WO2021180063A1 - Procédés de détection et de quantification de modifications biologiques dans des anticorps - Google Patents
Procédés de détection et de quantification de modifications biologiques dans des anticorps Download PDFInfo
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- WO2021180063A1 WO2021180063A1 PCT/CN2021/079731 CN2021079731W WO2021180063A1 WO 2021180063 A1 WO2021180063 A1 WO 2021180063A1 CN 2021079731 W CN2021079731 W CN 2021079731W WO 2021180063 A1 WO2021180063 A1 WO 2021180063A1
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6806—Determination of free amino acids
- G01N33/6812—Assays for specific amino acids
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4716—Complement proteins, e.g. anaphylatoxin, C3a, C5a
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
- G01N2440/24—Post-translational modifications [PTMs] in chemical analysis of biological material hydroxylation
Definitions
- the present invention relates to a hydroxyproline-containing therapeutic antibody or therapeutically active antigen binding fragment thereof which binds to C5aR, in particular to human C5aR, and a method for detecting and determining the site and the amount ofat least onebiologicalmodification within the antibody or therapeutically active antigen binding fragment.
- a hydroxyproline-containing antibody or therapeutically active antigen binding fragment with at least one proline partially modified by the attachment of a hydroxy group.
- the complement system is a crucial component of the host response to infection and tissue damage.
- the complement system can be activated via three pathways: the classic, the lectin and the alternative pathway, all of which converge at C3.
- Activation of the complement pathway produces a variety of biologically active fragments of complement proteins, such as anaphylatoxins including C3a, C4a and C5aand membrane attack complex (MAC) C5b-9, all of which maintain homeostasis in the body by, for example, defending the body from infection.
- the complex membrane attack complex (MAC) produces holes in the cell membrane, causing the destruction of target cells.
- Complement fragments such as C3a, C4a, and C5a trigger inflammation as anaphylatoxins and chemotactic factors (Takahiko Horiuchi &Hiroshi Tsukamoto, Inflammation and Regeneration, 2016, Vol 36, Article number: 11) .
- the complement system is a central component of innate immunity and bridges the innate to the adaptive immune response, abnormal complement activation leads to various inflammatory diseases. Modulation of the complement system has been recognized as a promising strategy in drug discovery, and a large number of therapeutic modalities have been developed.
- C5a The complement activation product, C5a, is a key factor for regulation of inflammatory responses, which function throughbindingtoits receptors expressed on a variety of cell surfaces including C5a receptor (C5aR) and C5a receptor-like 2 (C5L2) .
- C5a receptor also known as C5aanaphylatoxin chemotactic receptor 1 (C5aR1) or CD88 (Cluster of Differentiation 88) is a classical G-protein coupled receptors (GPCR) that bindsC5a with high affinity (K d ⁇ 1 nM) , whereas the functional roles of C5L2 remain controversial (Andreas Klos, et al, Mol Immunol.
- C5aR is expressed in various cell types. Highest C5aR expression levels are described for neutrophils. Low-to-moderate expression levels have been shown for macrophages/monocytes, dendritic cells, mast cells, eosinophils, lung vascular smooth muscle cells, astrocytes, microglia, osteoblasts, osteoclasts, epithelial and endothelial cells (Monk PN et al., Br J Pharmacol. 2007, 152: 429 –448; Wetsel RA, Immunol Lett. 1995, 44: 183 –187) . For human T cells, low expression levels have been observed (Nataf S, J Immunol. 1999, 162: 4018 –4023) .
- C5aR The interaction of C5aR with C5a has been described in many different disease settings; most of them being involved in inflammatory and autoimmune diseases (Morgan BP et al., Nat Rev Drug Discov. 2015, 14: 857 –877; Hawksworth OA et al., Mol Immunol. 2017, 89: 36 –43) .
- Some initial research has been performed to identify the underlying mechanisms by which C5a stimulates tumor growth (Markiewski MM et al., Nat Immunol. 2008, 9: 1225 –1235; Corrales L. et al, J Immunol. 2012, 189: 4674 –4683; Cho MS et al., Cell Rep. 2014, 6: 1085 –1095) .
- C5a increases cancer cell proliferation, intra-tumor angiogenesis and enhances tumor invasiveness and metastasis. More recent data also implicate a role for C5a/C5aR in the generation of immunosuppressive environments in the context of solid tumors (Sayegh ET et al., Cancer Med. 2014, 3: 747 –758; Darling VR et al., Expert Rev Clin Immunol. 2015, 11: 255 –263; Markiewski MM et al., Cancer Res.
- C1-INH have been the only clinical compounds to exert control of the initiation pathways controlling complement initiation.
- eculizumab acts at the opposite end of the complement cascade by controlling the terminal pathway of complement activation.
- This antibody is a humanized anti-C5 monoclonal antibody that binds to the ⁇ chain of C5, preventing C5 from being cleaved into C5a and C5b by C5 convertase, as well as the subsequent formation of the membrane attack complex C5b-9.
- PNH paroxysmal nocturnal hemoglobinuria
- aHUS atypicalhemolytic uremic syndrome
- Direct targeting of C5aR has a number of advantages over targeting C5 or C5a, respectively.
- direct C5aR targeting may provide pharmacodynamic advantages over inhibition of soluble C5a, due to its small molecular weight and its high turnover rate. Overall, there is strong interest in developing C5aR inhibitors, such as aptamers, peptides, and non-peptide small molecules being tested in pre-clinical and clinical trials.
- Therapeutic antibodies targeting C5aR has been considered for clinical development, for example, Neutrazumab (G2 therapies, Darlinghurst, NSW, Australia) is in preclinical development for RA and stroke.
- Neutrazumab G2 therapies, Darlinghurst, NSW, Australia
- the clinical development of the antagonistic C5aR specific antibody Neutrazumab, a humanized IgG4 mAb was stopped in Phase II clinical trials due to issues with immune cell depletion and immunogenicity (Daniluk S et al., Annals of the Rheumatic Diseases. 2014, 73: 684–685) . Since C5aR appears constitutively expressed on a variety of cell types, it is important that an antagonistic antibody does not induce any depletion of the target cells.
- NNC0215-0384 US2013/0295116 (NOVO NORDISK) ; clone 32F3A6GL
- This antibody is a human IgG1 antibody derived from transgenic mice and is currently under clinical development as IPH5401 in the field of cancer (Olivier Demaria et al., Innate Pharma 2017. Poster #B184. CRI-CIMT-EATI-AACR Mainz) .
- IPH5401 bears a silenced human IgG1 Fc region to eliminate the ability of the antibody to induce effector function.
- PTMs by host-cell enzymes are especially critical if theynegatively influence drug potency or drug safety. They need to be diminished or removed by manufacturing process optimizations or further protein engineering. Even ifPTMs just increase the product heterogeneity, they at least have to be monitored andcontrolled to demonstrate batch consistency or comparabilityof manufactured clinical material.
- An array of methods and separation techniques are used to characterize therapeuticantibodies products and identify the product variants and impurities. These include capillary electrophoresis and chromatography methodologies as ion exchange, reversed-phase, size-exclusion or hydrophobic-interaction (Beck A, Wagner-Rousset E, etal, Anal Chem 2012, 85: 715–736) . These methods are time-consuming, require several instrumental setups and specific sample preparation methodology. Due to the high complexity of biopharmaceuticals, it makes direct and fast measures of product quality in real time very challenging. Additionally, these methods are not able to quantify the product quality at molecular level, for example, detecting the site of PTM.
- MS mass spectrometry
- the present invention provides therapeutic antibodiesor antigen binding fragments thereof that target C5aR, and further provides a sensitive and precise MS-based methodthat allows the simultaneous detection, identification and quantification of PTM of the antibodies or antigen binding fragments, in particular, a hydroxylation modification at the molecular level. Analysis thisstructural signature of the antibody is important to evaluate theproduct and process quality in manufacturing of the antibody.
- hydroxyproline is an unusual amino acid that is not directly synthesized but results from post-translational hydroxylation of a proline resided within a peptide (Sadava D. and Chrispeels MJ. Biochemistry 1971, 10 (23) : 4290–4294) . Since hydroxyl groups are hydrophilic, addition of a hydroxyproline residue to an antibodyor a therapeutically active fragment may affect its structural integrity and water solubility, and further affect its drug efficacy and drug safety. In this way, extensive analytical methods are needed for in-depth characterization of the modified antibody or the therapeutically active fragment to ensure theproduct quality and process controlling in manufacturing.
- one aspect of the present invention provides a sensitive and reliable method for detecting and determining the site and the amount of at least one modification within anantibody or a therapeutically active antigen binding fragment thereof by liquid chromatography with subsequent mass spectrometry (LC-MS) .
- LC-MS liquid chromatography with subsequent mass spectrometry
- the present invention provides a method comprising the following steps of:
- the modification comprises co-translational modifications, post-translation modifications or substitutions.
- the modification is hydroxylation of one or more proline residues within the antibody or the therapeutically active antigen binding fragment.
- the antibody comprises a heavy chain having amino acid sequence of SEQ ID NO: 1 and a light chain having amino acid sequence of SEQ ID NO: 2, and the modification is hydroxylation of the proline resided at position 14 of SEQ ID NO: 2.
- the antibody comprises a heavy chain having amino acid sequence of SEQ ID NO: 4 and a light chain having amino acid sequence of SEQ ID NO: 2, and the modification is hydroxylation of the proline resided at position 14 of SEQ ID NO: 2.
- the antibody is an antibody mixture of the antibody having amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2, and the antibody having amino acid sequences of SEQ ID NO: 4 and SEQ ID NO: 2, and the modification is hydroxylation of the proline resided at position 14 of SEQ ID NO: 2.
- At least 90%of the antibody has amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2, preferably, at least 95%, more preferably, 95-98%of the antibody has amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2.
- At least 5 %of the proline resided at position 14 of SEQ ID NO: 2 is modified to hydroxyproline, preferably, 10-20 %of this proline is modified to hydroxyproline, more preferably, 13-15 %of this proline is modified to hydroxyproline.
- the antibody can be a human, humanized or chimeric antibody. In yet another preferred embodiment, the antibody is a humanized antibody.
- the antibody or a therapeutically active antigen binding fragment can be of any isotype.
- the antibody is a class of IgG1 antibody with a lambda-type light chain.
- the antibody is produced by CHO cells and purified via selected chromatography including affinity chromatography, ion-exchange chromatographyand size exclusion chromatography.
- the antibody prior to enzymatic digestion, is treated with denaturation, reduction and alkylation steps to unfold the antibody and facilitate enzymatic digestion.
- the antibody is treated with a digestive enzyme trypsin, or a combination of digestive enzymes, preferably a combination of endoproteinase Lys-C and trypsin, to promote a complete and reproducible digestion. After digestion, the antibodyis digested into a mixture of peptides.
- the peptide mixture is separated through a reversed-phase ultra-performance liquid chromatography (UPLC) , preferably, a Poroshell 120 SB-C18 column (Agilent) , with waterand acetonitrile as mobile phase at 0.4 ml/min flow rate.
- UPLC reversed-phase ultra-performance liquid chromatography
- the antibody is characterized by ESI-Q-TOF-MS/MS in connection with UPLC.
- the acquired MS data areanalyzed using Agilent MassHunter BioConfirm software bycomparing withthe theoretical sequences.
- a signature peptide having amino acid sequenceof SEQ ID NO: 3 with +15.995Da modification is identified and the +15.995 Da modification is localized at a proline at the 14 th residue of SEQ ID NO: 2.
- the +15.995Da modification is identified as hydroxylation of proline at position 14 of SEQ ID NO: 2, and the modified proline accounts for 14.3%of the total proline (modified and unmodified proline) at this position.
- the antibody is hydrolyzed by acid to analyze the amino acid composition of the antibody.
- the hydroxylated proline is then quantified by external standards as 0.42%of the total prolines within the antibody, which is consistent with the conclusion from the above-mentioned MS analysis.
- functional activity of the antibodywith modification is characterized using ⁇ -Arrestin Assay from DiscoverX. Results show that the antibody with modification can efficiently inhibit C5a induced C5aR activity in vitro.
- the antibody or therapeutically active antigen binding fragment and the corresponding detection and quantification methods are significant. To our knowledge, it is the first time identifying an anti-C5aR antibody with hydroxyproline modification, which can be a structural signature of the antibody. The method for detecting and determining the site and the amount ofhydroxylationof prolineis important for quality control and process monitoring in manufacturing the antibody or a therapeutically active antigen binding fragment thereof. Further, the results hold promise as a new treatment strategy.
- FIG. 1 showsMS/MS spectra of a signature peptide having amino acid sequence of SEQ ID NO 3.
- Upper panel A unmodified peptide;
- Bottom panel B modified peptide
- FIG. 2 showsthe structure of hydroxyproline.
- FIG. 3 shows EIC of modified and unmodified peptide.
- FIG. 4 shows the principle of ⁇ -Arrestin Assay.
- FIG. 5 shows the mechanism of the conversion from proline to hydroxyproline in biochemistry.
- FIG. 6 shows a Liquid chromatograph after acid hydrolysis of the antibody.
- FIG. 7 shows the amino acid sequences of SED ID NO: 1, SED ID NO: 2, SED ID NO: 3, SED ID NO: 4, and SED ID NO: 5.
- C5a receptor As used herein, “C5a receptor” , “C5aR” , or “C5aR 1” and variations thereof refers to a protein known as C5a anaphylatoxin chemotactic receptor 1 or CD88.
- An example of the amino acid sequence of a human C5aR is provided in SEQ ID NO: 5, however, as the skilled person will be aware there are naturally occurring allelic variants of this molecule which are also encompassed by the term “C5aR” .
- Human C5aR (Uniprot: P21730
- antibody refers to a protein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, which interacts with an antigen.
- Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
- Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
- VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR) , interspersed with regions that are more conserved, termed framework regions (FR) .
- CDR complementarity determining regions
- Each VH and VL is composed of three CDRs and four FR’s arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
- the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
- the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system.
- the term “antibody” includes for example, monoclonal antibodies, human antibodies, humanized antibodies, camelid antibodies and chimeric antibodies.
- the antibodies can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA and IgY) , class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.
- therapeutically active fragment refers to a variant of an antibody or one or more portions of an antibody that retain the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing spatial distribution) an antigen. These therapeutically active fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
- a “human antibody” or “human therapeutically active fragment” is an antibody and therapeutically active fragment having variable regions in which both the framework and CDR regions are from sequences of human origin.
- Human antibodies can also be isolated from synthetic libraries or from transgenic mice (e.g. Xenomouse) provided the respective system yield in antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region also is derived from such sequences.
- Human origin includes, e.g., human germline sequences, or mutated versions of human germline sequences or antibody containing consensus framework sequences derived from human framework sequences analysis, for example, as described in Knappik et al., (2000) J Mol Biol 296: 57-86) .
- a “humanized antibody” or “humanized therapeutically active fragment” is defined herein as an antibody molecule, which has constant antibody regions derived from sequences of human origin and the variable antibody regions or parts thereof or only the CDRs are derived from another species.
- a humanized antibody can be CDR-grafted, wherein the CDRs of the variable domain are from a non-human origin, while one or more frameworks of the variable domain are of human origin and the constant domain (if any) is of human origin.
- chimeric antibody or “chimeric therapeutically active fragment” is defined herein as an antibody molecule, which has constant antibody regions derived from, or corresponding to, sequences found in one species and variable antibody regions derived from another species.
- the constant antibody regions are derived from, or corresponding to, sequences found in humans
- the variable antibody regions are derived from sequences found in a non-human animal, e.g. a mouse, rat, rabbit or hamster.
- isolated antibody refers to an antibody or therapeutically active fragment that is substantially free of other antibodies or therapeutically active fragments having different antigenic specificities. Moreover, an isolated antibody or therapeutically active fragment may be substantially free of other cellular material and/or chemicals. Thus, in some aspects, antibodies provided are isolated antibodies, which have been separated from antibodies with a different specificity. An isolated antibody may be a monoclonal antibody. An isolated antibody may be a recombinant monoclonal antibody. An isolated antibody that specifically binds to an epitope, isoform or variant of a target may, however, have cross-reactivity to other related antigens, e.g., from other species (e.g., species homologs) .
- antibody mixture or “mixture of antibodies” , as used herein, refers to a composition of two or more different antibody molecules. In the context of the present invention, antibodies within the mixture have highly identical amino acid sequences.
- recombinant antibody includes all antibodies that are prepared, expressed, created or segregated by means not existing in nature. For example, antibodies isolated from a host cell transformed to express the antibody, antibodies selected and isolated from a recombinant, combinatorial human antibody library, and antibodies prepared, expressed, created or isolated by any other means that involve splicing of all or a portion of a human immunoglobulin gene, sequences to other DNA sequences or antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom.
- an animal e.g., a mouse
- such recombinant antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences.
- such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
- a recombinant antibody may be a monoclonal antibody (Rothe et al, J. Mol. Biol. (2008) 376, 1182-1200) .
- the antibody or a therapeutically active fragment is produced by Chinese Hamster Ovary (CHO) cells, wherein CHO cells are transfected with twovectors encoded with antibody light chain (LC) and heavy chain (HC) sequences respectively. Allcell cultures are harvested on day 14 posttransfectionand the supernatant of the cell culture containing expressed antibody are collected to be purified using selectedchromatography.
- CHO Chinese Hamster Ovary
- Mass spectrometry is a powerful tool to analyze proteins.
- an antibody or a therapeutically active fragment need to be treated with a digestive enzyme, such as a protease, to form a peptide mixture.
- a digestive enzyme such as a protease
- the antibody may be further treated with various buffers, denaturing agent, reducing agent, alkylating agent and even elevated temperatures for extended period of time.
- protease or digestive enzyme refers to an enzyme capable of cleaving or hydrolyzing peptides or proteins into fragments in either a specific or generic, random manner.
- Example protease or digestive enzyme includes, but not limited to, tyrosine, Chymotrypsin, endoproteinase Lys-C, endoproteinase Lys-N, Glu-C, rLys-C, Asp-N, rAsp-Nand non-specific protease pepsin, thermolysin, Elastase etc, and Trypsin enhancer.
- the modified antibody is a tightly folded protein, some places may be resistant to proteolysis due to inaccessibility of internal cleavage sites for protease.
- the modified antibody Prior to digestion, the modified antibody is treated with denaturing agent to overcome this challenge.
- the denaturing agent is Guanidine Hydrochloride (Gdn-HCL) at a concentration of 6.0 M.
- the modified antibody is further treated with a reducing agent to further facilitate protease digestion.
- the reducing agent is dithiothreitol (DTT) .
- DTT dithiothreitol
- the modified antibody after reduction by DTT, the modified antibodyis further cysteine-alkylated to prevent reformation of disulfide bonds.
- the alkylating agent isiodoacetamide.
- the modified antibody is digested by trypsin.
- the modified antibody is digested by two digestive enzymes sequentially, andthe two enzymes arepreferably endoproteinase Lys-C and trypsin.
- the resulting peptide mixture is analyzed using ultra performance liquid chromatography with subsequent tandem mass spectrometry (UPLC-MS/MS) .
- Mass spectrometry is a detection technique by measuring mass-to-charge ratio of ionic species.
- the procedure consists of different steps. First, a sample is injected in amass spectrometer and then evaporated. Second, species in the sample are charged by certain ionized methods, such as electron ionization (EI) , electrospray ionization (ESI) , chemical ionization (CI) , matrix-assisted laser desorption/ionization (MALDI) . Finally, the ionic species will be analyzed depending on their mass-to-charge ratio (m/z) in the mass analyzer, which is key to the sensitivity, resolution and mass accuracy of themass spectrometer.
- EI electron ionization
- ESI electrospray ionization
- CI chemical ionization
- MALDI matrix-assisted laser desorption/ionization
- Sensitivity describes an instrument’s ability to detect low-abundance peptides, resolution describes its ability to distinguish ions of very similar m/z values, and mass accuracy describes its ability to obtain mass measurements that are very close to the truth.
- mass analyzer There are several basic types of mass analyzer: quadrupole (Q) , ion-trap (IT) , time-of-flight (TOF) , Fourier transform ion cyclotron resonance (FTICR) , and the orbitrap.
- Q-TOF ion-trap
- TOF time-of-flight
- FTICR Fourier transform ion cyclotron resonance
- Different mass analyzers are commonly combined to achieve the best utilization as a single mass spectrometer (e.g., Q-TOF, Triple-Q) (Yuliya V. Karpievitch, et al Ann Appl Stat. 2010; 4 (4) : 1797–1823)
- the mass spectrometric identification is widely used together with chromatographic separation.
- Liquid chromatography (LC) has an efficient capacity of separation and MS has a high sensitivity and strong ability of structural characterization.
- the high-resolution mass spectrometry ( ⁇ 3 ppm RMS with external calibration) provides a high mass accuracy, allowing differentiation of components with the same nominal mass with high confidence, which is crucial for identifying post-translational modifications (PTMs) and substitutions (Chris Spahr et al, MABS 2017, 9 (5) : 812–819) .
- the inventor discloses a method for characterizing the antibody or a therapeutically active fragment with modificationby using liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (LC/ESI-QTOF-MS) .
- LC/ESI-QTOF-MS electrospray ionization quadrupole time-of-flight mass spectrometry
- ESI electrospray ionization quadrupole time-of-flight mass spectrometry
- chromatography refers to a process in which a mixturecarried by a liquid or gas is separated into components as a result of differential distribution andseparation of the chemical entities as they flow around or over a stationary liquid or solid phase.
- LC liquid chromatography
- Liquidchromatography comprises, but to limited to, high performance liquid chromatography (HPLC) , ultra-performance liquid chromatography (UPLC) , reverses-phase liquidchromatography (RPLC) , and high turbulence liquid chromatography (HTLC) , hydrophilicinteraction liquid chromatography (HILIC) , and normal phase liquid chromatography (NPLC) .
- HPLC high performance liquid chromatography
- UPLC ultra-performance liquid chromatography
- RPLC reverses-phase liquidchromatography
- HTLC high turbulence liquid chromatography
- HILIC hydrophilicinteraction liquid chromatography
- NPLC normal phase liquid chromatography
- modification refers to a change to, or replacement of, an amino acid in a protein or a peptide, e.g., by way of co-translational modification (CTM) , post-translational modification (PTM) or substitutions.
- CTM co-translational modification
- PTM post-translational modification
- substitutions are chemical changes that proteins experience as a result of their covalent attachment to functional groups of proteins and the cleavage of their peptide bonds. These modifications alter the structure of individual proteins and therefore, potentially affect their activity, stability, localization and/or interacting partner molecules. Hydroxylation of proline is a PTM in protein synthesis catalyzed by the enzyme prolyl hydroxylase.
- hydroxyproline being a critical quality attribute (CQA) has not been extensively discussed in the literature, potentially because of the lack of biotherapeutic agents with this modification selected to move on to late-stage development.
- CQA critical quality attribute
- hydroxyproline is a naturally-occurring non-proteinogenic amino acid, and therefore less likely to be immunogenic.
- the addition of a hydrogen bond forming hydroxyl group to the otherwise inert backbone can alter the protein structure (Chris Spahr, et al MABS, 2017, 9 (5) : 812–819) .
- hydroxyproline side chain could be involved in undesired hydrogen bonding with the fusion protein, thus leading to potential modification of the activity or biophysical properties of the partner protein. It has been suggested through crystal structure analysis that hydroxyproline stabilizes collagen fibrils via mediating direct contact between neighboring molecules (Berisio R, Vitagliano L, Mazzarella L, Zagari A. Biopolymers 2000, 56: 8-13; PMID: 11582572) .
- hydroxyproline being a CQA
- high level of hydroxyproline is never the less a significant concern with regard to product homogeneity, especially when it can easily be mistaken for oxidation modifications on residues such as methionine and tryptophan. Therefore, it requires careful analysis of proteins with hydroxylation modification of proline (Spahr C, Gunasekaran K, Walker KW, Shi SD. Mabs. 2017, 9 (5) : 812–819) .
- the modified antibody is digested into a peptide mixture to be separated by a suitable liquid chromatography, preferably, areversed-phase UPLC, more preferably, a Poroshell 120 SB-C18 column (Agilent) .
- a suitable liquid chromatography preferably, areversed-phase UPLC, more preferably, a Poroshell 120 SB-C18 column (Agilent) .
- UV absorbance at 214 nm is employed to monitor the separations process anddetect peptides.
- LC is followed by a mass spectrometry for characterizing the modified antibody or therapeutically active fragment, preferably, the MS is ESI-QTOF-MS technology, more preferably, a tandem MS is utilized.
- data acquired from LC-MS/MS are processed and interpreted using Agilent MassHunter BioConfirm software.
- a signature peptidewith modifications is identified and characterized by comparing the measured mass to the theoretical mass.
- hydroxylation refers to a process that introduces a hydroxyl group (-OH) into a compound or an amino acid.
- hydroxylation of proline is catalyzed by an enzyme called prolyl hydroxylases.
- structural signature refers to a set of characteristics that unequivocally identifies a protein from other proteins.
- the structural signature can be determined by one or more methods chosen from the group comprising of MALDI-MS, ESI- MS, CE, HPLC, FPLC, fluorometry, ELISA, chromogenic assays, colorimetric assays, NMR and other spectroscopic techniques.
- signature peptide refers to a unique peptide, such as a peptide with special chemical or biological modifications.
- a signature peptide usually shows experimentally advantageous chromatographic and /or advantageous mass spectrometric properties. As such, it can be identified by an experimental method.
- the inventor discloses thatmost detected fragment ions match the theoretical distributions of fragment ions, however, one peptide is not able to be identified by comparing the measured mass withthe theoretical mass of thispeptide sequence, and a +15.995 Da modification is identified withinthe signature peptide (SEQ ID NO: 3) .
- the +15.995 Da modification is localized at a proline at the 14 th residue of SEQ ID NO: 2.
- the proline at this position could be mutated to leucine and/or isoleucine, which is a mutation of proline, orcan be hydroxylated, which is a post translational modification of proline.
- the inventor discloses that the +15.995Da modification is attributed to the hydroxylation of proline based on the accurate mass (with error less than 10 ppm) .
- the measured molecular mass of the signature peptide of Seq ID NO: 3 is 1706.8571 Da which is only 1.4ppm error from the theoretical mass 1706.8588 Da of hydroxyproline peptide.
- the proline at the 14 th residue of SEQ ID NO: 2 is mutated to leucine, the theoretical molecular mass of the peptide of Seq ID NO: 3 should be 1706.8911 Da with a mass error of 19 ppm.
- the observed mass difference between the y4+ and y3+ ions of 113.0477 Da is found to be in perfect agreement with the presence of a hydroxyproline at the 14 th residue of SEQ ID NO: 2 (theoretical mass increment of 113.0477 Da) , that virtually ruled out alternative explanations of proline to leucine and/or isoleucine mutation (theoretical mass increment of 113.0841 Da) (See Figs. 1 and 2) .
- arelative quantification by extracted ion chromatograms (EIC) of the modified peptide and the unmodified peptide demonstratesthat the modified peptide is eluted out earlier than the unmodified peptide (See Fig. 3) , which is consistent with the previous conclusion that the +15.995Da variant is a hydroxyproline and not a proline to leucine and/or isoleucine mutationsince the hydroxylated peptide is more hydrophilic and eluted earlier from the reversed-phase LC while proline to leucine and/or isoleucine would make the peptide more hydrophobic and elute later than the unmodified peptide on reverse phase LC. Further, based on the peptide signal intensity and integration of peak area, a relative abundance of modified peptide is calculated to 14.3 % (in another words, a ratio of modified proline to the total proline at this position) (See Fig. 3) .
- the modified antibodywith hydroxylation modification of proline demonstrates a functional activity of blocking C5a interaction with C5aR, same as the original unmodified antibody. This modification does notaffect the drug efficiency and safety (data not shown) .
- this hydroxylation of proline in the present invention can be used as a structural signature to determine the amount of the modified antibody. A method of detecting and quantifying this structural signaturecan be used in quantity control and process monitoring in manufacturing.
- the functional activity of the antibody or therapeutically active fragment produced by CHO cell is characterized using ⁇ -arrestin assay from DiscoverX.
- CHO cells are engineered to co-express C5aR1 protein tagged with a small fragment of ⁇ -galactosidase enzyme.
- the interaction between C5a and C5aR1 can activate C5aR1 and recruit ⁇ -arrestin, which forces complementation of two ⁇ -galactosidase enzyme fragments to hydrolyze a substrate and generate a chemiluminescent signal.
- the functional activity of the modified antibody can be evaluated by analysis of the chemiluminescent signal. Results show that the modified antibody could block C5a/C5aR1 interaction.
- Hydrolysis of antibody by acid is further performed to confirm the relative abundance of hydroxyproline quantified byextracted ion chromatograms.
- Acid hydrolysis with constant-boiling HCI at 110 °C for more than 24 h has been widely accepted as a standard method for hydrolysis of protein (see TSUGITA et al., Eur. J. Biochem. 1982 (124) : 585-588) .
- the modified antibody of 0.1mg is hydrolyzed with HCl at a concentration of 6 M in a centrifugal tubeat 100 °C for 30 hours. After acid hydrolysis, the antibody is hydrolyzed to a mixture of amino acids.
- the hydrolyzed antibody is suspended with 20 mM HCl, and then derivatizedusing AccQ ⁇ TagTM Ultra Derivatization kit from Waters.
- the derivatives are separated using a C18 column by UPLC (Waters) , and then quantified with FLD spectroscopy. Then the hydroxylated proline is quantified by external standard as 0.42 %of total prolines within the modified antibody, results are consistent with the results of EIC analysis.
- CHO cells were transfected with mammalian expression vector encoding heavy chain and light chain of the desired antibody. All cell cultures were harvested on day 14 post transfection. Supernatant containing expressedantibodywas collected andclarified, and then purified using an affinity chromatography (ProteinA column) , followedbyan anion exchange chromatographyin flow-throughmodeanda cation exchange chromatographythroughelutionmode. Afterpurification, antibody was buffer exchanged into a formulation buffer and used as drug substance. The protein concentration was measured by UV spectrometry at 280nm. The overall purity characterized by CE-SDS analysis was above 95%.
- Example 1 The antibody produced in Example 1 was expected to reduce or inhibit the bindings ofC5a to C5aR1.
- This potential activity can be tested ⁇ -Arrestin Assay from DiscoverX.
- CHO cells are engineered to co-express specifically tagged GPCR ⁇ -arrestin components that, upon receptor activation and ⁇ -arrestin recruitment, will force complementation of two ⁇ -galactosidase enzyme fragments to hydrolyze a substrate and generate a chemiluminescent signal (see Fig. 4) .
- CHO cells expressing the engineered version of human C5aR, were mixed with cell plating reagent and seeded in a tissue culture plate and cultured for 16-18 hours at 37 °C in a humidified atmosphere containing 5%CO 2 .
- 2.8-fold serial dilutionsof antibody withastartingconcentrationat 0.1mg/ml were added to the plate and incubated for 30min, followed by incubation with 0.5ug/ml C5a for 4 hours.
- 60 ⁇ L of the detection regent was added to each well, and the cells were cultured for another 1hat room temperaturein dark. Results were expressed as chemiluminescent signal and detected by a plate reader (PerkinElmer) .
- the dose–response curves for this potential agonist were generated and the activity was evaluated usingPrism
- the antibodyneeds In order to produce signature peptides for analysis by mass spectrometry, the antibodyneeds to be treated with digestive enzyme.
- the purified antibody was denatured in 6M Gdn-HCl (Sigma) , 50mM Tris (pH 8) buffer. Reduction and alkylation of cysteine residues can improve peptide yield, sequence coverage and the identification of proteins with a high number of disulfide bonds.
- the denatured antibody was further reduced in 20 mM DTT (Sigma) for 30minutes at 56°C., and then the reduced antibodywas alkylated in 50 mM iodoacetamide (Sigma) for30minutes at roomtemperature in the dark.
- Buffer exchange (0.1 M Tris, pH 7.5) of the antibody was performed using a ultrafilter column (Millipore) prior to an efficient enzymatic digestion. Then, the resulting antibody wasdigested by trypsin (Promega) for24hours at 37°C, and thedigestion was terminatedusing formic acid (typically 0.1–1%final concentration or until a pH of ⁇ 2–3 is reached) . Then the digested peptides werefrozen prior to LC-MS analysis.
- proteolytic peptide mixtures was subjected to reversed-phase UPLC followed by MS/MS.
- MS/MS MS/MS
- ESI-Q-TOF mass spectrometric technology is developed.
- peptide analysis 2 ⁇ g of enzymatic-digested antibody was used as loading amount for analysis by LC-MS.
- a reversed-phase Poroshell 120 SB-C18 column (Agilent) was used with a flow rate of 0.4 ml/ml.
- the following LC conditions as shown in Table1 were used. UV chromatograms were acquired by a diode array detector detected at 214 nm.
- MS data was acquired using Agilent 6545XT AdvanceBio Q-TOF.
- the mass spectrometer was set for data dependent scanning full spectra, and the top 10ions were selected for subsequence MS/MS scan.
- the following MS conditions were used as shown in Table 2.
- ESI-Q-TOF MS/MSdata was based on the assignment of the peptide fragment m/z value to fragment ions.
- the most used ions were y+1 ions and b+1 ions, which were considered as the most valuable ions for the characterization of a PTM.
- the mass raw data including high resolution MS spectra and MS/MS spectra were analyzed quantitatively using the Agilent MassHunter BioConfirm software based on the theoretical sequence. Mass tolerance of 10 ppm was allowed. Peptide length was limited to70 amino acids. A full sequence coverage peptide map was obtained for identifying heterogeneities.
- the +15.995Da shift is suggested to be the hydroxylation of proline, because the measured molecular mass of the peptide of SEQ ID NO: 3 is 1706.8571Da which is only 1ppm error from the theoretical mass of hydroxyproline peptide of 1706.8588 Da. Otherwise, if the Proline at position 14 of SEQ ID NO: 2 is mutated to leucine, the theoretical molecular mass of the peptide is 1706.8911 Da and the measured peptide mass error would be 19 ppm.
- the +15.995 Da shift is further confirmed by extracted ion chromatogram (EIC) .
- An extracted ion chromatogram (EIC) is a signal trace where the intensity of ions from a defined m/z window is plotted versus retention time.
- the unmodified and +15.995Da modified peptide were separated on a mixed-mode hybrid C18 column. Results were shown in Fig 3.
- the +15.995 Da modification was observed eluting 1.35min earlier than the unmodified version, indicating that the modification decreased the hydrophobicity of the peptide, thereby, confirming the modification is hydroxylation, not leucine and/or isoleucine mutation.
- a relative abundance of modified peptide is calculated to 14.3 % (a ratio of modified proline to total proline at this position) (See Fig. 3) .
- hydroxyproline is formed by oxidation of the carbon in the gamma position of the proline pyrrolidine ring through the action of prolyl hydroxylase with existence of dioxygen and alpha-ketoglutarate (Fig. 5) (PNAS, 2006, 103 (26) , 9814-9819) .
- Hydrolysis of antibody by acid is further performed to confirm the relative abundance of hydroxyproline quantified by EIC.
- the modified antibody of 0.1 mg was dried and resuspended with 0.25mL of 6.0 M HCland then hydrolyzedat 100°C for 30 h. After cooling down, the acid is removed on a rotary evaporator with a water vacuum pump at 65°C andsample was suspended with 0.1mL 20mM HCl. After acid hydrolysis, the antibody is hydrolyzed to a mixture of amino acids.
- the hydrolyzed antibody is derivatized by using AccQ ⁇ Tag TM Ultra Derivatization kit from Waters.
- the derivatives were separated using a C18 column by UPLC (Waters) and quantified with UV spectroscopy. Data were processed using EmpowerSoftware.
- the hydroxylated proline is quantified by external calibration standard as 0.42%of total proline within the modified antibody, results are consistent with the results from EIC (See Fig. 6) .
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Abstract
La présente invention fournit un procédé pour détecter et déterminer le site et la quantité d'au moins une modification dans un anticorps ou un fragment thérapeutiquement actif de celui-ci se liant à un antigène, comprenant les étapes consistant à traiter l'anticorps ou le fragment thérapeutiquement actif se liant à un antigène avec une enzyme digestive pour former un mélange de peptides, analyser le mélange de peptides par chromatographie liquide suivie d'une spectrométrie de masse en tandem (LC-MS/MS) pour détecter un peptide signature avec au moins une modification, et déterminer le site et la quantité de la modification dans le peptide signature sur la base du spectre de masse. En outre, la présente invention concerne un anticorps qui se lie à C5aR ou à un fragment de liaison à l'antigène thérapeutiquement actif de celui-ci avec au moins une proline partiellement ou complètement modifiée par la fixation d'un groupe hydroxy.
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WO2022057910A1 (fr) * | 2020-09-17 | 2022-03-24 | I-Mab Biopharma Co., Ltd | Polythérapies ciblant des voies c5ar et pd-1/pd-l1 |
US11773179B2 (en) | 2020-01-13 | 2023-10-03 | Visterra, Inc. | Antibody molecules to C5aR1 and uses thereof |
US11912781B2 (en) | 2021-01-13 | 2024-02-27 | Visterra, Inc. | Humanized complement 5A receptor 1 antibodies and methods of use thereof |
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US20030049715A1 (en) * | 2001-08-31 | 2003-03-13 | Welsch Dean J. | Peptide biomarker and method of identification |
CN102482348A (zh) * | 2009-09-16 | 2012-05-30 | 盐野义制药株式会社 | 胶原新表位抗体 |
CN109541222A (zh) * | 2018-11-08 | 2019-03-29 | 新乡医学院 | 蛋白质棕榈酰化修饰位点的检测方法 |
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US20030049715A1 (en) * | 2001-08-31 | 2003-03-13 | Welsch Dean J. | Peptide biomarker and method of identification |
CN102482348A (zh) * | 2009-09-16 | 2012-05-30 | 盐野义制药株式会社 | 胶原新表位抗体 |
CN109541222A (zh) * | 2018-11-08 | 2019-03-29 | 新乡医学院 | 蛋白质棕榈酰化修饰位点的检测方法 |
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US11773179B2 (en) | 2020-01-13 | 2023-10-03 | Visterra, Inc. | Antibody molecules to C5aR1 and uses thereof |
WO2022057910A1 (fr) * | 2020-09-17 | 2022-03-24 | I-Mab Biopharma Co., Ltd | Polythérapies ciblant des voies c5ar et pd-1/pd-l1 |
US11912781B2 (en) | 2021-01-13 | 2024-02-27 | Visterra, Inc. | Humanized complement 5A receptor 1 antibodies and methods of use thereof |
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