analysis of overlapping peptides and comparison to known protein sequences.

[0061] "Top-down" sequencing involves measuring the mass of an intact protein or polypeptide and then fragmenting the entire protein by mass spectrometry into a series of product ions from which sequence information can be derived.

[0062] "Middle-down" sequencing involves splitting a protein into larger sized fragments (e.g., about 5-25 kDa), which can then be separated and further analyzed by top-down and / or bottom-up approaches, which typically use proteases to generate the fragments.

[0063] As used herein, "isolating" one or more protein fragments following an enzymatic cleavage reaction refers to at least partially separating the desired protein fragment from other fragments and / or at least partially separating the fragment from the enzyme such that sequence or structural analysis can be performed on the fragment without interference from contaminating proteins. In some cases, the desired fragments can be isolated during mass spectrometry, while in other cases they can be at least partially isolated by chromatography, filtration, or other methods.

[0064] "Liquid chromatography" or "LC" refers to a process of separating components of a sample by their respective interactions with a stationary phase (e.g., a column of particulate material) and a mobile phase (i.e., a fluid). LC may be performed in one dimension (1D-LC), meaning that one separation process is performed, or in two dimensions (2D-LC), meaning that the eluent of the first separation, or a portion thereof, is further separated in a second separation step using a different separation means, such as using a different mobile phase. LC encompasses, for example, HPLC and reversed-phase HPLC methods. "High performance liquid chromatography" or "HPLC" refers to a type of LC system in which a mobile phase is flowed under pressure through a stationary phase, such as a column. The HPLC system may be coupled to a detector, such as a mass spectrometer. HPLC processes can be performed in "normal phase" ("NP" or "NP-HPLC") or "reversed phase" ("RP" or "RP-HPLC" or "RPLC"). In RPLC processes, the stationary phase (e.g., column) is non-polar and the mobile phase is polar, such as a water / polar organic solvent mixture or gradient. In normal phase HPLC, the stationary phase (e.g., column) is polar and the mobile phase is non-polar. LC methods also include size exclusion chromatography (SEC) to separate polypeptides by size, as well as methods that use phases that separate by charge or isoelectric point (pI), such as hydrophobic interaction chromatography (HIC), strong cation exchange (SCX), strong anion exchange (SAX), WSX and other charge-variant interaction phases.

[0065] "Mass spectrometry" or "MS" refers to a technique that measures the mass-to-charge ratio (m / z) of one or more molecules in a sample. As used herein, "tandem MS" or "MS / MS" refers to a process in which a single ion, multiple ions, or the entire mass envelope (precursors) are transferred to a fragmentation chamber and then the fragmented products are sent to a mass analyzer. Depending on the design of the mass analyzer, the fragmentation events can occur before a single mass analyzer, between two or more different analyzers, or within a single mass analyzer.

[0066] There may be various options for MS analysis. In some embodiments, the MS instrument does not include a quadrupole analyzer. In some embodiments, the MS instrument includes at least one quadrupole. In some embodiments, the MS instrument includes at least two quadrupole analyzers. In some embodiments, the MS instrument includes at least three quadrupole analyzers. In some MS, the detector is an ion trap, a quadrupole, an orbitrap, or a TOF. In some embodiments, the MS instrument or method is multiple reaction monitoring (MRM), single ion monitoring (SIM), triple stage quadrupole (TSQ), quadrupole / time of flight (QTOF), quadrupole linear ion trap (QTRAP), hybrid ion trap / FTMS, time of flight / time of flight (TOF / TOF), an Orbitrap instrument, an ion trap instrument, parallel reaction monitoring (PRM), data dependent acquisition (DDA), data independent acquisition (DIA), multi-stage fragmentation, or time tandem MS / MS. antibody cleavage

[0067] In some embodiments herein, the antibody is cleaved by cathepsin L, cathepsin D, or a combination of cathepsin L or cathepsin D. In some embodiments, the antibody is digested only with cathepsin L and no other enzymes. In some embodiments, the antibody is digested only with cathepsin D and no other enzymes. In some embodiments, the antibody is digested with both cathepsin L and cathepsin D, but no other enzymes. In some embodiments, at least one other protease, such as IdeS, is also used to digest the antibody. When both cathepsin L and cathepsin D enzymes are used, the enzymes may be incubated simultaneously with the antibody (i.e., in a one-pot reaction) or sequentially alternating.

[0068] In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is a human IgG antibody, such as an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. In some embodiments, the antibody is a full-length IgG antibody. In some embodiments, the antibody is a full-length human IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. In some embodiments, the antibody has a full-length light chain and / or a full-length heavy chain. In other embodiments, the antibody does not have a full-length light chain and / or does not have a full-length heavy chain. In some such cases, the antibody comprises a light chain comprising a VL region and at least a portion of a CL region. In some such cases, the antibody comprises a VH region and at least a portion of a CH1 region, such as a CH1, a hinge and at least a portion of a CH2 segment, or only a CH1 and a hinge, or only a CH1, or only an N-terminal portion of a CH1. In some embodiments, the antibody is an antigen-binding fragment, such as, for example, a F(ab')2 fragment or a Fab fragment.

[0069] In some embodiments, the cleavage reaction is performed on the antibody in its native state, i.e., the native antibody folding pattern is maintained and any disulfide bonds are intact and not reduced. Thus, in some embodiments, the antibody has not been subjected to reduction or denaturation. However, in some embodiments, up to 50% organic solvent may be added to moderate the antibody conditions to some extent for optimal cleavage. For example, in some embodiments, 0-30% organic solvent may be added, such as 5-30%, 10-30%, 0-10%, 5-15%, 10-20%, 15-25%, or 20-30%. The organic solvent may include, for example, acetonitrile, methanol, ethanol, or isopropyl alcohol.

[0070] The antibody cleavage sites on human IgG1 and IgG2B antibodies are shown diagrammatically in FIG. 8. In the methods herein, both cathepsin L and cathepsin D can cleave the antibody at one or more locations, including between the VH and CH regions and between the VL and CL regions, thereby trimming off VH and VL antibody fragments from the antibody that contain at least a majority of the VH and a majority of the VL regions, respectively. This cleavage also leaves behind CL and CH fragments that contain the CL and CH regions C-terminal to the cleavage site. For example, in some embodiments where the antibody is a human IgG1, both cathepsin L and cathepsin D also cleave above the hinge region of the antibody to generate two F(ab') fragments (containing VL+CL and VH+CH1, respectively) and additional fragments from the heavy chain that contain the hinge region and additional constant region sequence segments from the CH2 and CH3 regions. If cleavage occurs at both of these positions, between the variable and constant regions and above the hinge, the set of fragments includes the VL fragment, the CL fragment, the VH fragment, the CH1 fragment, and the remaining heavy chain hinge and constant region sequences, and also the Fab fragment if the cleavage is incomplete. Again, depending on the exact position of the cleavage, the CH1 fragment may include amino acid sequences from the C-terminus of the VH region and may not extend all the way to the C-terminus of CH1 as defined by EU numbering. Thus, the fragment may include most of the CH1 region. In other embodiments where the antibody is a human IgG2B or IgG4 antibody, for example, cathepsin L or cathepsin D may not cleave above the hinge due to different disulfide bond structures in the antibody. (See FIG. 8.) Both cathepsin L and D may further cleave the IgG1 antibody between the CH2 and CH3 regions of the Fc portion, leaving CH2 and CH3 fragments on either side of the cleavage position, as shown in FIG. 8. In the methods herein, cathepsin D can also cleave just below the hinge region, resulting in the generation of an F'(ab)2 fragment, and an Fc fragment with all or most of the Fc region sequence below the cleavage site. (See FIG. 8.) When both enzymes are used to cleave a human IgG1 antibody, a series of fragments can be generated, corresponding for example to VL, VH, CL, CH1, CH2, CH3, CL+CH1 (disulfide bond) and F(ab')2 fragments. In some embodiments, cathepsin D and cathepsin L are combined to generate VL, VH, CL, CH1, CH2, CH3, CL+CH1 (bond) and F(ab')2 fragments from a human IgG1 antibody. In some embodiments, cleavage by cathepsin L or cathepsin D or both cathepsin D and cathepsin L leaves VH and VL fragments of about 8-16 kDa, e.g., about 10-16 kDa, 10-13 kDa, 10-12 kDa, 10-11 kDa or 11-12 kDa. In some cases, the VH and VL fragments are about 90-120 amino acids in length, e.g., about 95-110 amino acids in length. In some cases, simultaneous cleavage or miscleavage results in fragments that include various combinations of VL fragments, VH fragments, CL fragments, CH1 fragments, CH2 fragments, CH3 fragments, CL+CH1 fragments, and F(ab')2 fragments.

[0071] The Examples herein provide data showing cleavage of known antibodies by cathepsin L and / or cathepsin D. As shown in Table 5a, obinutuzumab was cleaved by cathepsin L at the end of the variable portion of the heavy chain, at VH CDR3. The enzyme then cleaved at several positions in the heavy chain hinge region. This resulted in VH fragments of amino acids 1-99 and 1-135, 1-136, 1-137, 1-138 and 1-139, e.g. with masses of 10-16 kDa, as well as longer light and heavy chain fragments, e.g. LC fragments of amino acids 1-206 and 1-218, HC fragments of amino acids 1-220 and LC+HC fragments with masses of 20-25 kDa and 45-50 kDa, respectively. (See Table 5a.) As shown in Table 5b below, exposure to cathepsin D cleaves obinutuzumab in the heavy chain hinge region. For example, this cleavage results in an LC+HC fragment and a F(ab')2 fragment of approximately 220-245 amino acids in length. Tables 7, 8 and 10 show the cleavage positions of trastuzumab, which, like obinutuzumab, is a human IgG1 antibody. Trastuzumab cleaved with cathepsin L produced heavy chain fragments of amino acids 1-140 and 1-102 (i.e., VH fragments) and light chain fragments of amino acids 1-213, 1-214 and 9-196, as well as a heavy chain fragment of amino acids 100-223 (including the CH1 region) and a light chain fragment of amino acid residues 117-214 of the CL region. (Table 8.) Cleavage with cathepsin D yielded a similar set of fragments, light chain fragments from residues 1-214, 1-213, and heavy chain fragments from 1-140 and 1-102, as well as heavy chain fragments containing residues 2-222 and 11-110. (Table 9.) Cleavage with both enzymes yielded several VL fragments containing residues 1-107, 1-111, and 1-116, a longer LC fragment from residues 1-202, and a VH fragment containing positions 1-115, as well as HC constant region fragments from amino acids 241-349 and 243-341. Fragments combining the LC and heavy chain CH1 regions were also produced, as well as F(ab') and F(ab')2 fragments. (Table 10.) Eculizumab, a human IgG2B antibody exposed to cathepsin L, like obinutuzumab, yields a VH fragment from amino acids 1-102 to 1-106 of the heavy chain sequence, terminating in the heavy chain CDR3.Table 6 shows that cathepsin D also cleaves eculizumab in the VH CDR3 and cleaves the F(ab')2 from the Fc below the hinge region.

[0072] Thus, in some embodiments, cathepsin L or / and D, acting on human IgG1 antibodies, cleaves between VH and CH and / or between VL and CL, particularly within or immediately after the HC and LC CDR3s, generating VH and VL fragments that end within or immediately after the CDR3s. In some embodiments, cathepsin L or / and D also cleaves the antibody between the first and second interchain disulfide bonds to generate F(ab'). In some embodiments, cathepsin D cleaves within or just below the heavy chain hinge region to give F(ab')2 fragments, which can then be further cleaved, for example, between the VH and CH1 portions and / or between the VL and CL portions. Furthermore, in some embodiments, both proteases also cleave between CH2 and CH3 to generate CH2 and CH3 fragments along the CH2 domain. Thus, in some embodiments, cleavage by one or both enzymes results in VH-containing and / or VL-containing fragments that contain the respective CDR1, CDR2 and CDR3.

[0073] In some embodiments, cleavage conditions are optimized for greater cleavage efficiency at one or more positions. Cleavage efficiency (also called digestion efficiency), when referring to the digestion of a protein or antibody, generally refers to the percentage of the starting antibody that is cleaved. Cleavage efficiency at specific positions or sites, such as after the VH and VL of an antibody, can also be evaluated. In some embodiments, a relatively low cleavage efficiency (e.g., at least 1% to 50%) at each expected cleavage site can be tolerated to yield a range of fragment sizes. Also, a low overall cleavage efficiency can be tolerated when there are relatively many antibodies to be analyzed. In other cases, a higher cleavage efficiency is desired, especially for the site between VH and CH and, for example, the site between VL and CL, to maximize the production of VH and VL antibody fragments. In some embodiments, the overall cleavage efficiency is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100%. In some embodiments, the cleavage efficiency for cleavage after VH and VL is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100%. Cleavage efficiency can be affected by factors such as reaction time, temperature, pH, and the presence of organic solvents, as well as the tertiary structure of the antibody.

[0074] The present disclosure encompasses cleavage reactions with cathepsin D and / or L that generate one or more fragments, including VL fragments, VH fragments, CL fragments, CH1 fragments, CH2 fragments, CH3 fragments, CH2-CH3 fragments, CL+CH1 (combined) fragments, F(ab') fragments, and F(ab')2 fragments, and the resulting compositions comprising those fragments. Thus, for example, the enzymatic cleavage reactions herein may generate VL and / or VH fragments that include a length of 90-150 amino acids, e.g., 95-140 amino acids, 100-140 amino acids, or 100-120 amino acids. Such VH and / or VL fragments may have a molecular weight of, for example, 10-16 kDa, e.g., 10-13 kDa, 10-12 kDa, 10-11 kDa, or 11-12 kDa. Depending on the efficiency of cleavage at various positions of the native antibody, larger fragments of 200-250 amino acids in size (e.g., combined light and heavy chain fragments) may also be generated. In some embodiments, F(ab') and / or F(ab')2 fragments may also be generated. In some cases, a mixture of all of these fragments may result. Thus, some cleavage reactions may result in a composition that includes at least two of the following ten types of fragments: VL fragment, VH fragment, CL fragment, CH1 fragment, CH2 fragment, CH3 fragment, CH2-CH3 fragment, CL+CH1 (combined) fragment, F(ab') fragment, and F(ab')2 fragment. Some cleavage reactions may result in a composition that includes at least three of the following ten types of fragments: VL fragment, VH fragment, CL fragment, CH1 fragment, CH2 fragment, CH3 fragment, CH2-CH3 fragment, CL+CH1 (combined) fragment, F(ab') fragment, and F(ab')2 fragment. Some cleavage reactions may result in a composition comprising at least two of the following four types of fragments: VL fragment, VH fragment, CL fragment, CH1 fragment, CH2 fragment, CH3 fragment, CH2-CH3 fragment, CL+CH1 (joint) fragment, F(ab') fragment, and F(ab')2 fragment. Some cleavage reactions may result in a composition comprising a VL fragment and / or a VH fragment of, for example, 90-150 amino acids and at least two of the following additional fragments: CL fragment, CH1 fragment, CH2 fragment, CH3 fragment, CH2-CH3 fragment, CL+CH1 (joint) fragment, F(ab') fragment, and F(ab')2 fragment.

[0075] For example, in some embodiments, the reaction is carried out at pH 2-8, pH 2-7, pH 2-6, pH 2-5, pH 3-6, pH 3-5, pH 3-4, pH 4-5, pH 2, pH 2.5, pH 3, pH 3.5, pH 4, pH 4.5, pH 5, pH 5.5, pH 6, pH 7, or pH 8. In some embodiments, the reaction is carried out at pH 3-5, such as pH 3, pH 3.5, pH 4, pH 4.5, or pH 5. In some embodiments, the reaction is carried out at pH 4. The choice of pH may also depend on whether cathepsin L, cathepsin D, or both cathepsin D and cathepsin L are used.

[0076] In some embodiments, the reaction is carried out at room temperature (about 18-25°C) to 50°C. In some embodiments, the reaction is carried out at a temperature of 30-50°C, 30-45°C, 30-37°C, 37-45°C, 40-50°C, or 37-50°C. For example, a higher temperature may allow for a shorter reaction time. In some embodiments, the reaction is carried out for 4-30 hours, for example, 4-12 hours, 12-24 hours, or 12-18 hours. In some embodiments, the reaction is carried out at pH 3-5, for example, pH 3, pH 3.5, pH 4, pH 4.5, or pH 5, and 37-50°C. In some embodiments, the reaction is carried out at pH 4, 37°C, and 12-24 hours.

[0077] In some embodiments, the cathepsin D is human cathepsin D, e.g., comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the cathepsin L is human cathepsin L, e.g., comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the cleavage reaction is performed with a ratio of cathepsin D and / or cathepsin L enzyme to protein of 1:20 to 1:2000. In some embodiments, ratios of 1:20 to 1:500, 1:50 to 1:500, 1:100 to 1:500, 1:200 to 1:1000, 1:200 to 1:2000, 1:500 to 1:2000, 1:1000 to 1:2000, or 1:20, 1:50, 1:100, 1:200, 1:300, 1:400, 1:500, or 1:1000 are used. Varying the ratio may, in some embodiments, alter the degree of cleavage and, in certain cases, affect the efficiency of different cleavage sites. For example, without being bound by theory, decreasing the amount of enzyme may not only result in a lower overall cleavage efficiency, but may also affect the resulting cleavage products by favoring certain more accessible cleavage sites over others. Thus, in some embodiments, varying the enzyme to protein ratio may affect the distribution of the resulting cleavage products.

[0078] The data herein unexpectedly showed that cathepsin L and cathepsin D cleavage depends on the tertiary structure of the antibody. For example, the cleavage efficiency at certain sites may vary depending on the folding of the molecule near those sites or due to disulfide bond patterns. The tertiary structure may block cleavage at a position that would be cleaved in a denatured antibody. For example, as shown in Figure 8, cathepsin D cleavage occurs under the hinge region of human IgG1 antibodies, but the cleavage does not occur in human IgG2B or IgG4 antibodies due to differences in disulfide bond patterns.

[0079] Thus, in some embodiments, the antibody is cleaved in its native state. An antibody in the "native state" is an antibody that retains its general native folded structure and disulfide bonds, i.e., the antibody is not denatured or unfolded, and the disulfide bonds have not been reduced. In contrast, bottom-up cleavage is typically performed by digesting the protein to be analyzed in a denatured state, so that small peptides can be easily generated from the protein. In some embodiments, as described above, an organic solvent can be added up to 50%, e.g., to relax the antibody conditions somewhat for optimal cleavage, but to avoid denaturing or unfolding the antibody so that the antibody remains in the native state. For example, in some embodiments, 0-30% organic solvent may be added, such as 5-30%, 10-30%, 0-10%, 5-15%, 10-20%, 15-25%, or 20-30%. The organic solvent may include, for example, acetonitrile, methanol, ethanol, or isopropyl alcohol. In some embodiments, the reaction buffer includes 50% or less organic solvent. In some embodiments, the reaction buffer comprises 30% or less organic solvent. In some embodiments, the reaction buffer comprises 10% or less organic solvent. Again, as noted above, in some embodiments, the antibody is not reduced to avoid interference with its native disulfide bonding pattern.

[0080] In some embodiments where both cathepsin D and cathepsin L are used, the enzymes are exposed to the antibody one after the other, while in other embodiments they are incubated with the antibody simultaneously under the same reaction conditions. In some embodiments, both enzymes are added simultaneously to the antibody in a buffer of pH 3-5, e.g., pH 3, pH 3.5, pH 4, pH 4.5 or pH 5, and at a temperature of 30-50° C., e.g., 37° C. In some such cases, 10-30% organic solvent is also added to the reaction mixture. Unexpectedly, up to 100% cleavage efficiency (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100%) between the VL and CL and between the VH and CH of an antibody was observed when both cathepsin D and cathepsin L were used simultaneously in a buffer of pH 3-5, e.g., pH 3, pH 3.5, pH 4, pH 4.5 or pH 5, and at a temperature of 30-50°C.

[0081] In some methods herein, for example, the antibody may remain in a native state during cleavage, since cathepsin L / D cleavage appears to be tertiary structure dependent. In some embodiments, the antibody remains in a native state during and after cleavage and MS analysis. In some methods herein, the antibody is not treated with a denaturing agent or an agent that reduces disulfide bonds during or after cleavage. In some methods herein, the antibody retains its disulfide bonds during or after cleavage. Some methods herein include performing mass spectrometry (MS) of one or more antibody fragments after cleavage, where the antibody remains in a native state and is not treated with a denaturing agent or an agent that reduces disulfide bonds prior to MS analysis. Thus, in some embodiments, in addition to the preferred selection of antibody fragments that may be generated by reaction with cathepsin L and / or cathepsin D, such as VL, VH, F(ab'), F(ab')2, or other fragments comprising CDR1, CDR2 and CDR3 of the heavy or light chain, MS analysis may be performed immediately after cleavage without denaturing or reducing disulfide bonds in the resulting antibody fragment.

[0082] After cleavage of the antibody, the antibody can be treated to remove the enzyme(s), exchange buffers, and / or isolate specific fragments. Optional post-disconnect processing

[0083] The antibody fragments generated during cleavage can be further processed in various ways prior to mass spectrometry.

[0084] In some embodiments, antibody fragments can be treated to reduce disulfide bonds. When disulfide bonds are reduced, certain fragments, for example, comprising two polypeptide segments linked by a disulfide bond, can dissociate into two separate species. In some cases, a portion of the cleaved antibody can be evaluated by mass spectrometry without reducing the SS bond, while another portion can be reduced before being evaluated, for example, allowing top-down analysis of both reduced and non-reduced fragments to obtain further data. In some embodiments, the resulting fragments can be alkylated or otherwise chemically modified.

[0085] In some embodiments, after cleavage, the resulting fragments can be isolated from the enzymatic reaction mixture in a variety of ways. For example, the fragments can be isolated by chromatography or size filtration. For example, the fragments can be separated via capillary electrophoresis or liquid chromatography. Or, the fragments can be separated from other components by filtration, such as using a molecular weight cutoff filter that retains higher molecular weight species but allows smaller species, such as the specific cleavage fragments, to flow through. In some embodiments, for example, if the desired fragment is less than, for example, 25 kDa, for example, about 10-14 kDa, a molecular weight cutoff filter, for example, of 20 kDa or 30 kDa, can be used to separate the desired fragment from the larger fragments, optionally followed by a step of concentrating the flow-through over a MW filter that retains the fragment of interest. If desired, a relatively low molecular weight cutoff filter can also be used to separate the desired fragment from smaller fragments or buffer contaminants, for example, a filter that retains molecules greater than 3 kDa or greater than 10 kDa.

[0086] Cleaved antibody fragments can also be isolated from the enzymatic reaction mixture by buffer exchange, for example to change pH and other buffer conditions. This can occur, for example, during a chromatography or filtration process. In some embodiments, the fragments can be analyzed by Edman degradation, optionally in combination with further enzymatic or MS cleavage, as an alternative means to obtain or verify their sequence.

[0087] Any combination of the above processes may be used in the embodiments herein. mass spectrometry

[0088] To obtain sequence or structural information about the antibody fragments, mass spectrometry can be performed on at least one fragment. In some embodiments, MS analysis is performed on the VH and / or VL fragments obtained from cleavage with cathepsin L or D or both cathepsins L and D. In some embodiments, a top-down analysis method is performed on at least one of the generated fragments. In some embodiments, a bottom-up approach is performed. In some embodiments, a combination of top-down and bottom-up approaches is used.

[0089] In some embodiments, the complete sequence of at least one of the fragments is obtained. In other embodiments, the sequence of at least a 10 amino acid stretch is obtained. In some embodiments, the sequence of at least a 15 amino acid stretch is obtained. In some embodiments, the sequence of at least a 25 amino acid stretch is obtained. In some cases, the sequence of at least a 50 amino acid stretch is obtained. In some cases, the sequence of a stretch of 10-50, 15-50, 25-50 or 10-25 amino acid stretch is obtained. In some cases, a CDR3 sequence is obtained for the VH fragment or the VL fragment. In some cases, a CDR1 sequence, a CDR2 sequence and / or a CDR3 sequence is obtained for the VH fragment or the VL fragment. In some embodiments, the sequence of the antibody fragment was unknown prior to the method, and thus the method obtains a sequence or a subsequence of the fragment de novo.

[0090] MS measures the mass-to-charge ratio (m / z) of one or more molecules in a sample. Tandem MS may be used in some embodiments and is a process in which a single ion, multiple ions, or the entire mass envelope (precursor) is transferred to a fragmentation chamber and then the fragmented products are sent to a mass analyzer. Depending on the design of the mass analyzer, the fragmentation event can occur before a single mass analyzer, between two or more different analyzers, or within a single mass analyzer.

[0091] There may be various options for MS analysis. In some embodiments, the MS instrument does not include a quadrupole analyzer. In some embodiments, the MS instrument includes at least one quadrupole. In some embodiments, the MS instrument includes at least two quadrupole analyzers. In some embodiments, the MS instrument includes at least three quadrupole analyzers. In some MS, the detector is an ion trap, a quadrupole, an Orbitrap™, or a time of flight (TOF). In some embodiments, the MS instrument or method is multiple reaction monitoring (MRM), single ion monitoring (SIM), triple stage quadrupole (TSQ), quadrupole / time of flight (QTOF), quadrupole linear ion trap (QTRAP), hybrid ion trap / FTMS, time of flight / time of flight (TOF / TOF), Orbitrap instrument, ion trap instrument, parallel reaction monitoring (PRM), data dependent acquisition (DDA), data independent acquisition (DIA), multi-stage fragmentation, or time tandem MS / MS. In some embodiments, the mass spectrometer comprises at least one quadrupole and uses a dissociation means selected from collision induced dissociation (CID), higher energy collision dissociation (HCD), electron capture dissociation (ECD), electron transfer dissociation (ETD), electron transfer / high energy collision dissociation (EThcD), or photodissociation, e.g. UV photodissociation (UVPD).

[0092] In some embodiments, one or more antibody fragments are analyzed using direct infusion or static spray infusion or flow injection analysis mass spectrometry. In some cases, the infusion is performed immediately after the enzymatic cleavage reaction or after a buffer exchange step after the cleavage reaction. In some cases, for example, the infusion is an alternative to isolating the desired fragments via techniques such as chromatography followed by mass spectrometry such as LC-MS. In the infusion method, a sample containing the antibody fragment(s) can be directly introduced into a mass spectrometer device for fragmentation by electrospray or nanospray ionization. The precursor ions can then be optionally separated by a quadrupole mass filter for mass measurement and analysis, and fragmented for amino acid sequence prediction. Top-down and / or bottom-up analysis

[0093] In some embodiments herein, mass spectrometry is used for top-down sequence analysis. For example, a mass spectrometer can be used to further fragment the cleaved antibody fragments into a series of product ions, for which sequence predictions can be made. An example is shown, for example, in Table 13 below. The predicted sequences can be aligned using associated software. An example is shown, for example, in Figures 14A and 14B. For example, in the case of VH or VL fragments, the fragments typically begin with the natural N-terminal amino acid of the antibody being analyzed, so the mass spectrometry sequence information can be deconvoluted and aligned starting from the N-terminus of the fragment. Thus, the N-terminus can serve as a reference point for the alignment of the product ions generated during mass spectrometry. For example, when analyzing VH or VL fragments, the fragments are at the N-terminus of the antibody polypeptide chain, so the product ions generated in the MS analysis can be aligned from the N-terminus. For this and other reasons, it is not necessary for the enzyme to specifically cleave at a single amino acid to form the C-terminus of the fragment.

[0094] A variety of software packages can be used to deconvolute and align sequence information from top-down MS analyses, examples include PEAKs™ (Bioinformatics Solutions, Inc.) and Byos™ and its component software (Protein Metrics, Cupertino, Calif., USA).

[0095] In some embodiments, the methods herein may be combined with additional middle-down and bottom-up methods for further sequence analysis. For example, cleavage with cathepsin L and / or cathepsin D may be combined with cleavage with other enzymes that cleave within or near the hinge region of an antibody, such as the protease Streptococcus pyogenes (IdeS) (e.g., FabRICATOR™ (Genovis, Inc.)) or GingisKhan™ (Genovis). In some embodiments, cathepsin L, cathepsin D, or a combination of cathepsin L and cathepsin D may be combined with IdeS or GingisKhan™ (Genovis). In some embodiments, other proteases, such as trypsin, papain, chymotrypsin, may be combined with cathepsin L, cathepsin D, or both cathepsin L and cathepsin D. The products from such cleavage reactions may then be analyzed, for example, by top-down or bottom-up processes.

[0096] In some embodiments, the fragments generated from the cathepsin L and / or cathepsin D cleavage reaction may be further analyzed by bottom-up methods. Thus, in some embodiments, for example, the resulting fragments may be reduced or denatured and then further cleaved with a protease such as trypsin or papain to generate smaller fragments for MS analysis. In some embodiments, top-down analysis may be combined with bottom-up analysis, for example, as a means to verify and cross-reference sequence information. De Novo Antibody Sequencing Workflow

[0097] In some embodiments, the methods herein can be used for partial or complete de novo sequence analysis of one or more antibody fragments. In general, determining a sequence stretch de novo requires complete fragmentation of that sequence stretch during mass analysis, i.e. fragmentation after each subsequent amino acid in the sequence stretch, so that fragmentation allows the mass of each consecutive amino acid residue to be determined and therefore the amino acid residues to be identified. Depending on the fragmentation method selected, reduction of disulfide bonds can be performed prior to mass analysis to ensure complete fragmentation of a particular amino acid stretch. For example, if an Orbitrap™ with high-energy collision dissociation (HCD) is used for fragmentation, reduction may be required to obtain fragmentation after each consecutive residue. In other cases, de novo sequencing can be performed without prior reduction of disulfide bonds. In other embodiments, a portion of the sample containing the antibody fragments can be analyzed without reduction and another portion of the sample can be analyzed after reduction.

[0098] For example, FIG. 14A shows a depiction of the trastuzumab light chain variable region after MS fragmentation. As shown in that figure, a span of contiguous y ions occurs from residues H91 to E105. Thus, the sequence of this portion of the VL, which corresponds approximately to the CDR3 region, can be determined by top-down analysis. Such top-down analysis can use commercially available software for determining amino acid sequence from mass data and fragment alignment, such as PEAKS™ (Bioinformatics Solutions, Inc.) or Supernovo™ (Protein Metrics) or open source programs. To determine the sequence of further portions of the antibody fragment, such as the VL fragment, further fragmentation frequencies can be used such that fragmentation occurs after all or nearly all amino acid residues in the fragment molecule. Additionally, antibody cleavage products can be treated to reduce disulfide bonds so that they do not interfere with fragmentation. To sequence larger segments of antibody fragments, commercially available software parameters can be adjusted to allow de novo determination of sequences of fragments, for example, up to about 12 kDa. For example, the total peptide length allowed within the program can be extended, the error tolerance allowed at the MS1 level can be increased, and the p-score or A-score can be modified, for example, to allow for the incorporation of disulfide rules. Additionally, in some embodiments, candidate de novo sequences can be validated by sequencing using more than one software program. Kits and Products

[0099] The disclosure also encompasses kits and articles of manufacture for performing optimized cathepsin L, cathepsin D, or cathepsin L and cathepsin D cleavage reactions on antibodies. In some embodiments, the kits include cathepsin L. In some embodiments, the kits include cathepsin D. In some embodiments, the kits include both cathepsin L and cathepsin D. In some embodiments, the kits include one or more additional enzymes, such as IdeS, papain, trypsin, chymotrypsin, or GingisKhan™, in addition to cathepsin L and / or cathepsin D. In some embodiments, the kits include one or more reaction buffers for cathepsin L and / or cathepsin D cleavage reactions. For example, in some embodiments, the reaction is carried out at pH 2-8, pH 2-7, pH 2-6, pH 2-5, pH 3-6, pH 3-5, pH 3-4, pH 4-5, pH 2, pH 2.5, pH 3, pH 3.5, pH 4, pH 4.5, pH 5, pH 5.5, pH 6, pH 7, or pH 8. In some embodiments, the reaction buffer is pH 3-5, such as pH 3, pH 3.5, pH 4, pH 4.5, or pH 5. In some embodiments, the reaction buffer is pH 4. In some embodiments, the reaction buffer comprises one or more organic solvents. In some embodiments, the organic solvent is methanol, ethanol, isopropyl alcohol, or acetonitrile. In some embodiments, the reaction buffer comprises 5-50% organic solvent, such as 5-30%, 10-30%, 0-10%, 5-15%, 10-20%, 15-25%, or 20-30%. In some embodiments, the reaction buffer comprises 50% or less organic solvent. In some embodiments, the reaction buffer comprises 30% or less organic solvent. In some embodiments, the reaction buffer comprises 10% or less organic solvent. In some embodiments, the article or kit also comprises instructions for use in performing a cathepsin D and / or cathepsin L cleavage reaction on the antibody composition. EXAMPLES

[0100] Example 1. Despite the great success of proteomics in protein sequencing, the complete sequencing of antibodies (Abs) still presents a considerable challenge [1,2]. It is performed with the aim of annotating and validating the position and order of all amino acids (and any variants), which currently requires the use of 4-5 different proteases to generate overlapping and ideal length peptides for liquid chromatography (LC)-mass spectrometry (MS) bottom-up proteomics analysis. This is a higher analytical standard compared to intact analysis, or the simple monoclonality check performed in protein validation, which generally uses a single enzyme for digestion. If the annotation is de novo, these mass spectra must be processed by specialized Ab sequencing programs that use information on the extracted mass shifts between product ion peaks. The assignment is made based on computational rules that limit the allowed mass error, and the success of such an analysis depends heavily on the excellent quality of the MS / MS spectra [3-7].

[0101] Alternatively, middle-down enzymatic proteomics approaches have begun to be explored in which antibodies are first cleaved above or below the hinge region to generate Ab protein fragments that are amendable for sequencing by top-down and / or middle-down proteomics [8-10]. These methods can selectively generate F(ab')2 fragments, F(ab') fragments, Fc fragments, Fd fragments, light chain (LC) fragments, and heavy chain (HC) fragments, depending on the protease or denaturant used, making the protease Streptococcus pyogenes (IdeS) (e.g., FabRICATOR™ (Genovis, Inc.)) in particular very popular [10-13]. IdeS is a protease that digests antibodies at a specific site just below the hinge to generate a homogenous pool of F(ab')2 fragments and Fc / 2 fragments [12,13].

[0102] The main difference of middle-down is the use of relatively high molecular weight (HMW) precursors (5-25 kDa) when compared to bottom-up approaches for sequencing. These protein fragment precursors provide the corresponding sequences to which all fragment ions can and should be mapped. Combined with MS1 intact information on the different fragments, top-down and middle-down proteomic approaches can be very powerful, especially when determined at high resolution [8-11, 14-21].

[0103] The combination of native mass spectrometry [22-28] with top-down proteomics [14, 16, 20, 29, 30] can provide additional advantages for the analysis of biotherapeutics. LC separation of digested complex mixtures is often insufficient, resulting in coelution of species, and under denaturing conditions, these masses often overlap, reducing signal-to-noise and limiting accurate mass deconvolution. Similarly, higher charge states result in increased higher collision energy dissociation (HCD) fragmentation efficiency in top-down analysis, but native mass spectrometry can allow the use of large separation widths that encompass multiple charge states as proteins move into less crowded m / z space [31, 32]. This can increase the signal-to-noise of product ions, resulting in higher coverage and simplifying the workflow. In general, top-down of large intact proteins (>25 kDa) with unreduced disulfide bonds lacks sufficient coverage for de novo application on the current MS “powerhouse” instruments found in industry, traditionally Orbitrap with time-of-flight or HCD instruments with collision-induced dissociation

[31] . These lack the highly efficient and orthogonal fragmentation approaches electron-induced dissociation [14, 18, 33-36], and ultraviolet photodissociation [16, 17, 30], which are also suitable for very large polypeptides. Without multiple fragmentation methods, the incomplete coverage limits the application of top-down and middle-down. Thus, there is an unmet need to establish more alternative middle-down workflows using less studied proteases to generate protein fragments more suitable for standard LC-MS / MS HCD experiments.

[0104] In the field of IgG analysis, the most commonly used middle-down proteolytic enzymes are the aforementioned IdeS, which cleaves preferentially at a single site below the hinge region [12,13], and GingisKHAN™ (Genovis, Inc.), which cleaves just above the hinge

[37] . Most alternative middle-down digestions have attempted to control the rate of indiscriminate enzymatic or chemical degradation such that the size of the polypeptide is related to the exposure time of the protein to the enzyme. More recently, Aspergillus saitoi acid proteinase has been immobilized on electrospray emitters for applications in online peptide mapping [38,39]. Investigative studies have generated peptides ranging from 3 to 15 kDa at a 1:5 ratio during exposure times of 0 to 2 min. Similarly, pepsin activity has been intentionally limited by deoptimization of pH to obtain middle-down size fragments and F(ab')2 domains

[40] . Furthermore, peptides generated by the proteases OmpT

[41] and Sap9

[42] are generally in the range of 1.5–15 kDa in size, which is larger than the average tryptic peptide size.

[0105] The commercially available lysosomal endopeptidases cathepsin L [43, 44] and D

[45] have been reported in the literature as two proteases with unpredictable cleavage sites. Cathepsins represent a family of enzymes found in lysosomes involved in protein degradation by hydrolysis of the protein backbone

[46] . Cathepsin L is a member of the peptidase C1 family and has been reported to cleave the P2 and P1 R sites after F, R or R [47-52], while cathepsin D, an aspartyl proteinase, has been reported to cleave at hydrophobic residues, specifically between Leu and Phe, although these preferences are inconsistent throughout the literature [47, 53-57]. These studies have been performed primarily on protein standards, extracts and peptides by SDS-PAGE, protein sequencers or peptide substrate microarrays. One consistency across these studies is that despite the promiscuity of the enzymes, they produce a surprisingly limited number of fragments. Protease degradation rates vary widely depending on the enzyme isoform, protein sequence, secondary and tertiary structure, buffer components (especially metals such as copper) and storage conditions

[58] .

[0106] The evaluation of new enzymes is challenging due to the large number of internal fragments that can be generated at any position and of any size found in the target protein, especially when there are few digestion site restrictions. This large computational space is further expanded when including possible disulfide bonds between chains or post-translational modifications such as glycans found on Fc. The work described herein to specifically map cathepsin digestion of Abs was made possible by utilizing the Intact Mass™ algorithm

[59] , which was updated in 2019 to include an automatic annotation feature for clipped species. This algorithm is described in further detail in the Methods section.

[0107] Here, we set out to explore the cleavage sites of cathepsins L and D when targeting three IgG1 antibodies (trastuzumab, rituximab, and obinutuzumab), one IgG2 / 4 antibody, eculizumab, and one IgG1 bispecific antibody (anti-Her2 / anti-CD3) containing knob-into-hole modifications using a single standard digestion condition. The Ab fragments formed were directly analyzed by high-resolution native mass spectrometry and denaturing LC-MS intact analysis. The cleavage sites of cathepsins L and D were explored under different pH, temperature, and denaturing (percent organic) conditions and then optimized to maximize cleavage between the variable and constant regions of the Ab. Our most interesting finding is that we can specifically generate a fragment of approximately 12 kDa encompassing the complementarity determining region (CDR) region of the Ab light chain. To our knowledge, this is the first demonstration of a middle-down technique directly targeting a hypervariable region of an Ab, and by fragmenting this 12 kDa fragment with standard HCD we were able to maximize coverage across this region. A. Results A.1. Mapping Cathepsin L and Cathepsin D Cleavage Sites Across Antibody Classes

[0108] Cathepsin L and cathepsin D cleavage sites were evaluated across five antibodies (Abs) to search for common digestion motifs and evaluate differences between IgG1 and IgG2 subtypes. To our knowledge, this is the first report of cathepsin L and cathepsin D digestion of intact Abs and the first concerted effort to examine sites spanning similar or dissimilar homologies.

[0109] Identification of protein fragments found in the digests was performed using Intact Mass and verified manually. Deconvoluted protein fragment peaks ranged in molecular weight from 9 to 98 kDa across all Abs for both cathepsin proteases (Tables 1-9, Figures 1-6). An example of the automated assignment of Rituximab when cleaved by Cathepsin D is shown in Figures 7 and 1. For the first chosen conditions (pH 7, 2 days of incubation), the enzymatic efficiency of Cathepsin L and Cathepsin D was found to be rather low, about 1%. Although the size distribution of protein fragments spanned a large molecular weight (MW) range, only four primary species were observed, corresponding to cleavage between VL and CL, between VH1 and CH1, between the first and second interchain bond CH1, and just after the hinge region (at a position almost identical to the IdeS cleavage site). At lower abundances, multiple cleavages were observed throughout the HC (Table 1).

[0110] Note that the masses shown in Figure 1 may vary slightly from the masses given in the tables disclosed herein (e.g., 9964.1 above is the same peak as 9963.45 in Table 2) because the exact average mass depends on the time, mass, resolution, signal to noise, and m / z range, as well as other parameters used in charge deconvolution. Similarly, the masses in Figures 2-6 may vary slightly from the masses given in the tables disclosed herein because the exact mass depends on the time, mass, and m / z range, as well as other parameters used in charge deconvolution.

[0111] When all low intensity signals were included, a large number of total peaks were deconvoluted, reflecting local versus global cleavage site diversity. For example, Figure 7B shows that cathepsin D can cleave at approximately 14 different but consecutive amino acids to generate F(ab'), many of which were generated with approximately equal likelihood based on their relative signal intensities (Tables 2 and 3). In this particular region, cleavage was shown to occur after C, D, K, T, or H. Further reflecting local cleavage diversity, certain products were found to be the result of asymmetric clipping, as opposed to simple single-site cleavage. For example, as shown in Figure 7A, the 97,538 Da peak is assigned to a complex of two LCs and one heavy chain (E1-F244) clipped with one amino acid difference to the second HC (E1-V243). Support for asymmetric cleavage assignment is provided in the top-down analysis section. Given that no amino acid enrichment was found across all identified peptides within the -4 to +4 cleavage site motif (Tables 1-3), this lends itself to the hypothesis that cathepsin D specificity may be influenced by secondary and tertiary structure as much as primary sequence, a role that will be discussed during digestion optimization experiments. As with Figures 1-6, the masses shown in Figure 7 may also vary slightly from those given in the tables disclosed herein, as the exact masses depend on the time, mass, and m / z ranges, as well as other parameters used for charge deconvolution.

[0112] Figure 8 provides a graphical overview of the observed clip positions when mapped onto schematics of antibodies with IgG1 and IgG2-B disulfide bond patterns. Although the site of the clip varied with the Ab and cathepsin protease used (Tables 1-9), several rules held without exception across all IgG1s: (1) cleavage occurs immediately after the HC and LC CDR3 by cathepsin L and cathepsin D, (2) cathepsin D cleaves the 1 番目 and 2 番目(3) the heavy chain hinge was subject to variable clipping by both proteases just below the hinge region to yield F(ab')2; and (4) there were cleavage sites identified for both cathepsin L and cathepsin D between CH2 and CH3.

[0113] When compared to IgG1, eculizumab showed the same pattern of F(ab') cleavage but apparently lacked a cleavage site within the hinge; thus, only F(ab')2 versus F(ab') was observed. This difference further supports the role of tertiary structure in mediating the digestion products of cathepsin D and cathepsin L. A.2. Quantitative optimization of digestion efficiency and fragment assignment

[0114] Although it was unpredictable whether cathepsin D and cathepsin L cleave proteins at specific sites or whether these enzymes are more promiscuous, we found that cathepsin L and cathepsin D specifically cleave Abs at four major sites. This specificity indicated the high feasibility of using cathepsin D and cathepsin L in a middle-down sequencing workflow. However, the digestion efficiency was rather poor under standard conditions. To improve the digestion efficiency, pH was selected as a digestion variable [60,61]. Similarly, the level of organic solvent was evaluated, since the tertiary structure of Abs is thought to affect cleavage. Finally, digestion was tested at temperatures of 37 °C or 50 °C. Three replicates were performed to compare the ratio of the charge state of intact trastuzumab to the total intensity of the digested fragments (Figure 9A). LC-UV-MS was used to minimize ion suppression from co-ionized species, improving relative quantification compared to nESI injection. To ensure that results were consistent, quantification was also performed by taking the ratio of UV peak area summed over the polypeptide elution region versus the Ab peak (Figure 9B).

[0115] The optimal pH efficiency for cathepsin L was determined to be pH 3 and for cathepsin D pH 5 (see Figures 9A and 9B), a trend supported by MS and UV data. Runs at 50% methanol showed increased efficiency for both enzymes but with significantly increased variability compared to the other conditions, likely due to the relative instability of the Abs under these conditions. Physical observations showed turbidity in some of the 50% methanol samples, likely due to the Abs precipitating but the protein fragments solubilizing, thus inflating the digestion ratio and increasing variability. At lower methanol levels (10 or 30%), no differences in digestion efficiency were observed by MS or UV compared to the control. Data from UV and MS directly contradicted each other for cathepsin L and cathepsin D data, and no conclusions could be made about the effect of temperature. Across all conditions, the protein fragments observed were in good agreement with those reported in the IgG1 control study. For example, a mass of 11,197 Da was detected across all IgG1 samples and is a low abundant species (<1e4) corresponding to the HC peptide E1-D102.

[0116] Based on the trends observed across conditions of pH, temperature, and percent organic matter, a single method for optimization was tested. A one-pot digest of trastuzumab with cathepsin L and cathepsin D was prepared at 37°C for 18 hours at pH 4. As shown in Figure 10, 100% digestion efficiency was achieved. The most abundant species in the sample corresponded to the loss of the CH2+CH3 region, generating F(ab')2 (approximately 98 kDa). Assignments were made within 2 Da for species less than 50 kDa and within 4 Da for species less than 100 kDa (corresponding to an average error of 31.8 ppm). The observed peptides appeared across all digestion replicates within 1 Da (N=6). The most abundant smaller MW peptides observed were mapped to the VH1, VL, and Cl regions (Table 10).

[0117] Interestingly, the 47 kDa species characterized in the control study (Figure 7B) was not observed in the LC-MS analysis of the optimized digest, but was observed later, during nESI injection for top-down analysis (Figure 11D). F(ab')2 has a broad elution width (~1 min) and shows a front, making F(ab') co-elution possible.

[0118] Highly specific cleavage sites were observed throughout the protein by LC-MS. This resulted in nine polypeptides, including a 98 kDa species, which comprised approximately 70% of the total intensity taken across all deconvoluted LC peaks (assigned + unassigned). The remainder of the observed but unassigned protein fragments belonged to low molecular weight species, mainly between 4 and 5 kDa. As the larger polypeptides provided 100% coverage of the antibody, a complete list is presented in Table 11, but no assignment of the 4-5 kDa species was attempted. Furthermore, at smaller molecular weights, the number of possible sequence assignments increased significantly due to the consideration of internal digestion products, and it was important to maintain the integrity of the identified sites.

[0119] To confirm the specificity of the nine identified peptides, Edman degradation was performed. Sequences were unambiguously confirmed for 7 / 9 identified protein fragments (Figure 12). Motifs beginning with "PT" and "APxxK" were observed, corresponding to a cross-linked mass resulting in a species of 22,517.13 Da. Although low abundance and overlapping gel bands precluded confirmation of the 12,488.77 mass, it was observed that at least the protein fragment beginning with Gly was identified at the expected molecular weight. A.3. Top-Down Analysis of Clipped Protein Fragments

[0120] A top-down analysis was performed to demonstrate the suitability of the polypeptide size and structure for fragmentation and provide further validation of the annotated trastuzumab protein fragments (Table 10). To enhance the signal-to-noise of the MS2 spectra, the optimized trastuzumab sample was infused into a Q Exactive UHMR by nESI to LC and precursors were averaged for at least 100 scans (Figure 11). Product ions were isotopically resolved at a resolution setting of 200,000 (Figure 13).

[0121] Product ion assignments were made in Freestyle 1.6 (Thermo Fisher Scientific, Bremen, Germany) by extracting monoisotopic masses with the Xtract algorithm and matching them to the respective predicted sequences in ProSight Lite

[62] (Table 10) within 10 ppm, with cysteines considered with H loss to account for the presence of disulfide bonds. Subsequently, an in-house database was constructed that accounts for y-ion NH3 loss and water loss, and matched the remaining extracted masses (Table 12). As shown in Figure 14A, good sequence coverage was obtained at the N- and C-termini of the 12,121.5 Da precursor, with fragmentation efficiency decreasing between disulfide bonds. Very large product ions were kept (Y104 and B109) to validate the sequence assignments. Overall, a sequence coverage of 29.4% was obtained.

[0122] For the 97,630 Da species, the assignment was complicated by the large number of disulfide bonds in the subunits. Although the inter- and / or intra-chains could be reduced to improve coverage, it was important to validate the polypeptide in its bound form to prove the presence of asymmetric HC cleavage (PAPELLG.G paired with PAPELLGG.PSV). As shown in Figure 14B, a significant number of y-ions corresponded specifically to each HC form. After the assignment of the standard y-, b-, ammonia- and water-loss ions, the presence of cross-linked species from the interconnected chains was evaluated (Table 13). For the LC, modifications constructed from the HC sequence NVNHKPSNTKVDKKVEPKSCDKTHT were considered, with C24 included in all subsequences as a cross-linking site. The N- and C-terminal bonds considered represented the start of the HC intra-chain disulfide (C148-C204) or the HC inter-chain disulfide (chain 1, C230 chain 2, C230), respectively. For HC, cross-linking modifications were considered starting from the LC sequence EVTHQGLSSPVTKSFNRGEC, which begins with the amino acid after the last inter-disulfide bond cysteine ​​and then cross-links at the C-terminus. Ions detected from these species confirmed the presence of the asymmetric C-terminus generated from the cathepsin L / D digest (Figure 14B). In total, 228 total ions were detected, giving a total coverage of 33.7%, with LC coverage of 26.6% and HC coverage of 40%. A.4 Application of optimized cathepsin L and D protocols to bispecific antibodies

[0123] Having established an optimized protocol, the application of cathepsins was further tested with a bispecific IgG1 antibody (anti-Her2 / anti-CD3). Bispecific antibodies have an identical disulfide bond structure to IgG1 but exhibit significant structural differences. By mutating various amino acids in the Fc region, a "knob-and-hole" structure was created, thereby enhancing the xHer2 and xCD3 chains and generating heterodimers

[63] . Primary structural changes facilitate this process, with multiple mutations found between amino acids 26–110 (variable region, CD3 binding), at positions 371 and 408 (knob / hole) and at 297 (preventing glycosylation). Furthermore, working with the newly expressed research antibody, we introduced a new source of variation (the presence of dimers at 4%) not found in most clinically used drug products.

[0124] Cathepsin proteases behaved as expected throughout the F(ab') (Table 4). In both xHer2 and xCD3 LC and HC, cleavage was observed immediately after CDR3 and outside the disulfide bond region. The LC and CH1 constant regions are homologous, assigning a single protein fragment as the digestion product of both chains. Cleavage after the hinge yielded five protein fragments, with asymmetric cleavage occurring along the highly preferred and conserved GGPSVFLFPPK sequence. Interestingly, no other cleavage was observed within the xCD3 CH2 or CH3 regions. In the anti-Her2 (knob) chain, digestion occurred after the hinge, yielding the F(ab')2 (AA1-239) and a few amino acids later the CH2+CH3 fragment (AA261-436). Multiple protein fragments in CH3 were also generated in the knob chain. However, the only Fc region cleaved on the xCD3 chain occurred simultaneously across both chains. Considering that cathepsin proteases are fairly promiscuous, the most likely explanation for this difference is the specific tertiary structure of the bispecific antibody. It is possible that the shape or structural rigidity of the hole, compared to the knob, makes it difficult for the cathepsin to "act" on this region until it is at least partially exposed by cleavage of the xHer2 chain. Validation of this particular knob / hole effect would require testing of a larger number of bispecifics, which is outside the scope of this study. These structural implications may also affect the digestion efficiency. The optimized protocol resulted in 100% digestion of trastuzumab, while 20% of the intact bispecific remained (based on deconvoluted peak intensity). This may reflect slower digestion as a result of the unique structure, or may be partially caused by slower digestion of the dimer, which decreased from 4 to 0% in the final digest. B. Discussion

[0125] The development of a middle-down approach that results in polypeptides of ideal size for MS sequencing is critical to advancing a top-down antibody sequencing workflow. Cathepsin L and cathepsin D proteases offer efficient, commercially available and inexpensive one-pot digests to directly enable this workflow.

[0126] Under all digestion conditions, peptides were observed individually giving coverage of the VL, VH, CL, CH1, CH2, CH3, CL+CH1 (combined) and F(AB') fragments. The combination of Edman degradation (N-terminal confirmation), intact mass (<40 ppm matching) and top-down data was clearly successful in sequencing the main cleavage products obtained from the digests, comprising 70% of the total protein signal in the optimized samples. The VL and VH regions were generated at high abundance and cleavage occurred immediately after CDR3. This region represents the most challenging part for sequencing due to its high variability over a short region. The ability to directly sequence long reads, which are the ideal size for top-down sequencing and encompass CDR1, CDR2 and CDR3, is a key feature of the presented middle-down approach.

[0127] Compared to the most widely adopted IdeS protocol, both procedures can directly generate F(ab') and the hinge cleavage site is nearly identical between the two protocols. Reducing reagents can be used in the IdeS protocol to generate free LC or HC, but these pose size-related challenges for full top-down sequencing by CID or HCD, whereas in the cathepsin protocol, the generation of protein fragments offers a straightforward approach. When combined with existing de novo bottom-up techniques, the generation of protein fragments allows for checking the mass of sequences locally on the Ab rather than the entire intact Ab. This provides an opportunity to rapidly find and localize problematic assignments. As computational tools develop for top-down de novo sequencing, the cathepsin approach solves many of the challenges of sequencing alignment, the digests stay within the size range, and high-quality product ion spectra are obtained.

[0128] HCD is known to result in limited sequence coverage of Abs across disulfide bonds

[20] . Alternative fragmentation techniques such as electron capture dissociation (ECD) can solve this sequencing challenge and allow direct analysis of digestion mixtures, but are not found on many mass spectrometers. Top-down HCD can achieve 100% coverage of smaller molecular weight species in the absence of disulfide bonds. Thus, the use of this method for Ab de novo sequencing, versus sequence verification as demonstrated in this paper, fractionation of the sample by either chromatography or molecular weight cutoff filters, followed by treatment and cleanup with guanidine hydrochloride, allows detailed sequencing while limiting the number of co-ionized peaks. Furthermore, application of guanidine transitions precursors to higher charge states, further improving fragmentation efficiency.

[0129] Evaluation of cathepsin L and cathepsin D across Abs showed remarkable consistency in cleavage sites across all IgG1s, especially when considered in the context of motif promiscuity. Across all antibodies tested (trastuzumab, rituximab, obinutuzumab, eculizumab, and bispecific antibodies), including combined L / D digests, 50 unique cleavage sites were identified. Cathepsin L cleaved at 27 sites and cathepsin D cleaved at 28 sites. While there was clear evidence that neutral sites were preferred for cleavage, otherwise both cathepsin proteases showed little preference for specific amino acids from p4 to p4' (Figure 15). The literature generally reported F and R as cleavage sites by cathepsin L, and although these ends were present, they constituted less than 10% of the total cleavage products. Compared to a 2011 study of cathepsin L activity on HEK293 protein extracts that found equal enrichment between 4 and 6 amino acids enriched per p3-p3' site, the Ab showed a significant reduction in motif preference, indicating that higher order structure versus secondary structure may be most important

[48] . This may be the reason why individual studies that have examined cathepsin L and cathepsin D protein activity identify many exceptions to these rules in their reported cleavage sites [47-57]. An alternative possibility is that those studies have used different cathepsin L and cathepsin D isoforms that are known to result in different products

[52] . Additionally, cathepsin L and cathepsin D have been shown to have the ability to cleave at proline, which is relatively rare across all proteases, with the protease EndoPro® being a notable exception

[64] .

[0130] To investigate the potential role of secondary and tertiary structure, cathepsin-induced cleavage sites on Abs were compared to their crystal structures (Figure 16). No cleavage was observed within any alpha helices, and most were located within random coils. The remainder of the cleavage sites were observed within a distance of 1–2 amino acids from the end of the random coil, but within the beginning of the beta sheet. The nature of these sites strongly suggests that cathepsin L and cathepsin D have a limited ability to cleave within ordered regions of Abs, but can bind to nearly any amino acid motif within flexible, disordered regions. The cleavage sites are further constrained by the disulfide bond pattern. Disulfide bonds impose significant constraints on the final tertiary structure, exhibiting rather low flexibility compared to non-bound regions

[65] . Although cathepsin enzymes may be promiscuous at the local amino acid level, local flexibility found outside the disulfide bonded regions may be required to position the antibody sequence to be cleaved inside the enzyme pocket. Interestingly, this conclusion is supported by a comparison of digestion efficiencies performed under different pH, thermal and organic conditions. Although different efficiencies were observed, the cleaved sites were found to be consistent across conditions. Despite possible denaturation at the secondary structure level, each treatment did not affect the disulfide bond pattern, indicating that this constraint is the major factor in determining the cleavage location. When digestion was extended to bispecific antibodies, CH2 / CH3 digestion in the anti-CD3 arm was prevented beyond the hinge region, in contrast to the standard IgG1 cathepsin L+D digestion pattern found across the anti-Her2 arm. Anti-Her2 cleavage occurred at locations with primary sequence and secondary structure homologous to the anti-CD3 chains, suggesting a role for local flexibility of sequence, domain orientation, or other tertiary (hole) effects in determining cathepsin L and cathepsin D digestion.

[0131] Standard conditions (pH7, 37 oComparison of the protein fragments identified in trastuzumab using optimized conditions (C, 2 days, 1:20 ratio) and optimized conditions showed interesting differences in their relative abundance (Tables 8-10). For example, at pH 7 the most abundant low molecular weight polypeptide is the 11,197 Da (HC:1-102 (YYCSRWGGD.F)) fragment, whereas at the optimal pH of 4 the most abundant fragment below 20 kDa is the 12,121 Da (LC:1-110 (RTVA.A)) product. One possibility is that cathepsin L and cathepsin D, when combined in a single pool, affect each other's activity by clipping the other enzyme when bound to Ab or through stoichiometric effects. No intact masses of cathepsin L or cathepsin D were observed during deconvolution. However, some of the observed masses of approximately 25 kDa may correspond to small clips of cathepsin L (30 kDa) or highly clipped forms of cathepsin D at approximately 45 kDa, which may explain some of the unidentified species that were deconvoluted in the clipping assessment.

[0132] The clipping patterns observed when using cathepsin L and cathepsin D suggest that cathepsin treatment may also be used as a relatively simple assay to check for disulfide bonds in Abs. Both cathepsins likely generate "single-arm" fragments of approximately 48 kDa for IgG1, but do not generate these fragments for IgG2-B Abs, which contain a distinct disulfide bond pattern. The IgG2-B pattern is difficult to confirm by non-reduced peptide mapping, most commonly done by Lys-C [66,67].

[0133] Here, we used high-resolution native MS and denaturing LC-MS in parallel on directly injected non-reduced Abs. This approach has the advantages of simplicity, minimal sample preparation, and mild source conditions with little or no in-source fragmentation. Reduction of disulfide bonds and deglycosylation can improve sensitivity and simplify mass spectra and data analysis, but add additional steps to the presented method. The use of software with the ability to automate intact assignments can help speed up the evaluation of new proteases and shows promise as a computational resource to evaluate native clipping within cells or antibody by-products. The method generates protein fragments of an ideal size for sequencing, achieves 100% coverage of Abs distributed across a limited number of protein fragments (9 species), and uses commercially available enzymes, making this workflow suitable as a robust and reproducible middle-down sequencing workflow. C. Method C.1. Chemicals and Materials

[0134] The therapeutic Abs, rituximab (MabThera®), obinutuzumab (Gazyva®) and eculizumab (Soliris®), were transfers from Genmab (Netherlands), while trastuzumab (Herceptin®) and anti-Her2 / anti-CD3 bispecifics were supplied in-house by Genentech, Inc. All Ab samples, except for the bispecifics, were from expired batches. Prior to use, Ab integrity was checked by native MS to confirm against any post-translational modifications or structural changes (checked by charge state distribution) and the Abs were expected to have high integrity. All amino acid sequences retrieved lacked the N-terminal signal peptide and are provided in Table 14. Dithiothreitol (DTT), iodoacetamide (IAA), ammonium acetate (AMAC), acetic acid, formic acid (FA), 8 M tris(hydroxymethyl)aminomethane (Tris), methanol (MeOH), and cathepsins L and D were purchased from Sigma-Aldrich (St Louis, MO), phosphate buffer was purchased from Lonza Group AG (Basel, CH), and acetonitrile (ACN) was purchased from Biosolve BV (North Brabant, NL) and Fisher Scientific (Hampton, NH). C.2. Antibody Digestion with Cathepsin L and Cathepsin D

[0135] To evaluate Ab motifs suitable for cleavage by cathepsin L and cathepsin D, digests were performed across all therapeutic Abs (rituximab, obinutuzumab, eculizumab, trastuzumab) in a single control condition. Abs were prepared at 5 μM in Milli-Q water and treated individually with cathepsin L or D at an enzyme-to-antibody ratio of 1:200. Samples were incubated at 37 °C for 2 days at neutral pH. Digested Ab samples were buffer exchanged into 150 mM aqueous AMAC (pH 7.5) by centrifugation using a 10 kDa cutoff filter (Merck Millipore, Burlington, MA). Final protein concentrations were measured by UV absorbance at 280 nm. Digests were adjusted to 2–3 μM and either used directly for native MS analysis or incubated overnight with 4 units of PNGase F using the standard native protocol

[22] . PNGase F-treated samples were buffer exchanged a second time into 150 mM AMAC (pH 7.5) prior to native MS measurements.

[0136] The cleavage efficiency of each cathepsin was then investigated under different digestion conditions using a single Ab, trastuzumab, in a stock solution of 2 mg / mL. Each cathepsin was resuspended in water at 1 mg / mL, and different pH, MeOH and temperature conditions were tested in triplicate (Table 15). Digests were prepared at an enzyme-to-protein ratio of 1:200 with a final Ab concentration of 0.2 mg / mL. For pH-adjusted preparations, a dilution buffer of 50 mM ammonium acetate at the desired pH was added, constituting 79% of the solution. For LC-UV-MS analysis, 15 μL was injected onto a 2.1×50 mm MAbPac™ RP HPLC column (80° C.) on an Ultimate 3000 LC coupled to a DAD detector (254 nm) and an Exactive™ EMR (Thermo Fisher Scientific, Waltham, MA). The flow rate was set at 300 μL / min, mobile phase A (MPA) was 99.88% water, 0.1% formic acid, and 0.02% trifluoroacetic acid, and B (MPB) was 90% ACN, 9.88% water, 0.02% trifluoroacetic acid, and 0.1% formic acid. MPB was increased from 5 to 20% in 1 min, to 65% in 9.5 min, to 90% in 10 min, and held at 90% for 2 min before re-equilibration.

[0137] The final one-pot reaction of L+D was evaluated for trastuzumab at pH 4.0 and temperature 37° C. for 18 hours. LC-UV-MS was used to assess the intact mass as described above. Top-down analysis was performed on a Q Exactive™ UHMR on samples buffer-exchanged directly into 50 mM ammonium acetate using a MicroBio-Spin column according to the manufacturer's instructions. Settings were adjusted as described in the static nESI MS section below. C.3. Static nESI MS of Cathepsin L and D Fragments

[0138] Samples were analyzed on a modified Exactive™ Plus Orbitrap instrument with extended mass range (EMR) (Thermo Fisher Scientific)

[68] or Q Exactive™ UHMR

[69] using an m / z range of either 500-12,000. The voltage offsets of the transport multipole and ion lenses were manually adjusted to achieve optimal transmission of protein ions at high m / z. Nitrogen was injected at 3-7 × 10 -10 A high-energy collision dissociation (HCD) cell was used at a gas pressure of 1 bar. MS parameters used were: spray voltage 1.2–1.3 V, source temperature 250 °C, source fragmentation and collision energies varied from 50 to 80 V, and resolution (m / z 200) 35,000 or 70,000 for all Abs. The instrument was mass calibrated using CsI solution as previously described

[22] . C.3. Edman Decomposition

[0139] Cathepsin-cleaved antibodies from the one-pot trastuzumab L+D digest were separated on 4-20% Novex™ Tris-Glycine SDS-PAGE gels (Thermo Fisher Scientific), electroblotted onto PVDF membranes, and then visualized with Coomassie Brilliant Blue R-250 staining. Bands of interest were excised and subjected to N-terminal sequence analysis using a 494 Procise™ sequencer (Applied Biosystems, Foster City, CA). The resulting mixture of sequences was analyzed using the sequencer-associated 610 software (Applied Biosystems, Foster City, CA) and manually verified. C.4. Antibody Digestion Product Data Analysis

[0140] Intact Mass™ v3.2-424 (October 2018) was used for charge deconvolution (Protein Metrics, San Carlos, CA). For the initial deconvolution, default parameters were used, including m / z range 600-9,000, m range 10,000-160,000, m / z spacing 0.04, m / z smoothing sigma 0.02, 0.2 charge spacing, and m spacing 0.5 and m smoothing sigma 3.0. For subsequent deconvolutions, parameters were adjusted to obtain the highest quality results. In all cases, the minimum mass peak difference was 15 and the peak detector sigma was 5 (1 / 3 of the minimum mass peak difference). A mass matching tolerance of 4 Da was applied for automatic peak annotation.

[0141] Details of the Intact Mass™ program, which became commercially available in 2019, are summarized here, as the clipped species matching algorithm has not been described in the scientific literature. After charge deconvolution [27,59], the algorithm picks peaks in the neutral mass spectrum, in descending order of intensity, using a “Mexican hat” peak detection filter to deprioritize masses found in the dense shoulders of peaks. Settings can be customized to specify the peak detector width (standard deviation of the positive part of the filter, default = 5 Da), mass range, maximum number, minimum mass interval, minimum percentage of base peaks, and minimum signal-to-noise ratio of the selected peaks.

[0142] The deconvoluted and selected peaks were matched to the theoretical mean isotopic mass calculated from the input amino acid sequence (including multiple chains) and a table of natural isotope abundances. 13 The C abundance (1.079%) was determined. The average mass was used to avoid off-by-one Dalton errors of the monoisotopic mass and to provide uniformity across mass spectra that may contain a mixture of isotopically resolved and unresolved masses.

[0143] All peptide bonds in either the light or heavy chain were considered potential clip sites, rather than restricting cleavage to preferred amino acids. In the matching algorithm, suffixes (sequences that include the C-terminus of the chain but not the N-terminus) starting with Q were not assumed to start with pyro-Glu, whereas prefixes (sequences that include the N-terminus of the chain but not the C-terminus) sequences were. C (cysteine) is assumed to be disulfide bonded by default, odd-numbered single Cs are left reduced, and Intact Mass subtracts about 1 Da without trying to predict the pattern. The algorithm is a simple greedy algorithm

[70] : each selected peak is matched to the closest theoretical mass within a set mass tolerance. Except for the special case of identical chains cleaved at the same position, Intact Mass calculates the prefix or suffix sequence by cleaving only a single chain and summing it with the other intact second chain. For intact mAbs, two LCs and two HCs were linked such that the intact mass generates a prefix and a suffix by clipping a single chain and leaving the other three chains intact. The software also considers two identical chains cleaved at the same position, e.g., F(ab')2 fragments generated by IdeS protease cleaving between the Gs in CPPCPAPELLG.GPS. "Half-Abs" formed by a single LC+HC are automatically considered for clipping.

[0144] All assignments were manually verified, and all unassigned species were manually assessed to determine whether the algorithm had missed an assignment. D. References 1.Vandermarliere, E., Stes, E., Gevaert, K. & Martens, L. (2016) Resolution of protein structure by mass spectrometry, Mass Spectrom Rev.35, 653-665. 2.Rathore,D.,Faustino,A.,Schiel,J.,Pang,E.,Boyne,M.&Rogstad,S.(2018)The role of mass spectrometry in the characterization of biologic protein products,Expert Review of Proteomics.15,431-449. 3.Muth,T.,Hartkopf,F.,Vaudel,M.&Renard,B.Y.(2018)A Potential Golden Age to Come-Current Tools,Recent Use Cases,and Future Avenues for De Novo Sequencing in Proteomics,Proteomics.18,1700150. 4.Standing,K.G.(2003)Peptide and protein de novo sequencing by mass spectrometry,Curr Opin Struct Biol.13,595-601. 5.(1999)De Novo Peptide Sequencing via Tandem Mass Spectrometry,J Comput Biol.6,327-342. 6.Ma,B.,Zhang,K.,Hendrie,C.,Liang,C.,Li,M.,Doherty-Kirby,A.&Lajoie,G.(2003)PEAKS:powerful software for peptide de novo sequencing by tandem mass spectrometry,Rapid Commun Mass Spectrom.17,2337-2342. 7.Seidler,J.,Zinn,N.,Boehm,M.E.&Lehmann,W.D.(2010)De novo sequencing of peptides by MS / MS,Proteomics.10,634-649. 8.Liu,P.,Zhu,X.,Wu,W.,Ludwig,R.,Song,H.,Li,R.,Zhou,J.,Tao,L.&Leone,A.M.(2019)Subunit mass analysis for monitoring multiple attributes of monoclonal antibodies,Rapid Commun Mass Spectrom.33,31-40. 9.Srzentic,K.,Nagornov,K.O.,Fornelli,L.,Lobas,A.A.,Ayoub,D.,Kozhinov,A.N.,Gasilova,N.,Menin,L.,Beck,A.,Gorshkov,M.V.,Aizikov,K.&Tsybin,Y.O.(2018)Multiplexed Middle-Down Mass Spectrometry as a Method for Revealing Light and Heavy Chain Connectivity in a Monoclonal Antibody,Anal Chem.90,12527-12535. 10.Pandeswari,P.B.&Sabareesh,V.(2019)Middle-down approach:a choice to sequence and characterize proteins / proteomes by mass spectrometry,RSC Advances.9,313-344. 11.Tsiatsiani,L.&Heck,A.J.R.(2015)Proteomics beyond trypsin,The FEBS Journal.282,2612-2626. 12.Vincents,B.,von Pawel-Rammingen,U.,Bjorck,L.&Abrahamson,M.(2004)Enzymatic Characterization of the Streptococcal Endopeptidase,IdeS,Reveals That It Is a Cysteine Protease with Strict Specificity for IgG Cleavage Due to Exosite Binding,Biochemistry.43,15540-15549. 13.von Pawel-Rammingen,U.,Johansson,B.P.&Bjorck,L.(2002)IdeS,a novel streptococcal cysteine proteinase with unique specificity for immunoglobulin G,The EMBO journal.21,1607-1615. 14.Fornelli,L.,Ayoub,D.,Aizikov,K.,Liu,X.,Damoc,E.,Pevzner,P.A.,Makarov,A.,Beck,A.&Tsybin,Y.O.(2017)Top-down analysis of immunoglobulin G isotypes 1 and 2 with electron transfer dissociation on a high-field Orbitrap mass spectrometer,J Proteomics.159,67-76. 15.Srzentic,K.,Zhurov,K.O.,Lobas,A.A.,Nikitin,G.,Fornelli,L.,Gorshkov,M.V.&Tsybin,Y.O.(2018)Chemical-Mediated Digestion:An Alternative Realm for Middle-down Proteomics?,J Proteome Res.17,2005-2016. 16.McCool,E.N.,Chen,D.,Li,W.,Liu,Y.&Sun,L.(2019)Capillary zone electrophoresis-tandem mass spectrometry with ultraviolet photodissociation(213 nm)for large-scale top-down proteomics,Analytical Methods.11,2855-2861. 17.Cannon,J.R.,Cammarata,M.B.,Robotham,S.A.,Cotham,V.C.,Shaw,J.B.,Fellers,R.T.,Early,B.P.,Thomas,P.M.,Kelleher,N.L.&Brodbelt,J.S.(2014)Ultraviolet photodissociation for characterization of whole proteins on a chromatographic time scale,Anal Chem.86,2185-2192. 18.Shaw,J.B.,Malhan,N.,Vasil’ev,Y.V.,Lopez,N.I.,Makarov,A.,Beckman,J.S.&Voinov,V.G.(2018)Sequencing Grade Tandem Mass Spectrometry for Top-Down Proteomics Using Hybrid Electron Capture Dissociation Methods in a Benchtop Orbitrap Mass Spectrometer,Anal Chem.90,10819-10827. 19.Shaw,J.B.,Li,W.,Holden,D.D.,Zhang,Y.,Griep-Raming,J.,Fellers,R.T.,Early,B.P.,Thomas,P.M.,Kelleher,N.L.&Brodbelt,J.S.(2013)Complete protein characterization using top-down mass spectrometry and ultraviolet photodissociation,J Am Chem Soc.135,12646-12651. 20.Catherman,A.D.,Skinner,O.S.&Kelleher,N.L.(2014)Top Down proteomics:facts and perspectives,Biochem Biophys Res Commun.445,683-693. 21.Tran,J.C.,Zamdborg,L.,Ahlf,D.R.,Lee,J.E.,Catherman,A.D.,Durbin,K.R.,Tipton,J.D.,Vellaichamy,A.,Kellie,J.F.,Li,M.,Wu,C.,Sweet,S.M.M.,Early,B.P.,Siuti,N.,LeDuc,R.D.,Compton,P.D.,Thomas,P.M.&Kelleher,N.L.(2011)Mapping intact protein isoforms in discovery mode using top-down proteomics,Nature.480,254-258. 22.Rosati,S.,Yang,Y.,Barendregt,A.&Heck,A.J.R.(2014)Detailed mass analysis of structural heterogeneity in monoclonal antibodies using native mass spectrometry,Nat Protoc.9,967-976. 23.Bailey,A.O.,Han,G.,Phung,W.,Gazis,P.,Sutton,J.,Josephs,J.L.&Sandoval,W.(2018)Charge variant native mass spectrometry benefits mass precision and dynamic range of monoclonal antibody intact mass analysis,Mabs-Austin.10,1214-1225. 24.Phung,W.,Han,G.,Polderdijk,S.G.I.,Dillon,M.,Shatz,W.,Liu,P.,Wei,B.,Suresh,P.,Fischer,D.,Spiess,C.,Bailey,A.,Carter,P.J.,Lill,J.R.&Sandoval,W.(2019)Characterization of bispecific and mispaired IgGs by native charge-variant mass spectrometry,Int J Mass spectrom.446,116229. 25.Caval,T.,Tian,W.,Yang,Z.,Clausen,H.&Heck,A.J.R.(2018)Direct quality control of glycoengineered erythropoietin variants,Nat Commun.9,3342. 26.Wohlschlager,T.,Scheffler,K.,Forstenlehner,I.C.,Skala,W.,Senn,S.,Damoc,E.,Holzmann,J.&Huber,C.G.(2018)Native mass spectrometry combined with enzymatic dissection unravels glycoform heterogeneity of biopharmaceuticals,Nat Commun.9,1713. 27.Campuzano,I.D.G.,Robinson,J.H.,Hui,J.O.,Shi,S.D.H.,Netirojjanakul,C.,Nshanian,M.,Egea,P.F.,Lippens,J.L.,Bagal,D.,Loo,J.A.&Bern,M.(2019)Native and Denaturing MS Protein Deconvolution for Biopharma:Monoclonal Antibodies and Antibody-Drug Conjugates to Polydisperse Membrane Proteins and Beyond,Anal Chem.91,9472-9480. 28.Marcoux,J.,Wang,S.C.,Politis,A.,Reading,E.,Ma,J.,Biggin,P.C.,Zhou,M.,Tao,H.,Zhang,Q.,Chang,G.,Morgner,N.&Robinson,C.V.(2013)Mass spectrometry reveals synergistic effects of nucleotides,lipids,and drugs binding to a multidrug resistance efflux pump,P Natl Acad Sci USA.110,9704-9709. 29.Yang,Y.,Liu,F.,Franc,V.,Halim,L.A.,Schellekens,H.&Heck,A.J.R.(2016)Hybrid mass spectrometry approaches in glycoprotein analysis and their usage in scoring biosimilarity,Nat Commun.7,13397. 30.Greisch,J.-F.,Tamara,S.,Scheltema,R.A.,Maxwell,H.W.R.,Fagerlund,R.D.,Fineran,P.C.,Tetter,S.,Hilvert,D.&Heck,A.J.R.(2019)Expanding the mass range for UVPD-based native top-down mass spectrometry,Chemical Science.10,7163-7171. 31.Compton,P.D.,Zamdborg,L.,Thomas,P.M.&Kelleher,N.L.(2011)On the scalability and requirements of whole protein mass spectrometry,Anal Chem.83,6868-6874. 32.McLuckey,S.A.&Stephenson,J.L.(1998)Ion / ion chemistry of high-mass multiply charged ions,Mass Spectrom Rev.17,369-407. 33.Ge,Y.,Lawhorn,B.G.,ElNaggar,M.,Strauss,E.,Park,J.-H.,Begley,T.P.&McLafferty,F.W.(2002)Top Down Characterization of Larger Proteins(45 kDa)by Electron Capture Dissociation Mass Spectrometry,J Am Chem Soc.124,672-678. 34.Reid,G.E.,Wu,J.,Chrisman,P.A.,Wells,J.M.&McLuckey,S.A.(2001)Charge-State-Dependent Sequence Analysis of Protonated Ubiquitin Ions via Ion Trap Tandem Mass Spectrometry,Anal Chem.73,3274-3281. 35. Zhang, H., Cui, W., Wen, J., Blankenship, R. E. & Gross, M. L. (2011) Native Electrospray and Electron-Capture Dissociation FTICR Mass Spectrometry for Top-Down Studies of Protein Assemblies, Anal Chem. 83, 5598 - 5606. 36. Syka, J. E. P., Coon, J. J., Schroeder, M. J., Shabanowitz, J. & Hunt, D. F. (2004) Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry, P Natl Acad Sci USA. 101, 9528 - 9533. 37. Moelleken, J., Endesfelder, M., Gassner, C., Lingke, S., Tomaschek, S., Tyshchuk, O., Lorenz, S., Reiff, U. & Molhoj, M. (2017) GingisKHAN™ protease cleavage allows a high-throughput antibody to Fab conversion enabling direct functional assessment during lead identification of human monoclonal and bispecific IgG1 antibodies, Mabs-Austin. 9, 1076 - 1087. 38.Zhang,L.,English,A.M.,Bai,D.L.,Ugrin,S.A.,Shabanowitz,J.,Ross,M.M.,Hunt,D.F.&Wang,W.-H.(2016)Analysis of Monoclonal Antibody Sequence and Post-translational Modifications by Time-controlled Proteolysis and Tandem Mass Spectrometry,Molecular&cellular proteomics:MCP.15,1479-1488. 39.Mao,Y.,Zhang,L.,Kleinberg,A.,Xia,Q.,Daly,T.J.&Li,N.(2019)Fast protein sequencing of monoclonal antibody by real-time digestion on emitter during nanoelectrospray,Mabs-Austin.11,767-778. 40.Jones,R.G.A.&Landon,J.(2002)Enhanced pepsin digestion:a novel process for purifying antibody F(ab’)2 fragments in high yield from serum,J Immunol Methods.263,57-74. 41.Wu,C.,Tran,J.C.,Zamdborg,L.,Durbin,K.R.,Li,M.,Ahlf,D.R.,Early,B.P.,Thomas,P.M.,Sweedler,J.V.&Kelleher,N.L.(2012)A protease for ’middle-down’ proteomics,Nat Methods.9,822-824. 42.Srzentic,K.,Fornelli,L.,Laskay,U.A.,Monod,M.,Beck,A.,Ayoub,D.&Tsybin,Y.O.(2014)Advantages of Extended Bottom-Up Proteomics Using Sap9 for Analysis of Monoclonal Antibodies,Anal Chem.86,9945-9953. 43.Smith,S.M.&Gottesman,M.M.(1989)Activity and deletion analysis of recombinant human cathepsin L expressed in Escherichia coli,J Biol Chem.264,20487-95. 44.Kirschke,H.,Kembhavi,A.A.,Bohley,P.&Barrett,A.J.(1982)Action of rat liver cathepsin L on collagen and other substrates,The Biochemical journal.201,367-372. 45.Press,E.M.,Porter,R.R.&Cebra,J.(1960)The isolation and properties of a proteolytic enzyme,cathepsin D,from bovine spleen,The Biochemical journal.74,501-514. 46.Brix,K.(2005)Lysosomal Proteases:Revival of the Sleeping Beauty.in Madame Curie Bioscience Database(Saftig,P.,ed),Landes Bioscience,Austin(TX). 47.Dunn,A.D.,Crutchfield,H.E.&Dunn,J.T.(1991)Thyroglobulin processing by thyroidal proteases.Major sites of cleavage by cathepsins B,D,and L,J Biol Chem.266,20198-204. 48.Biniossek,M.L.,Nagler,D.K.,Becker-Pauly,C.&Schilling,O.(2011)Proteomic Identification of Protease Cleavage Sites Characterizes Prime and Non-prime Specificity of Cysteine Cathepsins B,L,and S,J Proteome Res.10,5363-5373. 49.Gosalia,D.N.,Salisbury,C.M.,Ellman,J.A.&Diamond,S.L.(2005)High Throughput Substrate Specificity Profiling of Serine and Cysteine Proteases Using Solution-phase Fluorogenic Peptide Microarrays,Molecular&Cellular Proteomics.4,626-636. 50.Puzer,L.,Cotrin,S.S.,Alves,M.F.M.,Egborge,T.,Araujo,M.S.,Juliano,M.A.,Juliano,L.,Bromme,D.&Carmona,A.K.(2004)Comparative substrate specificity analysis of recombinant human cathepsin V and cathepsin L,Arch Biochem Biophys.430,274-283. 51.Hu,Y.,Morioka,K.&Itoh,Y.(2007)Existence of Cathepsin L and its Chracterization in Red Bulleye Surimi.,Pakistan Journal of Biological Sciences.10,78-83. 52.Luo,H.B.,Tie,L.,Cao,M.Y.,Hunter,A.K.,Pabst,T.M.,Du,J.L.,Field,R.,Li,Y.L.&Wang,W.K.(2019)Cathepsin L Causes Proteolytic Cleavage of Chinese-Hamster-Ovary Cell Expressed Proteins During Processing and Storage:Identification,Characterization,and Mitigation,Biotechnol Progr.35. 53.Tang,J.&Wong,R.N.S.(1987)Evolution in the structure and function of aspartic proteases,J Cell Biochem.33,53-63. 54.Sun,H.,Lou,X.,Shan,Q.,Zhang,J.,Zhu,X.,Zhang,J.,Wang,Y.,Xie,Y.,Xu,N.&Liu,S.(2013)Proteolytic Characteristics of Cathepsin D Related to the Recognition and Cleavage of Its Target Proteins,PLOS ONE.8,e65733. 55.van Noort,J.M.&van der Drift,A.C.(1989)The selectivity of cathepsin D suggests an involvement of the enzyme in the generation of T-cell epitopes,J Biol Chem.264,14159-14164. 56.Woessner Jr.,J.(1977)Specificity and biological role of cathepsin D.,Adv Exp Med Biol.95,313-327. 57.Bee,J.S.,Tie,L.,Johnson,D.,Dimitrova,M.N.,Jusino,K.C.&Afdahl,C.D.(2015)Trace levels of the CHO host cell protease cathepsin D caused particle formation in a monoclonal antibody product,Biotechnol Progr.31,1360-1369. 58.Zhang,Z.,Pan,H.&Chen,X.(2009)Mass spectrometry for structural characterization of therapeutic antibodies,Mass Spectrom Rev.28,147-176. 59.Bern,M.,Caval,T.,Kil,Y.J.,Tang,W.,Becker,C.,Carlson,E.,Kletter,D.,Sen,K.I.,Galy,N.,Hagemans,D.,Franc,V.&Heck,A.J.R.(2018)Parsimonious Charge Deconvolution for Native Mass Spectrometry,J Proteome Res.17,1216-1226. 60.Terra,W.R.,Dias,R.O.&Ferreira,C.(2019)Recruited lysosomal enzymes as major digestive enzymes in insects,Biochem Soc Trans.47,615-623. 61.Honey,K.&Rudensky,A.Y.(2003)Lysosomal cysteine proteases regulate antigen presentation,Nature Reviews Immunology.3,472-482. 62.Fellers,R.T.,Greer,J.B.,Early,B.P.,Yu,X.,LeDuc,R.D.,Kelleher,N.L.&Thomas,P.M.(2015)ProSight Lite:graphical software to analyze top-down mass spectrometry data,Proteomics.15,1235-1238. 63.Merchant,A.M.,Zhu,Z.,Yuan,J.Q.,Goddard,A.,Adams,C.W.,Presta,L.G.&Carter,P.(1998)An efficient route to human bispecific IgG,Nat Biotechnol.16,677-681. 64.van der Laarse,S.A.M.,van Gelder,C.A.G.H.,Bern,M.,Akeroyd,M.,Olsthoorn,M.M.A.&Heck,A.J.R.Targeting proline in(phospho)proteomics,The FEBS Journal.n / a. 65.Isenman,D.E.,Dorrington,K.J.&Painter,R.H.(1975)The Structure and Function of Immunoglobulin Domains,II The Importance of Interchain Disulfide Bonds and the Possible Role of Molecular Flexibility in the Interaction Between Immunoglobulin G and Complement.114,1726-1729. 66.Wypych,J.,Li,M.,Guo,A.,Zhang,Z.,Martinez,T.,Allen,M.J.,Fodor,S.,Kelner,D.N.,Flynn,G.C.,Liu,Y.D.,Bondarenko,P.V.,Ricci,M.S.,Dillon,T.M.&Balland,A.(2008)Human IgG2 antibodies display disulfide-mediated structural isoforms,The Journal of biological chemistry.283,16194-16205. 67.Resemann,A.,Liu-Shin,L.,Tremintin,G.,Malhotra,A.,Fung,A.,Wang,F.,Ratnaswamy,G.&Suckau,D.(2018)Rapid,automated characterization of disulfide bond scrambling and IgG2 isoform determination,Mabs-Austin.10,1200-1213. 68.Rose,R.J.,Damoc,E.,Denisov,E.,Makarov,A.&Heck,A.J.R.(2012)High-sensitivity Orbitrap mass analysis of intact macromolecular assemblies,Nat Methods.9,1084-1086. 69.van de Waterbeemd,M.,Fort,K.L.,Boll,D.,Reinhardt-Szyba,M.,Routh,A.,Makarov,A.&Heck,A.J.R.(2017)High-fidelity mass analysis unveils heterogeneity in intact ribosomal particles,Nat Methods.14,283-286. 70. Temlyakov, V. (2008) Greedy Approximation in Acta Numerica (Iserles, A., ed) pp. 235 - 409, Cambridge University Press, Cambridge, England. 71. Thomsen, M. C. F. & Nielsen, M. (2012) Seq2Logo: a method for construction and visualization of amino acid binding motifs and sequence profiles including sequence weighting, pseudo counts and two - sided representation of amino acid enrichment and depletion, Nucleic Acids Res. 40, W281 - W287. Table E [Table 1] [Table 2] [Table 3] [Table 4] JPEG2025011140000006.jpg 231170 JPEG2025011140000007.jpg 38170 [Table 5a] [Table 5b] [Table 6] [Table 7] [Table 8]

Table 9

Table 10

Table 11

Table 12

Table 13

Table 14

Table 15

Claims

1. 1. A method for cleaving an antibody, comprising: mixing the antibody with cathepsin L, cathepsin D, or a combination of cathepsin L and cathepsin D to obtain one or more antibody fragments, wherein the antibody comprises a light chain comprising a light chain variable region (VL) and a light chain constant region (CL), and a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region (CH), wherein the cathepsin L and / or the cathepsin D cleaves the antibody between the VL region and the CL region and / or between the VH region and the CH region to produce VL antibody fragments and / or VH antibody fragments, and CL antibody fragments and / or CH antibody fragments, to obtain one or more antibody fragments, and optionally isolating one or more of the antibody fragments after the cleavage.

2. 1. A method for analyzing an antibody sequence, comprising: a. cleaving the antibody with cathepsin L, cathepsin D, or a combination of cathepsin L and cathepsin D to obtain one or more antibody fragments, wherein the antibody comprises a light chain comprising a light chain variable region (VL) and a light chain constant region (CL), and a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region (CH), and the cathepsin L and / or the cathepsin D cleaves the antibody between the VL region and the CL region and / or between the VH region and the CH region to generate VL antibody fragments and / or VH antibody fragments and CL antibody fragments and / or CH antibody fragments; b. optionally, isolating one or more of said antibody fragments after said cleavage; and c. performing mass spectrometry (MS) of said one or more antibody fragments. A method comprising:

3. The method of claim 1 or 2, wherein the antibody is an IgG antibody, such as a human IgG1 antibody, an IgG2 antibody, an IgG2A antibody, an IgG2B antibody or an IgG4 antibody.

4. The method of claim 1 , 2 or 3, wherein the cleavage generates a VL fragment and / or a VH fragment.

5. The method of claim 4, wherein MS analysis is performed on the VL fragment and / or the VH fragment.

6. The method of any one of claims 1 to 5, wherein the heavy chain constant region comprises at least a CH1 region, and optionally further comprises a hinge, a CH2 region and / or a CH3 region.

7. The method of any one of claims 1 to 6, wherein the antibody heavy chain constant region comprises at least a CH1 region, a hinge and a CH2 region, and the cathepsin L and / or the cathepsin D further cleaves the antibody between the CH1 region and the hinge.

8. The method of any one of claims 1 to 7, wherein the antibody is cleaved by a combination of cathepsin L and cathepsin D.

9. The method of claim 8, comprising incubating the antibody with both cathepsin L and cathepsin D simultaneously.

10. 10. The method of any one of claims 1 to 9, wherein cleavage is performed to achieve at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% cleavage between the VL and CL regions and between the VH and CH regions.

11. 11. The method of any one of claims 1 to 10, wherein the severing is carried out to achieve at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% severing at or below the hinge.

12. 12. The method of any one of claims 1 to 11, wherein the cleavage is performed at a ratio of cathepsin L and / or cathepsin D to antibody of 1:20 to 1:2000, 1:20 to 1:500, 1:50 to 1:500, 1:100 to 1:500, 1:200 to 1:1000, 1:200 to 1:2000, 1:500 to 1:2000, 1:1000 to 1:2000, or 1:20, 1:50, 1:100, 1:200, 1:300, 1:400, 1:500, or 1:1000.

13. 13. The method of any one of claims 1 to 12, wherein the antibody is an IgG antibody and is cleaved by a combination of cathepsin L and cathepsin D, and wherein the cleavage results in a VL fragment, a VH fragment, a CL fragment, a CH1 fragment, a CH2 fragment, a CH3 fragment, a CH2-CH3 fragment, a CL+CH1 (combined) fragment, a F(ab') fragment and a F(ab')2 fragment.

14. 14. The method of any one of claims 1 to 13, wherein cleavage is carried out by incubating the antibody with cathepsin L, cathepsin D, or a combination of cathepsin D and cathepsin L at pH 2-8 (e.g. pH 2-7, pH 2-6, pH 3-6, pH 3-5, pH 2, pH 2.5, pH 3, pH 3.5, pH 4, pH 4.5, pH 5, pH 5.5, pH 6, pH 7 or pH 8), at a temperature between room temperature and 50°C, in the presence of up to 50% organic solvent (e.g. acetonitrile, methanol, ethanol or isopropyl alcohol), and wherein the antibody is in a native state.

15. 15. The method of claim 14, wherein the pH is between 3 and 5, such as 3, 3.5, 4, 4.5 or 5.

16. The method of claim 14, wherein the pH is from 3.5 to 4.

5.

17. 15. The method of claim 14, wherein the pH is 4.

18. The method of any one of claims 14 to 17, wherein no more than 30% organic solvent is present.

19. The method of claims 14 to 18, wherein the cleavage is carried out in the presence of 10-30% organic solvent (e.g. 10-30% acetonitrile, 10-30% methanol, 10-30% ethanol or 10-30% isopropyl alcohol).

20. The method of any one of claims 14 to 19, wherein no more than 10% organic solvent is present.

21. The method according to any one of claims 14 to 20, wherein the temperature is between 37°C and 50°C.

22. 1. A method for analyzing an antibody sequence, comprising: a. cleaving the antibody with a combination of cathepsin L and cathepsin D to obtain one or more antibody fragments comprising at least a light chain variable region (VL) fragment and / or a heavy chain variable region (VH) fragment; i. the antibody is in its native state; ii. the antibody comprises a light chain comprising a light chain variable region (VL) and a light chain constant region (CL) and a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region (CH), and the cathepsin L and the cathepsin D cleave the antibody between the VL region and the CL region and / or between the VH region and the CH region to produce a VL antibody fragment and / or a VH antibody fragment and a CL antibody fragment and / or a CH antibody fragment; iii. obtaining one or more antibody fragments, wherein the cleavage is carried out at a temperature between 25°C and 50°C and a pH between 3 and 5, in the presence of up to 30% organic solvent; b. optionally, isolating one or more of said antibody fragments after said cleavage; and c. performing mass spectrometry (MS) of said one or more antibody fragments. A method comprising:

23. 23. The method of claim 22, wherein the cleavage is performed at a ratio of cathepsin L and / or cathepsin D to antibody of 1:20 to 1:2000, 1:20 to 1:500, 1:50 to 1:500, 1:100 to 1:500, 1:200 to 1:1000, 1:200 to 1:2000, 1:500 to 1:2000, 1:1000 to 1:2000, or 1:20, 1:50, 1:100, 1:200, 1:300, 1:400, 1:500, or 1:1000.

24. 24. The method of any one of claims 1 to 23, wherein after cleavage, the one or more antibody fragments are subjected to one or more of buffer exchange, chromatography (e.g. liquid chromatography such as high performance liquid chromatography or capillary electrophoresis), filtration (e.g. molecular weight cut-off filtration), reduction of disulfide bonds, exposure to guanidine hydrochloride, or alkylation.

25. The method of any one of claims 1 to 24, wherein the one or more antibody fragments after cleavage are isolated by chromatography or filtration.

26. 26. The method of claim 25, wherein the one or more antibody fragments are isolated by liquid chromatography.

27. The method of any one of claims 2 to 26, wherein the mass spectrometry (MS) comprises LC-MS or LC-MS / MS.

28. The method of any one of claims 1 to 24, wherein the one or more antibody fragments are not isolated after cleavage.

29. The method of any one of claims 2 to 28, wherein the mass spectrometry comprises direct infusion mass spectrometry (DIMS), static spray infusion mass spectrometry, or flow injection mass spectrometry.

30. The method according to any one of claims 2 to 29, wherein said mass spectrometry data is used to determine the amino acid sequence of at least a 10 amino acid stretch of an antibody fragment.

31. 31. The method of claim 30, wherein the amino acid sequence of at least a 15 amino acid stretch of an antibody fragment is determined.

32. 32. The method of claim 31 , wherein the amino acid sequence of at least a 20 amino acid stretch of an antibody fragment is determined.

33. The method of any one of claims 30 to 32, wherein the amino acid sequence of at least one antibody CDR region is determined.

34. The method of claim 33, wherein the sequences of CDR1, CDR2 and CDR3 of the VH and / or the VL are determined.

35. The method according to any one of claims 2 to 34, wherein the complete amino acid sequence of at least one antibody fragment, such as the VH and / or VL fragment, is determined.

36. The method of any one of claims 30 to 35, wherein the sequence is determined by top-down analysis.

37. 37. The method of claim 36, wherein the sequence is further analyzed or confirmed by bottom-up analysis.

38. The method according to any one of claims 1 to 37, wherein the amino acid sequence of the antibody VH region and / or VL region is unknown.

39. The method of any one of claims 1 to 37, wherein the amino acid sequence of the antibody is unknown.

40. 40. The method of any one of claims 2 to 39, comprising performing MS on the CL region and / or the CH region of the antibody, e.g. the CL fragment, the CH1 fragment, the CH2 fragment and / or the CH3 fragment generated from the cleavage.

41. The method of any one of claims 1 to 40, further comprising performing Edman degradation on at least one antibody fragment.

42. The method of any one of claims 1 to 41, wherein the antibody remains in its native state after said cleavage.

43. The method of any one of claims 1 to 42, wherein the antibody is not treated with a denaturing agent or an agent that reduces disulfide bonds during or after the cleavage.

44. The method of any one of claims 1 to 43, wherein the antibody retains its disulfide bonds during or after the cleavage.

45. 45. The method of any one of claims 1 to 44, comprising performing mass spectrometry (MS) of one or more antibody fragments after said cleavage, wherein said antibody remains in its native state and is not treated with denaturing agents or agents that reduce disulfide bonds prior to said MS analysis.

46. A composition comprising an antibody fragment produced according to the method of any one of claims 1 to 45.

47. 1. A composition comprising an IgG antibody fragment generated by cleavage with cathepsin L, cathepsin D, or a combination of cathepsin L and cathepsin D, wherein the antibody fragment comprises one or both of a VH fragment and a VL fragment, and at least one, at least two, or at least three of the following fragments: a CL fragment, a CH1 fragment, a CH2 fragment, a CH3 fragment, a CH2-CH3 fragment, a CL+CH1 (joint) fragment, an F(ab') fragment, and an F(ab')2 fragment.

48. 48. The composition of claim 47, wherein the VH fragment and / or the VL fragment comprises a length of 90 to 150 amino acids, such as 95 to 140 amino acids, such as 100 to 140 amino acids, or such as 100 to 120 amino acids.

49. 48. The composition of claim 47, wherein the VH fragment and / or the VL fragment have a molecular mass of 10-16 kDa, such as 10-13 kDa, such as 10-12 kDa, such as 10-11 kDa, or such as 11-12 kDa.

50. A kit for digesting a protein with cathepsin L, cathepsin D, or a combination of cathepsin L and cathepsin D, comprising (a) cathepsin L and / or cathepsin D, (b) one or more reaction buffers, and optionally (c) instructions for use in digesting a protein.

51. A kit according to claim 50, capable of cleaving an antibody according to the method of any one of claims 1 to 45.

52. 52. The kit of claim 50 or 51, wherein the reaction buffer is pH 2-8, pH 2-7, pH 2-6, pH 2-5, pH 3-6, pH 3-5, pH 3-4, pH 4-5, pH 2, pH 2.5, pH 3, pH 3.5, pH 4, pH 4.5, pH 5, pH 5.5, pH 6, pH 7 or pH 8, and optionally the reaction buffer comprises one or more organic solvents (e.g. methanol, ethanol, isopropyl alcohol, or acetonitrile) at a concentration of 0-50%, 5-50%, 5-30%, 0-30%, 10-30%, 0-10%, 5-15%, 10-20%, 15-25%, or 20-30%.

53. 53. The kit of claim 50, 51 or 52, wherein the reaction buffer is at pH 3, 3.5, 4, 4.5 or 5.

54. 54. The kit of claim 53, wherein the reaction buffer is at pH 4.

Images