WO2023126821A1 - C1q binding assay - Google Patents

Abstract

The present disclosure provides a C1q binding assay. The assay disclosed here determines theC1q binding affinity of antibody by using Biolayer interferometry (BLI) technique. The disclosed method does not require any labelling of antibody, and thus is a simple and robust method for determining the binding affinity of antibody to C1q protein. The binding affinity of an antibody to a C1q protein involves use of biosensors such as anti-Human Fab-CH1 (FAB2G) biosensors to capture and functionalize the antibody.

Description

Clq Binding Assay

DETAILED DESCRIPTION

CROSS-REFERENCE TO RELATED APPLICATIONS

001] The present application claims priority benefit of Indian Provisional Patent Application No: 202141061460 entitled “Clq Binding Assay” filed on 29 December 2021 , hereby incorporated by reference in its entirety.

TECHNICAL FIELD

002] The present invention relates to Clq binding assay, in particular relates to the determination of binding affinity of an antibody to Clq protein using Biolayer interferometry (BLI).

BACKGROUND OF INVENTION

003] Monoclonal antibodies (mAbs) are successful therapeutics due to their specific and selective nature of binding to their target antigens. Upon binding they activate several Fab and Fc dependent immune effector mechanisms such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC). They have become established therapeutics and first line of treatment against wide array of diseases including cancer, autoimmune diseases like multiple sclerosis, inflammatory diseases like arthritis, Crohn’s disease, ulcerative colitis, osteoporosis.

004] World over, past decade has seen a surge in application and approval of biosimilars that are generic version of innovator biologic medicines originally approved by regulatory bodies such as U.S. Food and Drug administration (FDA), European Medical Agency (EMA) etc. for treatment of diverse diseases. As the name connotes, biosimilars are required to be highly similar to the innovator biologic in terms of structure, safety, purity, and potency. The innovator biologies are used as reference products while developing the biosimilar to compare and establish similarity. Whatever difference biosimilar may have should be minor and clinically insignificant. A biosimilar that yields the same outcome in terms of safety and effectiveness as that of reference product is termed as interchangeable and can be a ready substitute to the reference biologies. Biosimilars have gained prominence mainly due to their low cost and affordability. The rise of biosimilars is imminent as more and more reference biologies go off-patent in coming times. However, for the regulatory approval of the biosimilars, stringent characterization and parity in terms of safety and efficacy with the reference biologies is mandatory. Therefore, assessment of biosimilars requires accurate measures to compare and establish similarity with the reference product. A biosimilar that is highly similar to and has no clinically meaningful differences from an existing approved reference product has higher chances of getting regulatory approval without delay. Bioassays play a pivotal role in the development of biologic molecules. They provide an important tool for potency determinations and head-to-head comparison of a biosimilar molecule with reference biologies. They are expected to be highly specific, sensitive and pick minute structural and clinical differences. For successful development of biosimilars, it is imperative to have robust bioassays. The bioassays used for the characterization of biosimilars are of different types including ligand binding assays (LBA) for target proteins such as CD64, CD32 and CD 16 binding assays and complement binding assays, cell-based assays such as proliferation, neutralization and receptor binding assays, antibody- dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) assays. Complement Cl is a protein complex, physiologically involved in the complement cascade of the immune response. It facilitates the immune system to efficiently clear the microbes and damaged cells from the body. It consists of subcomponents Clq, Clr and Cis. Clq components bind to the Fc region of the IgG and IgM antibodies and activate the complement cascade leading to effector functions such as complement dependent cytotoxicity. The degree of Clq binding serves as one of the important measures to demonstrate antibody efficacy in-vitro. Therefore, a comparative determination of Clq binding between two different sets of antibodies (a reference and a test) would help determine their binding profile and establish similarity between the two. Consequently, in-vitro Clq binding assays have emerged as important assessment tools for determination of binding affinity of Clq binding antibodies such as IgG and IgM. Among the myriad of applications include, determination of binding affinity of antibodies present either in the biological samples or produced recombinantly and comparison of the same. One of the major applications of these assays is in the development of biosimilars where comparison of biosimilar antibody to that of reference antibody is done by comparing their binding affinities. ELISA based Clq binding assays are used routinely to determine the binding affinity of test antibodies. However, ELISA methods require labelling such as streptavidin, biotin and suffer disadvantages of dissociation of low binding antibodies during extended washing steps thereby impacting the sensitivity of the assay. Oil] Bio-layer interferometry (BLI) is an emerging technology, used to probe biomolecular interactions. It is an optical technique that measures interference pattern of white light when reflected from a layer of immobilized biomolecules. The shift in the interference pattern is measured as the antibody binds to the complement.

012] Zhou et al. 2018 reported characterization of antibody-Clq interactions using Bio-layer Interferometry (Anal. Biochem. 2018 May 15; 549:143-148.). However, the technique involved use of streptavidin-biotin labeling of the antibody where the antibody was chemically attached via amide linkage to biotin before assaying with Clq. Such covalent chemical labeling alters the structure of the antibody and impacts the binding sensitivity. Biotinylation of antibodies have also been known to generate high background noise due to non-specific binding leading to inaccurate determinations. Moreover, the simultaneous head- to-head comparison of antibodies cannot be performed with this technique as both the set of antibodies, reference and test, require separate labelling steps.

013] Based on the aforementioned discussion, there still remains an unmet need in the art to ameliorate or overcome the lacunae in the alternative methodologies employed to determine the binding affinity of therapeutic monoclonal antibodies (mAb) to Clq protein. More particularly, there is a need to provide a biolayer interferometry (BLI) based method to determine the binding affinity of therapeutic monoclonal antibodies (mAb) to Clq protein. The above discussion of documents is included solely for the purpose of providing a context for the present invention.

SUMMARY OF THE INVENTION

014] The following presents a simplified summary of the disclosure in order to provide a basic understanding to the reader. This summary is not an extensive overview of the disclosure. Further, the brief summary of the invention does not intend to limit the scope of the claimed subject matter. Its sole purpose is to give a prelude to the more detailed description that is presented later. A more complete appreciation of the present invention and the scope thereof can be obtained from the accompanying drawings which are briefly summarized below and the following detailed description of the presently preferred embodiments. An objective of the present disclosure is directed towards a biolayer interferometry (BLI) based method to determine binding affinity of an antibody to a Clq protein. It is another objective of the present disclosure to provide a biolayer interferometry (BLI) based method to determine binding affinity of an antibody to a Clq protein in a cost-effective and easy manner. It is yet another object of the present disclosure to provide a unique methodology to measure the interaction of antibody and Clq protein using BLI technique, wherein the antibodies are not subjected to any chemical modifications thereby enabling more accurate determination of the binding affinities in the native states. An objective of the present disclosure is directed to overcome the problem in the state of art by providing a simple and robust binding assay for determining binding affinity of antibody to Clq protein, wherein the determination is done using biophysical technique Biolayer interferometry (BLI) and Langmuir 1: 1 binding fit model. The antibody is captured on the biosensor without any chemical labelling or tagging such as but not limited to biotin, or streptavidin and dipped in the solution comprising Clq protein. 020] As the step of chemical labeling and tagging is eliminated, it becomes simple and faster technique for determining the binding affinity of an antibody to Clq protein. The elimination of chemical labelling also prevents the modification of the antibody and allows determination of native state interaction and binding affinity of the antibody to Clq protein. It is also possible to determine the real time interactions between the antibody and Clq protein and develop a high throughput assay.

021] In yet another objective of the present disclosure, the method is tag-free and does not involve use of tags or labels such as streptavidin and biotin that are covalently bonded to the antibody.

022] Furthermore, the objects and advantages of this invention will become apparent from the following description and the accompanying annexed drawings.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS

023] The following drawings form a part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein. Further, the brief description of drawings is described below, wherein like reference numerals have been used to designate like elements, and wherein:

024] FIG. 1 is a diagram 100 depicting a pictorial representation of the Clq binding assay steps in BLI method, in accordance with one or more exemplary embodiments.

025] FIG. 2 is a Flowchart 200 representing Clq binding assay steps in BLI method, in accordance with one or more exemplary embodiments. 026] FIG. 3 is a diagram 300 depicting evaluation of specificity of FAB2G biosensors to monoclonal antibody (mAb) in BLI method, in accordance with one or more exemplary embodiments.

027] FIG. 4 is a diagram 400 depicting evaluation of specificity of FAB2G biosensors to Clq protein in BLI method, in accordance with one or more exemplary embodiments.

028] FIG. 5 is a diagram 500 depicting prevention of non-specific interactions between FAB2G biosensors and Clq protein by a blocking agent, in accordance with one or more exemplary embodiments.

029] FIG. 6 is a diagram 600 depicting evaluation of buffers for loading monoclonal antibody mAb (IgGl) stability on biosensor surface (Assay buffers; A: IX HBS- EP+ buffer and B: IX PBS buffer), in accordance with one or more exemplary embodiments.

030] FIG. 7 is a diagram 700 depicting a graphical representation of optimization of assay temperature in BLI method, in accordance with one or more exemplary embodiments.

031] FIG. 8 is a diagram 800 depicting a graphical representation of optimization of loading threshold of monoclonal antibody (mAb) in BLI method, in accordance with one or more exemplary embodiments.

032] FIG. 9 is a diagram 900 depicting a graphical representation of optimization of association time in BLI method, in accordance with one or more exemplary embodiments. 033] FIG. 10 is a diagram 1000 depicting a graphical representation of optimization of shake speed in BLI method, in accordance with one or more exemplary embodiments.

034] FIG. 11 is a diagram 1100 depicting a representative sensorgram of method validation through Bevacizumab and Trastuzumab sample analysis (IgGl), in accordance with one or more exemplary embodiments.

035] FIG. 12 is a diagram 1200 depicting a representative sensorgram of Denosumab sample analysis (IgG2), in accordance with one or more exemplary embodiments.

DETAILED DESCRIPTION OF THE INVENTION

036] Those skilled in the art would be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.

037] All publications herein are incorporated by reference to the same extent as that clearly to be included by reference if each individual publication or patent application, and individually indicated. The following description includes information that may be useful for understanding the present invention. This does not admit that any of the information is prior art, or to be or being specifically related to the invention claimed herein.

Definitions

038] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are delineated here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below. The articles “a”, “an” and “The” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. The terms “comprise” and “comprising” are used in open sense, meaning that additional elements may be included. It is not intended to be construed as “consisting of only”. Throughout this specification, unless the context requires otherwise the word “comprise” and the variations “comprising” and “comprises” will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps. The term “including” is used to mean “including but not limited to”. “Including” and “including but not limited to” are used interchangeably. Ratios, concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. Furthermore, the list of abbreviations used in the present disclosure is given below.

°C : Degree Centigrade

% Percent p L : Micro Litre

ADCC : Antibody-dependent cell-mediated cytotoxicity

ADCP : Antibody-dependent cell-mediated phagocytosis

BLI : Biolayer Interferometry

Clq : Complement component Iq

CDC : Complement-dependent cytotoxicity

CV : Coefficient of variation

Da : Dalton

DP : Drug product

DS : Drug substrate

FAB2G : Anti-Fab 2^nd Gen biosensors

HBS-EP : HEPES Buffered Saline EDTA, Surfactant P20

IRS : Internal reference standard k_a : Association rate constant kd : Dissociation rate constant

KD : Equilibrium dissociation constant kDa : Kilo Daltons mAb/mAbs : Monoclonal Antibody/ Monoclonal antibodies mL : milli Litre nM : nano Molar nm : Nanometres PBS : Phosphate -buffered saline

RBA : Relative Binding Affinity

RMP : Reference Medicinal Product

RPM : Revolutions per minute

SDT : Set dissociation time

Sec. or s : Seconds Clq is the first subcomponent of Cl complex. Clq together with Clr and Cis forms the C 1 complex. C 1 complex initiates the classical complement pathway through Clq mediated binding to the Fc portion of antibody. BLI (Bio layer interferometry) is an optical technique that measures the interference pattern of white light reflected by surfaces at the tip of the biosensor. It effectively monitors binding events in real time without the requirement of supplementary and costly labelling. It can analyze the interaction between two different molecules such as ligand and an analyte. Usually, the ligand is immobilized on the biosensor and the analyte is kept in solution. The term tags and labels are used interchangeably and refer to chemical or protein molecule attachment of additionally attached to the antibody through chemical bonding. The non-limiting examples of tags or labels are biotin, streptavidin. The terms tagging and labeling refer to attachment of the chemical or protein molecule to the antibody. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference. The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally equivalent products, compositions, and methods are clearly within the scope of the disclosure, as described herein. Conventionally used methods to determine the binding affinity of Clq binding assay includes end point assays where the results can be analyzed only at the end of the assay. Also, they require tagging of the antibody. Both these aspects make the previous methods cumbersome and costlier. Present disclosure provides a method to determine the binding affinity of antibody to Clq protein using BLI technology, which is based on shift in the light interference (nm) and equilibrium binding constant (KD) is determined based on the measured kinetic binding rate constants (ka, kd). Here, a robust method for analyzing the binding of antibody to Clq is provided. The method does not involve tagging or chemical labeling of the antibody or ligand. When the antibody is used without any label, analyzing the true binding of the antibody with Clq protein in native state becomes possible. Further, the method developed can be used for a variety of antibodies present in the biological samples or recombinantly expressed. Thus, the disclosure provides a platform for analyzing the binding affinity of antibodies to Clq protein. It provides an economical and real time method to determine the binding affinity of immunoglobulin to Clq protein. In an aspect of the present disclosure, a method to determine the binding affinity of an antibody or antibody conjugates thereof to a Clq protein is disclosed. In an aspect of the present disclosure, the antibody binds to Clq. 056] In an aspect of the present disclosure, the antibody is IgG or IgM.

057] In an aspect of the present disclosure, the antibody is a monoclonal antibody.

058] In an aspect of the present disclosure, a method to determine the binding affinity of an antibody to a Clq protein involves use of biosensors such as anti-Human Fab-CHl (FAB2G) biosensors to capture and functionalize the antibody.

059] In a preferred aspect of the present disclosure, there is provided a method to determine binding affinity of immunoglobulin IgG to Clq protein, wherein the said IgG does not have any label such as but not limited to streptavidin or biotin and binding affinity is determined using bio-layer interferometry.

060] In an aspect of the present disclosure, there is provided a method to determine the binding affinity of antibody to Clq protein, wherein the antibody does not have any label or tag such as but no limited to streptavidin or biotin and binding affinity is determined using bio-layer interferometry.

061] In yet another aspect of the invention, the antibody is a monoclonal antibody and is expressed recombinantly or present in biological samples.

062] In an aspect of the present disclosure, the recombinant monoclonal antibody is IgG or IgM.

063] In an aspect of the present disclosure, the monoclonal antibody present in biological samples is IgG or IgM.

064] In one of the preferred aspects of present disclosure, there is provided a method to determine the binding affinity of monoclonal antibody (IgGl/IgG2) to human Clq protein, wherein the method involves capturing of label or tag free monoclonal antibody on the biosensor, blocking the additional unbound surface on the biosensor with human Fab fragment, dipping the captured antibody into varying concentration of the solution comprising Clq and the determining the binding affinity using biolayer interferometry and Langmuir 1:1 binding fit model. The main aspect of the disclosure is the determination of the binding affinity of label free antibody to Clq protein, which makes the method simple and economical. The biosensor used in the present disclosure is a FAB biosensor. The biosensor used in the present disclosure is anti-Human Fab-CHl 2nd generation or anti-Human Fab-CHl (FAB2G) biosensors. In an embodiment, anti-Human Fab-CHl (FAB2G) biosensors are used for capturing and functionalizing the antibody. In an embodiment of the present disclosure, there is provided a method to determine the binding affinity of antibody to human Clq protein, wherein the said binding is determined by BLI technology. In another embodiment of present disclosure, there is provided a method to determine the binding affinity of antibody to Clq protein, wherein the said binding is determined by BLI technology. In an embodiment of present disclosure, there is provided a method to determine the binding affinity of antibody to Clq protein, wherein the thickness of bio layer is analyzed by the analysis of interference pattern of white light from two surfaces. In an embodiment of the present disclosure, there is provided a method to determine the binding affinity of antibody to Clq protein using BLI technology wherein the sensor used is a biosensor. In an embodiment of the present disclosure, there is provided a method to determine the binding affinity of antibody to Clq protein, wherein said biosensor is anti-Human Fab-CHl (FAB2G) biosensor. In an embodiment of the present disclosure, there is provided a method to determine the binding affinity of antibody to Clq protein, wherein anti-Human Fab-CHl (FAB2G) biosensors were hydrated in assay buffer followed by capturing antibodies (analyte) on anti-Human Fab-CHl (FAB2G) biosensors, blocking unbound surfaces on the biosensors with human IgG Fab fragment and dipping the anti-Human Fab-CHl (FAB2G) biosensors with captured antibodies into a solution comprising Clq protein. In an embodiment of the present disclosure, there is provided a method to determine the binding affinity of antibody to Clq protein, wherein the binding affinity of the antibody to human Clq protein is determined by Langmuir 1: 1 binding fit model. In an embodiment of the present disclosure, there is provided a method to determine the binding affinity of antibody to Clq protein, wherein the method is devoid of chemical labelling or tagging of the antibody and binding is determined directly without any modification of the antibody. In an embodiment of the present disclosure, there is provided a method to determine the binding affinity of IgG to human Clq protein using BLI technology, wherein fixed concentration of IgG was captured on anti-Human Fab-CHl (FAB2G) biosensor, blocking unbound surfaces on the anti-Human Fab-CHl (FAB2G) biosensor with human IgG Fab fragment and dipping the anti-Human Fab-CHl (FAB2G) biosensor with the captured IgG into human Clq protein solution. Thereafter, the binding affinity of IgG to human Clq binding complex was determined by Langmuir 1 : 1 binding fit model. In an embodiment of the present disclosure, there is provided a method to determine the binding affinity of IgGl/IgG2 to human Clq protein using BLI technology, wherein fixed concentration of IgGl/IgG2 was captured on anti- Human Fab-CHl (FAB2G) biosensor, blocking unbound surfaces on the anti- Human Fab-CHl (FAB2G) biosensor with human IgG Fab fragment and dipping the anti-Human Fab-CHl (FAB2G) biosensor with the captured IgGl/IgG2 into human Clq protein solution. Thereafter, the binding affinity of IgGl/IgG2 to human Clq binding complex was determined by Langmuir 1: 1 binding fit model.

077] In an embodiment of the present disclosure, there is provided a method of determining the binding affinity of Bevacizumab to human Clq protein.

078] In an embodiment of the present disclosure, there is provided a method of determining the binding affinity of Trastuzumab to human Clq protein.

079] In an embodiment of the present disclosure, there is provided a method of determining the binding affinity of Denosumab to human Clq protein.

080] In an embodiment of the present disclosure, the assay buffer used in the BLI based method to determine the binding affinity of an antibody to Clq protein is selected from HEPES buffer, Phosphate buffer, Tris buffer and the likes. Optionally, chelating agents, detergents, reducing agents, non-ionic surfactants either individually or in combinations thereof may be added to the HEPES buffer, Phosphate buffer, Tris buffer and the likes to provide an assay buffer at pH 7.4± 0.2.

081] In a more specified embodiment, the assay buffer used in the BLI based method to determine the binding affinity of an antibody to Clq protein is 1XHBS-EP+ buffer (pH 7.4).

082] In an embodiment of the present disclosure, the regeneration buffer used in the BLI based method to determine the binding affinity of an antibody to Clq protein is selected from lmM-100 mM Glycine-HCl and the likes. 083] In a more specified embodiment, the regeneration buffer used in the BLI based method to determine the binding affinity of an antibody to Clq protein is preferably 10 mM Glycine-HCl (pH 2.0).

084] In an embodiment of the present disclosure, the blocking agent used in the based method to determine the binding affinity of an antibody to Clq protein is selected from IgG Fab fragment and the likes.

085] In a more specified embodiment, the blocking agent used in the BLI based method to determine the binding affinity of an antibody to Clq protein is preferably Human IgG Fab fragment (50 pg/ml).

086] In an embodiment of the present disclosure, the BLI based method to determine the binding affinity of an antibody to Clq protein is carried out at a temperature ranging from 25 °C and 30°C.

087] In a more specified embodiment, the BLI based method to determine the binding affinity of an antibody to Clq protein is preferably is carried out at a temperature of 30°C.

088] In an embodiment of the present disclosure, the BLI based method to determine the binding affinity of an antibody to Clq protein is carried out at a pH ranging from 7.0 to 8.0.

089] In a more specified embodiment, the BLI based method to determine the binding affinity of an antibody to Clq protein is preferably is carried out using an assay buffer at pH 7.4 and a regeneration buffer at pH 2.0.

090] In an embodiment of the present disclosure, the BLI based method to determine the binding affinity of an antibody to Clq protein is carried out at a loading threshold of the antibodies ranging from 0.3nm to 2.5 nm. 091] In a more specified embodiment, the BLI based method to determine the binding affinity of an antibody to Clq protein is preferably is carried out at antibody loading threshold ranging from 1.5nm to 2.5nm.

092] In an embodiment of the present disclosure, the BLI based method to determine the binding affinity of an antibody to Clq protein is carried out at an association time ranging from 60 secs to 90 secs.

093] In a more specified embodiment, the BLI based method to determine the binding affinity of an antibody to Clq protein is preferably is carried out at an association time more than 60 secs.

094] In an embodiment of the present disclosure, the BLI based method to determine the binding affinity of an antibody to Clq protein is carried out at a shake speed/flow rate for evaluating the effect of association and dissociation ranging from 200 rpm to 300 rpm.

095] In a more specified embodiment, the BLI based method to determine the binding affinity of an antibody to Clq protein is preferably is carried out at a shake speed/flow rate of 300 rpm and 200 rpm for association and dissociation respectively.

096] In an embodiment, an in-vitro assay kit comprising reagents for determining binding affinity of an antibody to a Clq protein using a biolayer interferometry (BLI) based method, wherein the method uses FAB biosensors such as FAB2G biosensors.

097] In an embodiment, the in-vitro assay kit also comprises a buffer, the antigen of the antibody (assuming that the antigen should not be biotinylated) and/or any compound that is suitable for implementing the method of the invention, such as albumin, a surfactant and/or a preservative, as disclosed above in the description. 098] In an embodiment, the reagents of the in-vitro assay kit are contained in one or several container(s), preferably several containers.

099] In an embodiment, the in-vitro assay kit comprises an adsorbent irradiated plastics multi- well tray as a base surface.

100] In another embodiment, the binding affinity of antibodies to Clq protein disclosed. The antibodies used the biolayer interferometry (BLI) based method to determine binding affinity of an antibody to a Clq protein are selected from Bevacizumab, Trastuzumab, Cetuximab, Rituximab, Tocilizumab, Omalizumab, Golimumab, Denosumab, Ustekinumab, Vedolizumab, Ipilimumab, Brentuximab-Vedotin, Trastuzumab-emtansine, Pertuzumab, Ocrelizumab, Daratumumab, Secukinumab, Evolocumab, Atezolizumab, Adalimumab, Ramucirumab, Olaratumab, Infliximab or Siltuximab.

101] Although the subject matter has been described in considerable detail with reference to certain examples and applications thereof, other applications are possible.

EXAMPLES

102] The disclosure will now be described with the working examples. Unless defined otherwise all the technical and scientific terms used herein have the same meaning as commonly understood to a person having ordinary skill in the art. It is further stated that the present disclosure is not limited to any particular methods and experiments described herein. The examples are for exemplary purposes only and variations to the particular examples are possible and encompassed. The examples provided herein will help in better understanding of the present invention.

103] The present disclosure provides a tag-free biolayer interferometry (BLI) based method to determine the binding affinity of therapeutic monoclonal antibodies (mAbs) to Clq protein. The method does not use tags or label such as biotin- streptavidin that aid in measuring the real time, native interactions among protein molecules. In the present study, mAbs were captured on the biosensor surface followed by blocking the leftover gaps on biosensor surface with human Fab. Clq binding to mAbs was measured by immersing mAb-captured biosensors in different dose ranges of Clq solution. In order to reuse the biosensors’, functionalized surfaces are regenerated at the end of each cycle by removing the residual bound mAb-Clq complex or mAb (FIG.l and FIG.2). Response is measured in real-time as spectral shift (nanometers - nm) that is displayed on a sensorgram. Furthermore, kinetic constants like rate of association and dissociation constants (k_a, kd), and equilibrium dissociation constant (KD) are derived from the spectral shift.

A. BLI method development

The tag-free BLI method was developed through multilevel optimization of following factors viz. a. Evaluation of specificity of FAB2G biosensor to mAb

The specificity of FAB2G biosensors to mAb was evaluated by exposing the FAB2G biosensors directly to mAbs (IgG). In the process of evaluation, FAB2G biosensors and other biosensors HISK and SAX were immersed in 100 pg/mL mAbs solution for 90 sec. The binding of mAbs to biosensors were recorded in terms of binding response (nm). As shown in FIG.3, significant and stable binding response (nm) was observed only in FAB2G biosensors but not in HISK and SAX biosensors. The results sates that the interaction between FAB2G biosensors and mAbs was specific. In the FIG.3 however, HISK (green) and SAX (orange) biosensors were superimposed and not distinguishable. b. Evaluation of specificity of FAB2G biosensor to Clq protein

The specificity of FAB2G biosensors to Clq protein was evaluated by exposing the FAB2G biosensors directly to Clq protein. In the process of evaluation, FAB2G biosensors and other irrelevant biosensors HISK and SAX, were immersed in 50 nM of Clq protein solution for 90 sec. The binding of Clq protein to biosensors was recorded in terms of binding response (nm). As shown in FIG.4, significant binding response (nm) was observed in FAB2G, HISK and SAX biosensors and the binding of Clq protein on biosensors surfaces were found to be unstable at baseline2. The interactions between biosensors and Clq protein were non-specific. This could be due to the complexity of Clq protein structure (Hetero-trimer). c. Non-specific interactions between FAB2G biosensors and Clq protein prevented by blocking

In this method, initially mAbs were captured on surface of biosensors. The preoccupied mAbs minimize the direct interaction between surface of FAB2G biosensor and Clq protein (Table 1). However, there was a possibility for non-specific interactions between leftover gaps on mAbs captured biosensors and Clq protein. These minimal interactions were prevented by blocking the leftover gaps on mAbs captured biosensors with Fab (IgGl molecule contains only Fab portion without Fc). This blocking step was introduced between loading and baseline2 steps (FIG.5 and Table 1) and that completely prevented the interactions between FAB2G sensor surface and Clq protein.

Table 1: Binding response of biosensors with and without blocking

Optimization of experimental conditions and parameters a. Selection of buffer

Stability of loading or capture molecules on biosensors surface played an important role in the assay to get stable, accurate and consistent results. Two buffers viz. IX HBS-EP+ buffer (pH 7.4) and IX PBS (pH 7.4), were evaluated for the stability of loading mAb on sensors surface. An undesired drift (indicating with black arrow in FIG.6B) in the baseline2 was observed immediately after mAb capture with IX PBS buffer. Contrastingly, capture was stable on biosensors surface when using in IX HBS-EP+ buffer at basline2 (indicating with black arrow in FIG.6A). In conclusion, 1XHBS-EP+ buffer was found to be optimum for assay. a. Optimization of assay temperature

Temperature is one of the most critical parameters which affects the binding kinetics significantly. To obtain the optimum temperature, the assay was performed at 25°C and 30°C. As shown in FIG.7, better curve fitting was observed at 30°C compared to 25 °C. Therefore, 30°C was considered as optimum temperature to study the binding kinetics of mAb with Clq. b. Optimization of loading threshold

Loading of threshold mAb (IgGl) levels on anti-Fab 2nd Gen (FAB2G) biosensors is one of the important parameters for binding kinetic studies. For stable, consistent, and accurate results, single loading threshold level should be fixed for all biosensors. To find the optimum loading threshold level, different loading levels viz. 0.3, 0.5, 1.0 and 1.5 nm were evaluated. At 0.3 and 0.5 nm loading levels (indicated with green horizontal bar in FIG.8A and FIG.8B), the response on biosensors went beyond the target levels quickly and showed response > 1.0 nm (indicated with blue horizontal bar in FIG.8A and FIG.8B). This could be due to high molecular weight (-148 kDa) of mAb. At 1.0 and 1.5 nm loading levels (indicated with green horizontal bar in (FIG.7C and FIG.7D), a controlled uniform response was observed in all biosensors and showed > 1.0 nm and > 1.5 nm response (indicated with blue horizontal bar in FIG.8C and FIG.8D), respectively. At loading levels 0.3, 0.5 and 1.0 nm, all the biosensors showed minimal response of ~ 1.0 nm (indicated with blue horizontal bar in FIGS.8A-8C). The threshold loading levels should be greater than the minimal response (-1.0 nm). Therefore, higher capture level 1.5 nm was selected as optimum loading threshold. c. Optimization of association time

The association time (sec) may have a significant role in the binding of ligand to the captured analyte. Optimum association time is required to establish maximum and stable analyte-ligand complexes on biosensors. Therefore, assay association times of 60 and 90 sec (FIG.9A and FIG.9B, respectively) were evaluated. A 90 sec association time was found to be optimum over 60 sec because the response reached saturation point and an improved the curve fitting was also observed. d. Optimization of shake speed

The shake speed/flow rate may have a significant effect on the association and dissociation during kinetics experiment. Therefore, assay shake speed/flow rate of 200, 300 and 300-200 rpm were evaluated with 90 sec association time. Based on the results (FIG.10A, FIG.10B and FIG.10C), association and dissociation at 300 rpm and 200 rpm, respectively as found to be optimum and with better curve fitting. ] The experiments performed with optimized conditions as mentioned in Table 2 and Table 3. The list of equipment, materials and reagents used in study were mentioned in Table 4.

Table 2: Assay conditions for determining the binding affinity of mAb (IgGl/IgG2) to Clq

Table 3: Program steps for determining the binding affinity of mAb (IgGl/IgG2) to Clq

Table 4: List of equipment, materials and reagents used in studies

Example 1 Method to determine binding affinity of Bevacizumab to Clq protein Bevacizumab - Clq binding affinity was determined by using anti-human Fab- CH1 (FAB2G) biosensor. No chemical modification viz. labelling, or tagging was done to the recombinantly expressed and purified IgG monoclonal antibody Bevacizumab. As a result of elimination of the chemical modification step the method provided here became a simple and robust to determine the Clq binding. The anti-Human Fab-CHl (FAB2G) biosensors were hydrated in assay buffer for 10 min prior to run which greatly reduced the non-specific interactions, followed by capturing of fixed concentration of Bevacizumab. The unbound surfaces of the biosensor were blocked with human Fab fragments (prepared inhouse). The antibody so bound to the biosensor was assayed by dipping in varying concentrations (1.5 - 50 nM) of Clq molecule. The binding affinity of Bevacizumab to Clq protein was determined by Langmuir 1 : 1 binding fit model. The relative binding affinity was determined with the reference to the standard. In this experiment, fixed concentration of Bevacizumab, 100 pg/ml was captured on anti-Human Fab-CHl (FAB2G) biosensors and unbound surfaces of biosensor were blocked with human Fab fragment. The binding of differential concentrations of human complement component Clq protein was analysed by dipping the antibody coated biosensor in ligand solution comprising Clq in concentration range of 1.5 - 50 nM (working concentrations). The working concentration range was prepared from a Clq protein solution with stock concentration ~1.0 mg/mL. Kinetic constants were determined based on association and dissociation of Bevacizumab-Clq protein through Langmuir 1 : 1 binding fit model. The active surfaces of the sensor were regenerated at the end of each cycle for reuse by removing the bound ligand and residual analyte-ligand complex using 10 mM Glycine-HCl (pH 2.0). Test procedure: The binding affinity analysis was performed with no ligand as control and with five different concentrations (1.5 - 50 nM) of the ligand Clq analyte as test. 100 pg/ml of the recombinantly expressed monoclonal antibody Bevacizumab was captured on the biosensor and unbound surfaces were further blocked with human Fab fragment. The concentration range of Clq ligand used was 1.5 - 50 nM for binding analysis. The binding assays were performed at constant temperature of 30 °C. The rest of the assay conditions steps and instrument, reagents used were as provided in the Table 2, Table 3 and Table 4.

Results KD represents the equilibrium dissociation constant between the antibody and its antigen. KD is the ratio of the antibody dissociation rate constant (kd), i.e., rate of dissociation from its antigen to the antibody association rate (k_a), i.e., rate of binding to its antigen. Affinity is the strength of binding of a single molecule to its ligand. It is typically measured and reported by the equilibrium dissociation constant (KD), which is used to evaluate and rank order strengths of bimolecular interactions. Method repeatability was used to describe the variation in successive measurements of the same variables taken under same conditions in a short period of time. The repeatability assessment is required to understand the reliability of an experiment. Table 5 given below provides results for method repeatability and reliability.

Table 5: Results from method repeatability of IgGl (Bevacizumab) with Clq

The calculated Coefficient of Variance (% CV) of equilibrium dissociation constant KD (M) for repeatability experiments from different days i.e., day 1 and day 2 was 1, 1 respectively. Inter assay precision (%CV) for average (n=10) KD of individual preparations on different days is < 5. The percent CV 20 for inter assay precision is well accepted for biological assays. In the present study percent CV for all runs was 5, thus the assay is found to be repeatable. Method suitability for drug product sample analysis - In order to analyze the suitability of the present method for the determination of binding affinity, a comparative binding analysis with reference drug product was done. The results provided in the Table 6 (given below) suggest that the given method is suitable to determine the binding affinity of the Bevacizumab to Clq protein. Table 6 and FIG.11 provides the summary of binding affinity of Bevacizumab and percent relative binding affinity values. Percent relative binding affinity is calculated by using the formula

, . ■ r, - > ■ Mean KD of internal reference standard ... „ „

%RBA (Relative Binding Affinity) = -

Mean KD of individual sample X100

Table 6: Summary of binding affinity of Bevacizumab

113] As described above binding affinity is measured and reported by the equilibrium dissociation constant KD which is used to evaluate the strength of bio molecular interactions. Smaller the KD value the greater the binding affinity. The results showed that binding affinity of Bevacizumab were found within the range of 97 -103. Thus, the binding assay provided in the present assay has a greater accuracy to determine the binding affinity of the Bevacizumab. The method provided in the present disclosure has high precision in providing the binding affinity for the Bevacizumab. This disclosure provides a method to determine the binding affinity which is economical and easy to analyze.

Example 2

Method to determine binding affinity of Trastuzumab to Clq protein

114] Trastuzumab - Clq binding affinity was determined by using anti -human Fab- CH1 (FAB2G) biosensor. No chemical modification viz. labelling, or tagging was done to the recombinantly expressed and purified IgG monoclonal antibody Trastuzumab. As a result of elimination of the chemical modification step the method provided here became a simple and robust to determine the Clq protein binding. The anti-Human Fab-CHl (FAB2G) biosensors were hydrated in assay buffer for 10 min prior to run which greatly reduced the non-specific interactions, followed by capturing of fixed concentration of Trastuzumab. The unbound surfaces of the biosensor were blocked with human Fab fragments. The antibody bound to the biosensor was assayed by dipping in varying concentrations (1.5 - 50 nM) of Clq protein solution. The binding affinity of Trastuzumab to Clq protein was determined by Langmuir 1: 1 binding fit model. The relative binding affinity was determined with the reference to the standard.

115] In this experiment, fixed concentration of Trastuzumab, 100 pg/ml was captured on anti-Human Fab-CHl (FAB2G) biosensors and unbound surfaces of biosensor were blocked with human Fab fragment. The binding of differential concentrations of human complement component Clq protein was analysed by dipping the antibody coated biosensor in ligand solution comprising Clq in concentration range of 1.5 - 50 nM. Kinetic constants were determined based on association and dissociation of Trastuzumab-Clq protein through Langmuir 1:1 binding fit model. The active surfaces of the sensor were regenerated at the end of each cycle for reuse by removing the bound ligand and residual analyte-ligand complex using 10 mM Glycine-HCl (pH 2.0). Test procedure: The binding affinity analysis was performed with no ligand as control and with five different concentrations (1.5-50 nM) of the ligand Clq analyte as test. 100 pg/ml of the recombinantly expressed monoclonal antibody Trastuzumab was captured on the biosensor and unbound surfaces were further blocked with human Fab fragment. The concentration range of Clq ligand used was 1.5 - 50 nM for binding analysis. The binding assays were performed at constant temperature of 30 °C. The rest of the assay conditions and steps used were as provided in the Table 2 and Table 3.

Results KD represents the equilibrium dissociation constant between the antibody and its antigen. KD is the ratio of the antibody dissociation rate constant (kd), i.e., rate of dissociation from its antigen to the antibody association rate (k_a), i.e., rate of binding to its antigen. Affinity is the strength of binding of a single molecule to its ligand. It is typically measured and reported by the equilibrium dissociation constant (KD), which is used to evaluate and rank order strengths of bimolecular interactions. Method repeatability was used to describe the variation in successive measurements of the same variables taken under same conditions in a short period of time. The repeatability assessment is required to understand the reliability of an experiment. Table 7 given below provides results for method repeatability and reliability.

Table 7: Results from method repeatability of IgGl (Trastuzumab) with Clq

The calculated Coefficient of Variance (%CV) of equilibrium dissociation constant KD (M) for repeatability experiments from different days i.e., Day 1 and Day 2 was 3 and 1, respectively for Trastuzumab. Inter assay precision (%CV) between KD (n=10) of individual preparations on different days is <5. A %CV of < 20 for inter assay precision is well accepted for biological assays. In the present study %CV for all runs was <5. Therefore, the assay is found to be repeatable. Method suitability for drug product sample analysis - In order to analyze the suitability of the present method for the determination of binding affinity, a comparative binding analysis with reference drug product was done. The results provided in the Table 9 (given below) suggest that the given method is suitable to determine the binding affinity of the Trastuzumab to Clq protein. Table 8 and FIG.11 provides the summary of binding affinity of Trastuzumab and percent relative binding affinity values.

121] Percent relative binding affinity is calculated by using the formula

Table 8: Summary of binding affinity of Trastuzumab

122] As described above, binding affinity is measured and reported by the equilibrium dissociation constant KD which is used to evaluate the strength of bio molecular interactions. The smaller the KD value, the greater its binding affinity. The binding affinity is affected by the presence of other molecules. The results showed that binding affinity of Trastuzumab were found within the range of 101 -102. Thus, the binding assay provided in the present assay has a greater accuracy to determine the binding affinity of the Trastuzumab. The method provided in the present disclosure has high precision in providing the binding affinity for the Trastuzumab. This disclosure provides a method to determine the binding affinity which is economical and easy to analyze.

Example 3

Method to determine binding affinity of IgG2 (Denosumab) to Clq protein

123] Denosumab - Clq binding affinity was determined by using anti -human Fab- CH1 (FAB2G) biosensor. No chemical modification viz. labelling, or tagging was done to the recombinantly expressed and purified IgG monoclonal antibody Denosumab. As a result of elimination of the chemical modification step the method provided here became a simple and robust to determine the Clq binding. The Anti-Human Fab-CHl (FAB2G) biosensors were hydrated in assay buffer for 10 min prior to run which greatly reduced the non-specific interactions, followed by capturing of fixed concentration of Denosumab. The unbound surfaces of the biosensor were blocked with human Fab fragments. The antibody so bound to the biosensor was assayed by dipping in varying concentrations (3.125-100 nM) of Clq molecule. The binding affinity of Denosumab to Clq protein was determined by Langmuir 1: 1 binding fit model. The relative binding affinity was determined with the reference to the standard. In this experiment, fixed concentration of Denosumab, 100 pg/ml was captured on anti-Human Fab-CHl (FAB2G) biosensors and unbound surfaces of biosensor were blocked with human Fab fragment. The binding of differential concentrations of human complement component Clq protein was analysed by dipping the antibody coated biosensor in ligand solution comprising Clq in concentration range of 3.125 - 100 nM. Kinetic constants were determined based on association and dissociation of Denosumab-Clq protein through Langmuir 1 : 1 binding fit model. The active surfaces of the sensor were regenerated at the end of each cycle for reuse by removing the bound ligand and residual analyteligand complex using 10 mM Glycine-HCl (pH 2.0). Test procedure: The binding affinity analysis was performed with no ligand as control and with five different concentrations (3.125-100 nM) of the ligand Clq analyte as test. 100 pg/ml of the recombinantly expressed monoclonal antibody Denosumab was captured on the biosensor and unbound surfaces were further blocked with human Fab fragment. The concentration range of Clq ligand used was 3.125-100 nM for binding analysis. The binding assays were performed at constant temperature of 30 °C. The rest of the assay conditions and steps used were as provided in Table 2 and Table 3.

Results KD represents the equilibrium dissociation constant between the antibody and its antigen. KD is the ratio of the antibody dissociation rate constant (kd), i.e., rate of dissociation from its antigen to the antibody association rate (k_a), i.e., rate of binding to its antigen. Affinity is the strength of binding of a single molecule to its ligand. It is typically measured and reported by the equilibrium dissociation constant (KD), which is used to evaluate and rank order strengths of bimolecular interactions. Method repeatability was used to describe the variation in successive measurements of the same variables taken under same conditions in a short period of time. The repeatability assessment is required to understand the reliability of an experiment. Table 9 provides results for method repeatability and reliability.

Table 9: Results from method repeatability of IgG2 (Denosumab) with Clq

128] The calculated Coefficient of Variance (% CV) of equilibrium dissociation constant KD (M) for repeatability experiments from different days i.e., day 1 and day 2 was 10, 7 respectively. Inter assay precision (%CV) for average (n=10) KD of individual preparations on different days is < 15. The 20% CV for inter assay precision is well accepted for biological assays. In the present study, the percentage CV for all runs was <12, thus the assay is found to be reproducible.

129] Method suitability for drug product sample analysis - In order to analyze the suitability of the present method for the determination of binding affinity, a comparative binding analysis with reference drug product was done. The results provided in Table 10 suggest that the given method is suitable to determine the binding affinity of the Denosumab to Clq protein. Table 10 and FIG.12 provides the summary of binding affinity of Denosumab and percent relative binding affinity values.

Table 10: Summary of binding affinity of Denosumab

130] As described above, %RBA is measured by the equilibrium dissociation constant KD which is used to evaluate the strength of bio molecular interactions. The smaller the KD value, the greater the binding affinity. The binding affinity is affected by the presence of other molecules. The results showed that %RBA of Denosumab was found to be within the range of 96-102. The method provided in the present disclosure has high precision in providing the binding affinity for IgG2 with Clq. This disclosure provides a method to determine the binding affinity which is economical and easy to analyze.

131] In an embodiment of the present disclosure, a biolayer interferometry (BLI) based method to determine binding affinity of an antibody to a C1Q protein comprising: a FAB biosensor; and capturing a fixed concentration of a tag-free antibody on the FAB biosensor, wherein determining the binding affinity of the tag- free antibody to the Clq protein does not involve use of tags such as biotinstreptavidin.

132] In an embodiment of the present disclosure, the biolayer interferometry (BLI) based method to determine binding affinity of an antibody to a C1Q protein, wherein the FAB biosensor is FAB2G biosensor.

133] In an embodiment of the present disclosure, a biolayer interferometry (BLI) based method to determine binding affinity of an antibody to a Clq protein comprising: a) capturing a threshold of antibody on a FAB biosensor by immersing a FAB biosensor in an antibody solution; b) blocking unbound spaces on the FAB biosensor by immersing the FAB biosensor in a blocking agent such as FAB fragment; c) measuring the binding of the Clq protein to the antibody captured on a surface of the FAB biosensor by immersing the FAB biosensor in a Clq protein solution; and d) measuring the binding affinity of the antibody to the Clq protein by Langmuir 1:1 binding fit model and measuring kinetic association constant (ka), dissociation constants (kd), and equilibrium dissociation constant (KD). 134] In an embodiment of the present disclosure, wherein the method is tag-free and does not involve use of chemical tags such as biotin-streptavidin for capturing or functionalizing the antibody on the FAB biosensor.

135] In an embodiment of the present disclosure, wherein the antibody is an IgG or an IgM antibody.

136] In an embodiment of the present disclosure, wherein the antibody is a monoclonal antibody.

137] In an embodiment of the present disclosure, wherein the monoclonal antibody is an IgGl or an IgG2 antibody.

138] In an embodiment of the present disclosure, wherein the IgGl or the IgG2 antibody is human, recombinant, humanized, chimeric, murine and combinations thereof.

139] In an embodiment of the present disclosure, a biolayer interferometry (BLI) based method to determine binding affinity of a monoclonal antibody to a Clq protein comprising: a) Measuring of a baseline by immersing a FAB biosensor in an assay buffer; b) Capturing a threshold of the monoclonal antibody on the FAB biosensor by immersing the FAB biosensor in a monoclonal antibody solution; c) Blocking unbound spaces on the FAB biosensor by immersing the FAB biosensor in a blocking agent; d) Measuring of a baseline 2 by immersing the FAB biosensor in the assay buffer; e) Immersing the FAB biosensor in a Clq protein solution; f) Measuring the binding of the Clq protein to the monoclonal antibody captured on a surface of the FAB biosensor; g) Measuring a dissociation of bound Clq protein from the monoclonal antibody on the surface of the FAB biosensor by immersing the FAB biosensor in the assay buffer; and h) Determining the binding affinity of the monoclonal antibody to the Clq protein is determined by Langmuir 1: 1 binding fit model by measuring kinetic association constant (ka), dissociation constants (kd), and equilibrium dissociation constant (KD).

140] In an embodiment of the present disclosure, wherein the monoclonal antibody is an IgGl or an IgG2 antibody.

141] In an embodiment of the present disclosure, wherein the IgGl or the IgG2 antibody is selected from Bevacizumab, Trastuzumab, Cetuximab, Rituximab, Tocilizumab, Omalizumab, Golimumab, Denosumab, Ustekinumab, Vedolizumab, Ipilimumab, Brentuximab-Vedotin, Trastuzumab-emtansine, Pertuzumab, Ocrelizumab, Daratumumab, Secukinumab, Evolocumab, Atezolizumab, Adalimumab, Ramucirumab, Olaratumab, Infliximab or Siltuximab.

142] In an embodiment of the present disclosure, wherein the assay buffer is selected from but not limited to phosphate buffer, Tris buffer, HEPES buffer and combinations thereof.

143] In an embodiment of the present disclosure, wherein the assay buffer optionally includes chelating agent, detergent, reducing agent, salts, amino acids either individually or combinations thereof.

144] In an embodiment of the present disclosure, wherein the assay buffer has a pH ranging from 7.0 to 8.0. 145] In an embodiment of the present disclosure, wherein the assay buffer islXHBS- EP+ (pH 7.4).

146] In an embodiment of the present disclosure, wherein the blocking agent is human IgG Fab fragment diluted in the assay buffer.

147] In an embodiment of the present disclosure, wherein the blocking agent has a concentration ranging from 50 pg/ml to 100 pg/ml.

148] In an embodiment of the present disclosure, wherein the Clq protein solution is a two-fold serially diluted human Clq protein solution.

149] In an embodiment of the present disclosure, wherein the human Clq protein solution has a concentration ranging from 1.5 - 50 nM (IgGl) or 3.125 - 100 nM (IgG2).

150] In an embodiment of the present disclosure, wherein the method is performed at constant temperature ranging from 25 °C to 30°C.

151] In an embodiment of the present disclosure, wherein the FAB biosensor is hydrated in assay buffer for 10 min prior to run to reduce the non-specific interactions.

152] In an embodiment of the present disclosure, wherein removal of a residual monoclonal antibody-Clq protein complex and a residual monoclonal antibody from the surface of the FAB biosensor is performed by immersing the FAB biosensor in a regeneration buffer such as 10 mM Glycine-HCl (pH 2.0) followed by the assay buffer.

153] In an embodiment of the present disclosure, an in-vitro assay kit comprising reagents for determining binding affinity of an antibody to a Clq protein using biolayer interferometry (BLI) based method, wherein the method uses FAB sensors such as FAB2G biosensor and is devoid of use of chemical tags such as biotin-streptavidin. The present disclosure has been described in terms of certain preferred embodiments and illustrations thereof, other embodiments and modifications to preferred embodiments may be possible that are within the principles and spirit of the invention. The above descriptions and figures are therefore to be regarded as illustrative and not restrictive. Thus, the scope of the present disclosure is defined by the appended claims and includes both combinations and sub combinations of the various features described herein above as well as variations and modifications thereof, which would occur to persons skilled in the art upon reading the foregoing description.

Claims

CLAIMS:

What is claimed is:

1) A biolayer interferometry (BLI) based method to determine binding affinity of an antibody to a C IQ protein comprising: a FAB biosensor; and capturing a fixed concentration of a tag-free antibody on the FAB biosensor, wherein determining the binding affinity of the tag- free antibody to the Clq protein does not involve use of tags such as biotin-streptavidin.

2) The biolayer interferometry (BLI) based method to determine binding affinity of an antibody to a C1Q protein of claim 1, wherein the FAB biosensor is FAB2G biosensor.

3) A biolayer interferometry (BLI) based method to determine binding affinity of an antibody to a Clq protein comprising: a) capturing a threshold of antibody on a FAB biosensor by immersing a FAB biosensor in an antibody solution; b) blocking unbound spaces on the FAB biosensor by immersing the FAB biosensor in a blocking agent such as FAB fragment; c) measuring the binding of the Clq protein to the antibody captured on a surface of the FAB biosensor by immersing the FAB biosensor in a Clq protein solution; and d) measuring the binding affinity of the antibody to the Clq protein by Langmuir 1:1 binding fit model and measuring kinetic association constant (ka), dissociation constants (kd), and equilibrium dissociation constant (KD). 4) The method as claimed in claim 3, wherein the method is tag-free and does not involve use of chemical tags such as biotin-streptavidin for capturing or functionalizing the antibody on the FAB biosensor.

5) The method of claim 5, wherein the antibody is an IgG or an IgM antibody.

6) The method of claim 3, wherein the antibody is a monoclonal antibody.

7) The method of claim 5, wherein the monoclonal antibody is an IgGl or an IgG2 antibody.

8) The method of claim 7, wherein the IgGl or the IgG2 antibody is human, recombinant, humanized, chimeric, murine and combinations thereof.

9) A biolayer interferometry (BLI) based method to determine binding affinity of a monoclonal antibody to a Clq protein comprising: a) Measuring of a baseline by immersing a FAB biosensor in an assay buffer; b) Capturing a threshold of the monoclonal antibody on the FAB biosensor by immersing the FAB biosensor in a monoclonal antibody solution; c) Blocking unbound spaces on the FAB biosensor by immersing the FAB biosensor in a blocking agent; d) Measuring of a baseline 2 by immersing the FAB biosensor in the assay buffer; e) Immersing the FAB biosensor in a Clq protein solution; f) Measuring the binding of the Clq protein to the monoclonal antibody captured on a surface of the FAB biosensor; g) Measuring a dissociation of bound Clq protein from the monoclonal antibody on the surface of the FAB biosensor by immersing the FAB biosensor in the assay buffer; and h) Determining the binding affinity of the monoclonal antibody to the Clq protein is determined by Langmuir 1: 1 binding fit model by measuring kinetic association constant (ka), dissociation constants (kd), and equilibrium dissociation constant (KD).

10) The method of claim 9, wherein the monoclonal antibody is an IgGl or an IgG2 antibody.

11) The method of claim 9, wherein the IgGl or the IgG2 antibody is selected from Bevacizumab, Trastuzumab, Cetuximab, Rituximab, Tocilizumab, Omalizumab, Golimumab, Denosumab, Ustekinumab, Vedolizumab, Ipilimumab, Brentuximab- Vedotin, Trastuzumab-emtansine, Pertuzumab, Ocrelizumab, Daratumumab, Secukinumab, Evolocumab, Atezolizumab, Adalimumab, Ramucirumab, Olaratumab, Infliximab or Siltuximab.

12) The method of claim 9, wherein the assay buffer is selected from but not limited to phosphate buffer, Tris buffer, HEPES buffer and combinations thereof.

13) The method of claim 9, wherein the assay buffer optionally includes chelating agent, detergent, reducing agent, salts, amino acids either individually or combinations thereof.

14) The method of claim 9, wherein the assay buffer has a pH ranging from 7.0 to 8.0.

15) The method of claim 9, wherein the assay buffer islXHBS-EP+ (pH 7.4).

16) The method of claim 9, wherein the blocking agent is human IgG Fab fragment diluted in the assay buffer.

17) The method of claim 9, wherein the blocking agent has a concentration ranging from 50 pg/ml to 100 pg/ml. 18) The method of claim 9, wherein the Clq protein solution is a two-fold serially diluted human Clq protein solution.

19) The method of claim 19, wherein the human Clq protein solution has a concentration ranging from 1.5 - 50 nM (IgGl) or 3.125 - 100 nM (IgG2). 20) The method of claim 9, wherein the method is performed at constant temperature ranging from 25°C to 30°C.

21) The method of claim 9, wherein the FAB biosensor is hydrated in assay buffer for 10 min prior to run to reduce the non-specific interactions.

22) The method of claim 9, wherein removal of a residual monoclonal antibody- Clq protein complex and a residual monoclonal antibody from the surface of the FAB biosensor is performed by immersing the FAB biosensor in a regeneration buffer such as 10 mM Glycine-HCl (pH 2.0) followed by the assay buffer.

23) An in-vitro assay kit comprising reagents for determining binding affinity of an antibody to a Clq protein using biolayer interferometry (BLI) based method, wherein the method uses FAB sensors such as FAB2G biosensor and is devoid of use of chemical tags such as biotin-streptavidin.