FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
AND THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See Section 10; Rule 13)
“METHOD FOR DETERMINING POTENCY OF BIOLOGICAL PRODUCTS”
Name of the Applicant:
SERUM INSTITUTE OF INDIA PRIVATE LIMITED
5 Address of the Applicant:
SERUM INSTITUTE OF INDIA PRIVATE LIMITED 212/2, Off Soli Poonawalla Road, Hadapsar, Pune - 411028, Maharashtra, India
10
Nationality: IN
The following specification particularly describes the invention and the manner in which it is to be performed.

TECHNICAL FIELD
The present disclosure relates to bio-analytical methods. Particularly, the present disclosure relates to methods for determining potency of biological products.
BACKGROUND OF THE DISCLOSURE
5 All publications herein are incorporated by reference to the same extent as if each individual
publication or patent application was specifically and individually indicated to be incorporated
by reference. The following description includes information that may be useful in
understanding the present invention. It is not an admission that any of the information provided
herein is prior art or relevant to the presently claimed invention, or that any publication
10 specifically or implicitly referenced is prior art.
Biologicals are a diverse group of medicines which includes vaccines, growth factors, immune
modulators, monoclonal antibodies, as well as products derived from human blood and plasma.
What distinguishes biologicals from other medicines is that these are generally proteins
purified from living culture systems or from blood, whereas other medicines are considered as
15 ‘small molecules’ and are either made synthetically or are purified from plants.
Potency of a biological product/ preparation is the quantitative measurement of the biological
activity of a given product/ preparation. Biological activity is a critical quality attribute, and
therefore, potency testing is an essential component of quality control. Potency test is a quality
assurance tool that is linked to the safety and efficacy of a biological product/ preparation at
20 administered doses. Potency testing measures the functional integrity of the biological product
and is intended to ensure that it retains immunocompetence, i.e., the ability to stimulate the desired immune response, in its final formulation throughout the shelf life of the products.
Monitoring potency of biological products is required to ensure its clinical effectiveness. The
potency assays should be designed to measure the binding of the antigen receptors on host
25 cells, the inhibition of antigen entry, reflecting the biological activity of the biological product
in vivo. Further, potency determination demonstrates that only those products that meet defined specifications/ acceptance criteria are administered during all phases of clinical investigation and after approval.
Potency tests, along with a number of other tests, are performed as part of product conformance
30 testing, comparability studies, and stability testing. These tests are used to measure product
2

attributes associated with product quality and manufacturing controls, and are performed to
assure identity, purity, strength (potency), and stability of products used during all phases of
clinical study. Similarly, potency measurements are used to demonstrate that only product lots
that meet defined specifications or acceptance criteria are administered during all phases of
5 clinical investigation and following market approval.
Potency (expressed in units) is the quantitative measure of biological activity based on the attribute of the product which is linked to the relevant biological properties, whereas, quantity (expressed in mass) is a physicochemical measure of protein content.
The generally accepted method for testing potency is the animal challenge test, wherein the
10 animal is inoculated with the recommended dose and the ability of the product to reduce the
incidence of the disease after a prescribed time is measured. Alternative methods include animal substitution and serological substitution.
Potency assays should provide a quantitative measure of the activity (or activities) of the
biological product (such as monoclonal antibody/ mAb) relevant to its mechanism of action.
15 Potency assays should be sufficiently sensitive to detect any differences in the mAb of potential
clinical importance. Potency assays are also an important measure of manufacturing consistency and should be sensitive enough to detect changes in the mAb that may impact its activity and function(s), such as binding affinity.
Potency assays can be one of the two types: ligand-binding (measure interaction of drug with
20 its target) or functional assays (measure a biological response). There are several different
assays that are used to measure immune responses to vaccination, including calculating titers via cell-based methods, such as the Rapid Fluorescent Focus Inhibition Test (RFFIT), the fluorescence antibody virus neutralization test (FAVN), the simplified fluorescence inhibition microtest and pseudotype virus micro-neutralization assays etc.
25 The RFFIT has been considered as the gold standard test for assessing serum viral neutralizing
antibodies. The test has many advantages; however, the bioassay has different variants such as use of biological materials like BHK-21 cells and virulent rabies challenge virus, which may not be feasible in any virology laboratory, and a greater coefficient of variation limit criterion is required for this viral neutralization assay. Further, the usage of an international standard
30 reference sample requirement for each sample run makes the assay costly.
3

The fluorescent antibody virus neutralization (FAVN) test is useful for detecting antibodies.
However, it has several limitations like it is costly and labor intensive, requires fixed virus,
challenge virus standard strain, cell lines, cold acetone, monoclonal antibody, expensive
fluorescence microscope and anti-mouse fluorescein isothiocyanate (FITC) conjugate. Also,
5 live virus poses a risk to the laboratory staff. Also, the anti-mouse FITC-conjugate imported
from overseas can be suddenly difficult to import for several reasons.
Currently, three tests are approved by WHO for the estimation of neutralizing antibodies after vaccination: the mouse neutralization test (MNT), the rapid fluorescent focus inhibition test (RFFIT), and the fluorescent antibody virus neutralization (FAVN) test.
10 In vivo methods require the sacrifice of large numbers of animals and often suffer from long
turn-around times, and a lack of robustness and reproducibility. Many regulatory agencies and manufacturers prefer in vitro potency tests for lot release.
Moreover, it is important for biopharma companies to characterize the quality attributes of therapeutic mAbs to ensure product safety, potency and consistency.
15 It has also become apparent that mAbs are more heterogeneous than previously thought or
reported. During manufacture, various forms of microheterogeneity in size, charge and other parameters occur due to enzymatic processes or spontaneous degradation and modifications. mAbs undergo chemical degradation via several different mechanisms, including oxidation, deamidation, isomerization and fragmentation, that result in the formation of various charge
20 variants and heterogeneity, thus modifying their isoelectric pH (pI) values.
Therapeutic mAbs are produced by cell culture followed by purification of the product and its formulation and storage. mAbs are produced along with their variants, many of which are removed at various stages of processing. These variants could be conformational isomers, charge variants, glycan variants, or size variants.
25 Charge variants having pI lesser than that of main mAb are called acidic variants, whereas
those with pI greater than that of the main molecule are called basic variants. Charge variants have been identified as critical quality attributes that must be assessed throughout development and the commercial product lifecycle to meet the regulatory requirements. The process and analytical control for charge variants can be challenging due to the heterogeneity from both
30 post translational modifications (PTMs) such as glycosylation and C-terminal lysine clipping
as well as chemical modifications such as oxidation and deamidation. These post translational
4

modifications can alter the charge distribution on the surface of the mAb and result in charge variants. Such charge variants impact the quality, stability, and potency of a mAb.
Typical acidic variants are species with deamidation, glycation, the sialylated glycan, trisulfide,
etc. Basic variants include C-terminal unprocessed lysine, C-terminal amidation, isomerization
5 of aspartate residues, and so on. Those protein charge variants can be measured and
characterized by charge indicating analytical assays, imaged capillary isoelectric focusing (icIEF), ion-exchange chromatography (IEC), or liquid chromatography/mass spectrometry (LC/MS).
Particularly, charge heterogeneity in the mAb is known to impact its interactions, structure,
10 and activity. It has been reported that shifts of approximately one isoelectric point (pI) or more
and charge variants resulting from chemical modification potentially affect the tissue
distributions and pharmacokinetics (PK) profiles of mAbs (Igawa Tet al 2010; Sharifi J, et al
1998). Further, charge variant patterns, traditionally monitored using cation exchange
chromatography or capillary isoelectric focusing, act as a fingerprint of the manufacturing
15 process and deviations in peak areas or the appearance of new peaks require in depth
characterization to ensure that no additional or undesired PTM has been introduced. Such
alterations in the molecular surface charge distribution can affect antigen or receptor binding
or may cause conformational changes that may increase the risk of aggregate formation and
potential induction of immunogenicity (Florian Füssl et. al. 2018). Also, the analysis of charged
20 variants is a regulatory requirement for bio-therapeutic protein. (ICH Q6B)
Rabishield (Rab1) contains 60-95 % main mAb whereas charge variants constitute 10-30% (acidic, basic).
There are multiple analytical techniques like ion exchange chromatography (IEX), capillary
isoelectric focusing (cIEF) or imaged capillary isoelectric focusing (icIEF) used for charge
25 variant analysis. However, these techniques only help in isolating & subsequently estimating
the amount of charge variants present in mAb but cannot be used for determining the potency of charge variants in mAb.
As far as mAb potency testing is concerned, existing assays like RFFIT (rapid fluorescent focus
inhibition test) and the FAVN (fluorescent antibody virus neutralization) test exhibit several
30 drawbacks, as they are less easily standardized, are costly, labour-intensive, require the use of
5

biological material MNK /BHK 21 cells, use of virulent live Rabies Virus, containment facilities (BSL 2/3), and skilled professionals.
Therefore, there is an unmet urgent need to develop and standardize simple, safe, rapid, cost-
effective, platform technique for potency determination of biological products, in particular
5 charge variants of monoclonal antibodies (as a potential alternative to existing RFFIT and
FAVN assays).
SUMMARY OF THE DISCLOSURE
The present disclosure provides a method for determining potency of a biological product by
ELISA comprising the steps of:
10 - coating a protein or an antigen to an ELISA plate;
- blocking unbound sites in the ELISA plate;
- detecting the protein or the antigen by;
(i) adding a detection antibody-enzyme conjugate, or
(ii) adding a primary antibody and adding a secondary antibody-enzyme
15 conjugate; and
- adding a substrate for the enzyme for eliciting a measurable signal.
In some embodiments, the present disclosure provides a method for determining potency of biological products by indirect ELISA.
The method for determining potency of biological products of the present disclosure provides
20 a platform, reliable, rapid, safe and cost-effective method for determining the potency of
monoclonal antibodies and also of charge variants of monoclonal antibodies, which can be used as an alternative to existing RFFIT, FAVN potency testing methods. The method uses inactivated strains of virus as antigens and hence avoids use of BSL3 facility, sacrifice of mice, and live virus handling for potency determination.
25 The present disclosure also provides a method for determining potency of charge variants of
rabies monoclonal antibodies, the method comprising:
a. identifying charge variants in rabies monoclonal antibody;
b. separating the charge variants; and
c. determining potency of an acid charge variant and potency of a basic charge variant of the
30 rabies monoclonal antibody by the method as described above.
6

The present disclosure further provides an ELISA assay kit to determine potency of rabies monoclonal antibodies, comprising:
a. rabies antigen,
b. rabies monoclonal antibodies,
5 c. horse radish peroxidase,
d. tetramethylbenzindine (TMB), and
e. optionally mouse anti human antibodies;
wherein the rabies monoclonal antibodies comprise one or more of main form, acid
charge variants and basic charge variants of the rabies monoclonal antibodies; and
10 wherein the potency is determined by the method as described above.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
In order that the disclosure may be readily understood and put into practical effect, reference
will now be made to exemplary embodiments as illustrated with reference to the accompanying
15 figures. The figures together with detailed description below, are incorporated in and form part
of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure where:
Figure-1 illustrates the graphical representation of delta absorbance versus log concentration of the primary antibody (rabies monoclonal antibody);
20 Figure-2a illustrates the Isoelectric Focusing (IEF) images for rabies monoclonal antibody
from upstream process and downstream process;
Figure-2b illustrates a magnified IEF image of rabies monoclonal antibody showing two distinct bands for the main form and the charge variant (isoform);
Figure-3 illustrates the charge variant separation process using cation exchange
25 chromatography;
Figure-4 illustrates the potency of acidic and basic charge variants of rabies monoclonal antibody using in accordance with the method of present disclosure;
Figure-5 illustrates the specificity of rabies monoclonal antibody for rabies antigen; and
7

Figure-6: Capillary isoelectric focusing (cIEF) of acidic and basic variants along with main form of rabies mAb overlay with rabies mAb IHRS positive control.
OBJECTS OF THE DISCLOSURE
Some of the objects of the present disclosure, which at least one embodiment herein satisfies,
5 are as follows:
An object of the present disclosure is to ameliorate one or more problems of the prior art or to at least provide a useful alternative.
Another object of the present disclosure is to provide a method for quantitative measurement of the biological activity of biological products.
10 Yet another object of the present disclosure is to provide a method for determining the potency
of biological products.
Yet another object of the present disclosure is to provide a method for determining the potency of monoclonal antibodies.
Yet another object of the present disclosure is to provide an ELISA based method for
15 determining the potency of monoclonal antibodies.
Yet another object of the present disclosure is to provide an ELISA based method for determining the potency of charge variants (acidic and basic) of monoclonal antibodies.
Yet another object of the present disclosure is to provide a reliable, rapid, safe, cost-effective, platform method for determining the potency of monoclonal antibodies.
20 Still another object of the present invention is to provide reliable, rapid, safe and cost-effective
ELISA based method for determining the potency of charge variants of Rabies monoclonal antibodies, Rabies monoclonal antibodies cocktail and other monoclonal antibodies.
Other objects and advantages of the present disclosure will be more apparent from the following description, which is not intended to limit the scope of the present disclosure.
25 DETAILED DESCRIPTION OF THE DISCLOSURE
Although the present disclosure may be susceptible to different embodiments, certain embodiments are shown in the drawing and following detailed discussion, with the
8

understanding that the present disclosure can be considered an exemplification of the principles of the disclosure and is not intended to limit the scope of disclosure to that which is illustrated and disclosed in this description.
Embodiments are provided so as to thoroughly and fully convey the scope of the present
5 disclosure to the person skilled in the art. Numerous details are set forth, relating to specific
components, and processes, to provide a complete understanding of embodiments of the
present disclosure. It will be apparent to the person skilled in the art that the details provided
in the embodiments should not be construed to limit the scope of the present disclosure. In
some embodiments, well-known composition, well-known processes, and well-known
10 techniques are not described in detail.
The terminology used, in the present disclosure, is only for the purpose of explaining a particular embodiment and such terminology shall not be considered to limit the scope of the present disclosure. As used in the present disclosure, the forms “a”, “an”, and “the” may be intended to include the plural forms as well, unless the context clearly suggests otherwise.
15 With respect to the use of substantially any plural and/or singular terms herein, those having
skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity. The use of the expression “at least” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may
20 be in the embodiment of the disclosure to achieve one or more of the desired objects or results.
The terms “comprise”, “comprising”, “including”, and “having” are open ended transitional
phrases and therefore specify the presence of stated features, integers, steps, operations,
elements, modules, units and/or components, but do not forbid the presence or addition of one
or more other features, integers, steps, operations, elements, components, and/or groups
25 thereof. The particular order of steps disclosed in the process of the present disclosure is not to
be construed as necessarily requiring their performance as described or illustrated, unless stated otherwise. It is also to be understood that additional or alternative steps may be employed.
The terms first, second, third, etc., should not be construed to limit the scope of the present
disclosure as the aforementioned terms may be only used to distinguish one element,
30 component, region, layer or section from another component, region, layer or section. Terms
9

such as first, second, third etc., when used herein do not imply a specific sequence or order unless clearly suggested by the present disclosure.
It is understood that each feature or embodiment, or combination, described herein is a non-
limiting, illustrative example of any of the aspects of the invention and, as such, is meant to be
5 combinable with any other feature or embodiment, or combination, described herein. For
example, where features are described with language such as “one embodiment”, “some embodiments”, “certain embodiments”, “further embodiment”, “specific exemplary embodiments”, and/or “another embodiment”, each of these types of embodiments is a non-limiting example of a feature that is intended to be combined with any other feature, or
10 combination of features, described herein without having to list every possible combination.
Such features or combinations of features apply to any of the aspects of the invention. More particularly, as regards the embodiments characterized in this specification, it is intended that each embodiment be read independently as well as in combination with another embodiment. For example, in case of an embodiment 1 reciting 3 alternatives A, B and C, an embodiment 2
15 reciting 3 alternatives D, E and F and an embodiment 3 reciting 3 alternatives G, H and I, it is
to be understood that the specification clearly and unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically
20 mentioned otherwise.
The term “about” as used herein encompasses variations of +/-5% and more preferably +/-2.5%, as such variations are appropriate for practicing the present invention.
The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect
to certain embodiments herein is intended merely to better illustrate the disclosure and does
25 not pose a limitation on the scope of the disclosure otherwise claimed.
Definitions:
In order for the present disclosure to be more readily understood, certain terms are first defined
below. Additional definitions for the following terms and other terms may be set forth through
30 the specification.
10

As used herein, ‘monoclonal antibody’ refers to a type of protein that is made in the laboratory and can bind to certain targets in the body, such as antigens on the surface of target cells (e.g., cancer cells). There are many kinds of monoclonal antibodies, and each monoclonal antibody is made so that it binds to only one antigen.
5 As used herein, ‘monoclonal antibody isoform’ refers to different forms/ variants of a protein
that arise due to various structural modifications or variations. Isoforms may exhibit structural differences by differing in amino acids sequence, post-translation modifications (PTMs), and other modifications. Isoforms may have different biophysical properties by having distinct isoelectric point (pI), stability and binding affinity. Isoforms can exhibit varying biological
10 activities. Some examples of isoforms in monoclonal antibodies include ‘glycosylation’:
different glycoform patterns can lead to isoforms with altered charge properties; ‘C- terminal lysine clipping’: removal of C-terminal lysine can result in isoforms; ‘cysteine related modifications’: presence of reduced cysteine, alternative disulfide bond linkage, and trisulfide bonds can contribute to isoforms; and ‘sialylated glycans’: difference in sialylated glycans can
15 result in different isoforms.
As used herein, ‘monoclonal antibody charge variants’ or ‘mAb charge variant’ refers to existence of multiple variants with differences in either charge, molecular weight or other properties of monoclonal antibodies. Charge variants are specific types of isoforms that differ primarily in their net charge. These charge variants are generally referred to as acidic or basic
20 compared with the main species. Charge variants may exhibit charge difference due to
modifications like deamidation, glycation and lysine clipping. Changes in charge can affect biological activities, such as, binding affinity, stability, and effector functions. Examples of charge variants in monoclonal antibodies include deamidation of asparagine to aspartate or isoaspartate leading to decreased mAb pI; removal of C-terminal lysine affecting charge
25 profiles; and Lysine or N-terminus glycation resulting in charge variants. The terms “isoform”
and “charge variant/ variant” are interchangeably used throughout the present disclosure. Isoforms encompass a broader range of structural variations, while charge variants specifically focus on differences in net charge. Both are critical quality attributes (CQAs) for mAbs, impacting safety, potency, and consistency throughout development and commercial use.
30 As used herein, the terms ‘main form’ and ‘main variant’ refer to the monoclonal antibody that
is not a charge variant as described above. These terms are interchangeably used throughout the present disclosure.
11

As used herein, ‘potency’ refers to the specific ability or capacity of the product, as indicated
by appropriate laboratory tests or by adequately controlled clinical data obtained through the
administration of the product in the manner intended, to effect a given result. Potency of bio
therapeutic monoclonal antibodies is measured by the half maximal inhibitory concentration
5 (IC50). IC50 is a quantitative measure that indicates how much of a particular inhibitory
substance (e.g. monoclonal antibody) is needed to inhibit antigen (virus) in vitro, a given
biological process or biological component by 50%. The IC50 (half-maximal inhibitory
concentration) It represents the concentration of a substance required to inhibit a specific
biological response by 50%. Indirect ELISA method as per the present disclosure may be used
10 to measure potency of biotherapeutic molecules by determining the relative potency of the
molecule with respect to the standard. The method of the present disclosure directed to determining potency of biological product(s) does not comprise or require any therapeutic, treatment or diagnostic steps/processes.
As used herein, ‘ELISA’ or ‘enzyme-linked immunosorbent assay’ refers to immunochemical
15 technique used to assess the presence of specific protein (antigen or antibody) in the given
sample and its quantification.
As used herein ‘capture antigen’ refers to the antigen/ antibody having specificity towards the target molecule.
As used herein, the expressions ‘secondary antibody-enzyme conjugate’ or ‘enzyme-linked
20 secondary antibody’ are used interchangeably and refer to enzyme conjugated or bound to
secondary antibody.
The present disclosure envisages a method for determining the potency of a biological product
(such as charge variants of monoclonal antibodies, monoclonal antibodies) by coating a plate
with a solution of a protein (such as an antigen); blocking unbound sites on the plate; detecting
25 the protein bound to the target; and quantifying the target through colour change.
In an aspect of the present disclosure, there is provided a method for determining the potency of a biological product. The method uses labelled immunoassay, wherein antibody-antigen complexes are formed to produce a measurable result.
In some embodiments of the present disclosure, the potency of the biological product is
30 determined using Enzyme-linked immunosorbent assay (ELISA). ELISAs are typically
performed in polystyrene plates (mostly having 96-well) with the plates coated to bind protein
12

very strongly. Depending on the ELISA type, testing requires a primary and/or a secondary
antibody, analyte/antigen, coating antibody/antigen, buffer, wash, and substrate/chromogen.
The primary antibody is a specific antibody that only binds to the protein of interest, while
the secondary antibody is an enzyme-conjugated antibody that binds to the primary antibody
5 (primary antibody is not enzyme-conjugated).
Accordingly, the present disclosure provides a method for determining the potency of a biological product by ELISA comprising the following steps:
- coating a protein or an antigen to an ELISA plate;
- blocking unbound sites in the ELISA plate; 10 - detecting the protein or the antigen by:
(i) adding a detection antibody-enzyme conjugate, or
(ii) adding a primary antibody and adding a secondary antibody-enzyme conjugate; and
- adding a substrate for the enzyme for eliciting a measurable signal.
In some embodiments of the present disclosure, the method for determining the potency of a
15 biological product by ELISA comprising the following steps:
- coating a protein or an antigen to an ELISA plate;
- blocking unbound sites in the ELISA plate;
- detecting the protein or the antigen by adding a detection antibody-enzyme conjugate; and
20 - adding a substrate for the enzyme for eliciting a measurable signal.
In some embodiments, the detection antibody is the same as the primary antibody described in detail in the subsequent embodiments.
In some embodiments of the present disclosure, indirect ELISA is used to determine the
potency of the biological product. Broadly, indirect ELISA (iELISA) comprises coating (with
25 an antigen); blocking; detection and reading.
Accordingly, in some embodiments of the present disclosure, there is provided a method for determining the potency of a biological product by indirect ELISA comprising the following steps:
- coating an antigen to an ELISA plate;
30 - blocking unbound sites in the ELISA plate;
- adding a primary antibody;
13

- adding a secondary antibody-enzyme conjugate; and
adding a substrate for the enzyme for eliciting a measurable signal.
In iELISA, a primary antibody is added that is known to bind the antigen. This is followed by
a wash step, to remove unbound material. Thereafter, there is an incubation with a secondary
5 antibody which is pre-conjugated to a tag/ enzyme. In iELISA, the presence of a binding
reaction is indicated by a secondary molecule not directly bound to the antigen on the plate.
Thus, the iELISA method uses an un-labelled primary antibody in combination with a labelled secondary antibody. The labelled secondary antibody is directed against all antibodies of a given species, it can be used with a wide variety of primary antibodies.
10 Accordingly, in some embodiments of the present disclosure, there is provided a method for
determining the potency of a biological product by indirect ELISA comprising the following steps:
- coating an antigen to an ELISA plate, followed by incubating and washing;
- blocking unbound sites in the ELISA plate by employing a blocking buffer, followed 15 by incubating and washing;
- adding a primary antibody in assay buffer, followed by incubating and washing;

- adding a secondary antibody-enzyme conjugate, followed by incubating and washing; and
- adding a substrate for the enzyme for eliciting a measurable signal.
20 In some embodiments of the present disclosure, the biological product may be selected from
therapeutic proteins and antibodies.
In some embodiments of the present disclosure, the biological product is an antibody selected
from the group comprising monoclonal antibody, charge variants of monoclonal antibody,
humanized antibody, chimeric antibody human antibody, bispecific antibody, multivalent
25 antibody, multi-specific antibody, antigen binding protein fragments, polyclonal, diabodies,
nanobodies, monovalent, bispecific, hetero-conjugate, multi-specific, autoantibodies, single chain antibodies, Fab fragments, F(ab)'2, fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, epitope-binding fragments and CDR-containing fragments and combinations thereof.
30 In some embodiments of the present disclosure, the biological product is a monoclonal
antibody selected from recombinant, chimeric antibodies, and human antibodies.
14

In some exemplary, non-limiting embodiments of the present disclosure, the biological product is a monoclonal antibody or charge variants of the monoclonal antibody.
In some embodiments of the present disclosure, the isoelectric point (pI) of the monoclonal
antibody is in the range of about 2 to 9, including values and ranges therebetween, such as
5 about 2 to 9, about 3 to 9, about 4 to 9, about 5 to 9, about 6 to 9, about 7 to 9, about 8 to 9,
about 2 to 8, about 3 to 8, about 4 to 8, about 5 to 8, about 6 to 8, about 7 to 8, about 2 to 7,
about 3 to 7, about 4 to 7, about 5 to 7, about 6 to 7, about 2 to 6, about 3 to 6, about 4 to 6,
about 5 to 6, about 2 to 5, about 3 to 5, about 4 to 5, about 2 to 4, about 3 to 4, about 2, about
3, about 4, about 5, about 6, about 7, about 8, or about 9, including values and ranges
10 therebetween.
In some preferred, non-limiting embodiments of the present disclosure, the isoelectric point (pI) of the monoclonal antibody is in the range of about 7 to 9.
In some other preferred, non-limiting embodiments of the present disclosure, the isoelectric point (pI) of the monoclonal antibody in the range of about 8 to 9.
15 Accordingly, in some embodiments, the present method comprises determining potency of a
therapeutic protein or a monoclonal antibody (mAb) or charge variants of monoclonal antibody (mAb) by ELISA, for e.g., indirect ELISA by the steps described in any one of the embodiments above. Said method of detecting antibody-antigen binding can serve as a potency assay for therapeutic protein or monoclonal antibody (mAb) samples to be used in further
20 applications (for ex. at the time of manufacturing or testing of a manufactured lot).
Accordingly, the present method comprising determining potency of a therapeutic protein or a
mAb or charge variants of mAb by ELISA employs said therapeutic protein or mAb or charge
variants of mAb as the primary antibody. In some embodiments, the present method comprising
determining potency of a therapeutic protein or a mAb or charge variants of mAb by indirect
25 ELISA employs said therapeutic protein or mAb or charge variants of mAb as the primary
antibody.
In accordance with some embodiments of the present disclosure, the monoclonal antibody
binds to an antigen selected from the group including, but not limited to, rabies virus, dengue
virus, respiratory syncytial virus (RSV), influenza virus, zika virus, West Nile virus, yellow
30 fever virus, chikungunya virus, herpes simplex virus (HSV), cytomegalovirus (CMV), Middle
East respiratory syndrome (MERS), Severe acute respiratory syndrome (SARS), Ebola virus,
15

Epstein-Barr virus, Varicella-Zoster virus, mumps virus, measles virus, polio virus, rhino virus,
adenovirus, hepatitis A virus, Hepatitis B virus, hepatitis C virus, Norwalk virus, Togavirus,
alpha virus, rubella virus, human immunodeficiency virus (HIV) virus, Marburg virus, Human
papilloma virus (HPV), polyoma virus, metapneumovirus, coronavirus, Vesicular stomatitis
5 virus (VSV), Venezuelan equine encephalitis virus (VEE), Staphylococcus aureus,
Streptococcus pyogenes, Clostridia species, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, Diphtheria tetani, Bacillus anthracis, Campylobacter jejuni, Acinetobacter baumannii, and the like.
In accordance with some embodiments of the present disclosure, the antigen is inactivated
10 strains of virus.
The method of determining potency in accordance with the present disclosure may be applicable for any monoclonal antibody, provided suitable capture antigen is available.
One of the critical steps in the iELISA method is to optimize concentrations of antigen and
antibody. This can be carried out by serial dilutions of antigen and antibody. The optimum
15 concentrations are identified by the sensitivity of the enzyme-substrate reaction and detection.
The concentration with the highest OD value and the least amount of protein is selected.
Once the antigen and antibody concentrations are optimized, the antigen is bound/ adsorbed to the plate surface. In some embodiments of the present disclosure, the antigen is bound/ adsorbed to plate surface (solid phase carriers) by coating the antigen to an ELISA plate.
20 Any suitable plate routinely used for ELISA assays may be used for potency determination in
accordance with the present disclosure. The ELISA plate may be made of any suitable substance, including polyvinyl chloride, polystyrene, polyacrylamide, cellulose, and the like. The form may be a flat plate, a test tube, a bead or the like. In some non-limiting embodiments of the present disclosure, the plate may be a polystyrene 96 wells ELISA plate.
25 The antigen is diluted in a coating buffer prior to coating on to the plates. Coating buffer allows
immobilizing proteins or antibodies on microplates.
Antigen coating takes place through passive adsorption of the antigen to the ELISA plate. The process occurs though hydrophobic interactions between the ELISA plate and non-polar protein residues of the antigen.
16

In accordance with the embodiments of the present disclosure, the coating buffer may be selected from but not limited to:
- Carbonate-Bicarbonate Buffer: this buffer is widely used for coating proteins/ antigens
onto ELISA plates;
5 - Phosphate Buffered Saline (PBS): PBS is a commonly used buffer for coating ELISA
plates;
- Bicarbonate Buffer: this buffer can be and 630 nm.
21) The method as defined in any of the embodiments 3 to 20, wherein the washing is
carried out using a wash buffer selected from a group comprising Tween 20, phosphate
buffered saline (PBS), phosphate-buffered saline with Tween 20 (PBST), Polysorbate
80, and Tris;
20 wherein pH of the wash buffer ranges from about 6.5 to 8.0;
wherein the wash buffer is 0.1% Tween 20 or 0.1 % PBST; or
wherein the washing of the ELISA plates is carried out with wash buffer for about 5 times to 20 times.
22) The method as defined in any of the embodiments 3 to 21, wherein each incubation step
25 is carried out at a temperature in the range of about 2 ℃ to 40 ℃ and for a time period
of about 10 minutes to 18 hours.
42

23) The method as defined in any of the embodiments 1 to 22, wherein the method
comprises determining the potency of a monoclonal antibody or charge variants of the
monoclonal antibody comprising steps of:
- coating vaccine (or an antigen thereof) diluted in carbonate-bicarbonate coating
5 buffer to an ELISA plate followed by incubating at about 2°C to 8°C for about
14 to 18 hours, and washing the ELISA plate about 5 times with PBST buffer;
- blocking unbound sites in the ELISA plate by employing about 5% milk powder
solution prepared in about 0.1% PBST, followed by incubating at about 36°C
to 38°C for about 1 hour and washing the ELISA plate about 5 times with PBST
10 buffer;
- adding monoclonal antibody or charge variants of the monoclonal antibody as
the primary antibody in about 1% milk powder in about 0.1% PBST, followed
by incubating at about 36°C to 38°C for about 1 hour and washing the ELISA
plate about 5 times with PBST buffer;
15 - adding mouse anti-human IgG Fc-HRP conjugate as the secondary antibody-
enzyme conjugate, wherein the secondary antibody is diluted to about 1: 10000¬fold in about 1% milk powder in about 0.1% PBST, followed by incubating at about 36°C to 38°C for about 1 hour and washing the ELISA plate about 5 times with PBST buffer;
20 - adding TMB substrate for the HRP enzyme and incubating for about 30 minutes
for eliciting a measurable signal;
- adding a stop solution for quenching the enzyme-substrate reaction; and
- measuring absorbance at wavelengths of about 450 and 630 nm, to determine the potency of the monoclonal antibody or the charge variants of monoclonal
25 antibody.
24) The method as defined in any of the embodiments 1 to 23, wherein the method
comprises determining the potency of rabies monoclonal antibody or charge variants of
rabies monoclonal antibody comprising steps of:
- coating rabies vaccine (RABIVAX-S) diluted in carbonate-bicarbonate coating
30 buffer to an ELISA plate followed by incubating at about 2°C to 8°C for about
14 to 18 hours, and washing the ELISA plate about 5 times with PBST buffer;
- blocking unbound sites in the ELISA plate by employing about 5% milk powder
solution prepared in about 0.1% PBST, followed by incubating at about 36°C
43

to 38°C for about 1 hour and washing the ELISA plate about 5 times with PBST buffer;
- adding rabies monoclonal antibody or charge variants of rabies monoclonal
antibody as the primary antibody in about 1% milk powder in about 0.1% PBST,
5 followed by incubating at about 36°C to 38°C for about 1 hour and washing the
ELISA plate about 5 times with PBST buffer;
- adding mouse anti-human IgG Fc-HRP conjugate as the secondary antibody-
enzyme conjugate, wherein the secondary antibody is diluted to about 1: 10000¬
fold in 1% milk powder in about 0.1% PBST, followed by incubating at about
10 36°C to 38°C for about 1 hour and washing the ELISA plate about 5 times with
PBST buffer;
- adding TMB substrate for the HRP enzyme and incubating for about 30 minutes for eliciting a measurable signal;
- adding a stop solution for quenching the enzyme-substrate reaction; and
15 - measuring absorbance at wavelengths of about 450 and 630 nm, to determine
the potency of the rabies monoclonal antibody or the charge variants of rabies monoclonal antibody.
25) A method for determining potency of charge variants of rabies monoclonal antibodies,
the method comprising:
20 - identifying charge variants in rabies monoclonal antibody;
- separating the charge variants; and
- determining potency of an acid charge variant and potency of a basic charge variant of the rabies monoclonal antibody by the method as defined in any one of preceding embodiments.
25 26) An ELISA assay kit to determine potency of rabies monoclonal antibodies, comprising:
a. the rabies antigen,
b. the rabies monoclonal antibodies,
c. horse radish peroxidase,
d. tetramethylbenzindine (TMB), and
30 e. optionally mouse anti human antibodies,
wherein the rabies monoclonal antibodies comprise one or more of main form, acid charge variants and basic charge variants of the rabies monoclonal antibodies;
44

and wherein the potency is determined by the method as defined in any one of the embodiments 1-25.
EXAMPLES:
5 The method for determining potency of biological products in accordance with the present
disclosure comprises the following broad steps, which are further explained in detail in the succeeding paragraphs:
- coating a protein or an antigen to an ELISA plate;
- blocking unbound sites in the ELISA plate;
10 - detecting the protein or the antigen by adding a detection antibody-enzyme conjugate;
and
- adding a substrate for the enzyme for eliciting a measurable signal.
Absorbance: the absorbance in the following examples is expressed as Delta Absorbance. Delta
absorbance is calculated by subtracting absorbance at 630nm (A630nm) from absorbance at
15 450nm (A450nm). A450nm detects the antigen antibody complex concentration i.e. signal of
the assay, whereas at A630nm the nonspecific binding as background value i.e. noise of the assay is detected.
Delta absorbance = A450nm – A630 nm.
Biological material used in the present disclosure:
20 Antigen: Licensed rabies vaccine (RABIVAX-S) S (2.5 IU/mL) manufactured by Serum
Institute of India Pvt. Ltd. Pune, Maharashtra, India.
Primary antibody: Rabies Monoclonal antibody (Rabishield) manufactured by Serum Institute of India Pvt. Ltd. Pune, Maharashtra, India.
Standard: In-house reference standard (IHRS) from Serum Institute of India Pvt. Ltd. Pune,
25 Maharashtra, India.
Secondary antibody: Mouse anti human IgG antibody Fc specific HRP conjugate. Source: Southern Biotech, USA.
Substrate: TMB Micro well Peroxidase Substrate System. It is a two component system:
3,3’,5,5’- tetramethyl benzidine (TMB) [C16H20N2] at a concentration 0.4 g/L in an organic
30 base and KPL Peroxidase Substrate Solution B containing H2O2 at a concentration of 0.02 %
45

in a Citric Acid Buffer; equal volumes of both solutions were mixed before use. Source: Sera Care, USA.
Example-1: Optimization studies
5 Coating buffer
Coating buffer is required to stabilize the biological product (antigen/ antibody/ protein) which
is used to coat the multiwell plate, in order to promote its passive adsorption to the microplate
surface without changing the epitopes. This results in maximizing adsorption to the plate and
optimizing interactions with the detection antibody. The biological product passively adsorbs
10 to the polystyrene surface through hydrophobic interaction. The optimum pH of the adsorption
of the biological product onto the polystyrene surface is 9-10. It is important that no other proteins are included in the coating buffer as they will compete with the biological product for binding to the plate.
Different coating buffers were studied to check the suitability by keeping other assay conditions
15 constant. According to Lambert Beer Law, the buffer which gives maximum absorbance value
on its lambda max wavelength is selected as the coating buffer. Different buffers studied as coating buffer for determining potency of biological products in accordance with the present disclosure along with the Delta absorbance is provided in Table-2.

Table-2: Delta Absorbance values for different coating buffers
Sr. No. Name of Coating Buffer Highest Delta Absorbance Lowest Delta Absorbance
1. Phosphate Buffered Saline (pH 7.4) 0.578 0.00
2. Phosphate Buffered Saline (pH 6.5) 0.547 0.00
3. Dulbecco′s Phosphate Buffered Saline (pH 7.4) 0.646 0.087
4. Carbonate Bicarbonate Buffer (pH 9.6) 1.405 0.255
5. Carbonate Bicarbonate Buffer (pH 10) 0.9010 0.00
20 The antigen (Rabies) and antibody (rabies monoclonal antibody) interaction was measured by
iELISA. The intensity of color developed is proportional to the amount of antigen-antibody binding. Highest delta absorbance indicates more antibody binding to antigen, and lowest delta absorbance indicate poor binding of antigen and antibody. Higher difference between the highest and lowest delta absorbance indicate better assay range and sensitivity.
46

Compared to other coating buffers Carbonate bicarbonate buffer with pH 9.6 gave better delta absorbance and hence was selected as the coating buffer for further studies.
It is seen from Table-2 that carbonate bicarbonate buffer with pH 9.6 gives maximum delta
absorbance value; indicating maximum binding of antibody to antigen and increased sensitivity
5 and specificity of the assay by this buffer.
Washing buffer
Assays, such as, ELISA use surface binding for separation, hence wash steps are repeated
between each step to remove unbound materials. The wash steps are a critical part of the
process and involve filling the wells entirely with the washing buffer. Use of washing buffer
10 increases the binding efficiency between the molecules, such as binding of a specific antibody
with an antigen, reduces background noise, and generates quality results.
Washing is repeated 3-5 times between each step to remove all the unbound material from the
wells. Excess washing buffer is removed to prevent dilution of the reagents in the subsequent
stages. Washing buffers may be removed by tapping the washed plate upside down on an
15 absorbant paper or by careful aspiration.
Different washing buffers were studied for determining potency of biological products in accordance with the present disclosure. Details of the washing buffers along with the Delta absorbance is provided in Table-3.

Table-3: Delta Absorbance values for different washing buffers
Sr. No. Name of Washing Buffer Highest Delta Absorbance Lowest Delta Absorbance
1. 0.1 % Tween 20 in PBS 1.985 0.034
2. 0.05 % Tween 20 in PBS 1.752 0.148
2. Only PBS 1.171 0.083
3. Only Tween 20 NA NA
4. Only WFI NA NA
20 It is seen from Table-3 that 0.1% Tween 20 in PBS as washing buffer gave minimum lowest
delta absorbance (background value) and maximum delta absorbance value which indicate maximum binding of antibody to antigen.
Further, “0.05 % Tween 20 in PBS” gave highest delta absorbance of 1.752 and lowest delta
absorbance of 0.148 while "0.1 % Tween 20 in PBST" gave highest delta absorbance of 1.985
25 and lowest delta absorbance of 0.034. Though the highest delta absorbance for both was
47

similar, “0.05 % Tween 20 in PBS” gave higher background value (as indicated by the values
at lowest delta absorbance). Increasing the amount of Tween 20 results in increased detergent
action resulting in washing of the antigen-antibody complex to improve the sensitivity, and
hence “0.1 % Tween 20 in PBST” was chosen as the washing buffer. However, if higher
5 amount of Tween 20 amount is used then the antigen-antibody complex may be washed off.
Washing buffers should be able to separate bound and unbound reagents and remove excess reagents from the multiwell plate, without damaging the antigen-antibody complex. It is seen from Table-3 that only PBS and only WFI (Water for Injection) were not capable of complete removal of excess reagents from the multiwell plate.
10 Protein concentration or antigen-antibody binding concentration was measured at 450 nm
wavelength. Further, the background absorbance was measured at 630 nm. It is possible for presence of non-specific binding of other molecules (e.g., secondary antibodies) to the plastic surface of 96 well plate, leading to a background signal, which may affect the accuracy of the assay by contributing to the overall absorbance reading.
15 However, it is seen from Table-3 that Tween 20 in PBST wash buffer was able to effectively
remove excess material from microtiter plate without damaging the antigen antibody complex.
It is also seen from Table-3 that use of 0.1 % tween 20 in PBS as washing buffer, aids in
minimizing the background without affecting the antigen-antibody complex, as seen from the
higher delta absorbance value. The use of Tween 20 widened the gap between highest and
20 lowest delta absorbance values. 0.1 % Tween 20 provided minimum background noise, as
compared to 0.05 % Tween 20. However, if a higher % amount of Tween 20 is used, there are chances of antigen -antibody complex being washed off.
Assay buffer
Different assay buffers were studied for determining potency of biological products in
25 accordance with the present disclosure. Details of the assay buffers along with the delta
absorbance is provided in Table-4.

Table-4: Delta Absorbance values for different assay buffers
Sr. No. Name of Assay Buffer Highest Delta Absorbance Lowest Delta Absorbance
1. 1% Milk Powder in 0.1 % PBST 1.985 0.162
2. 1% Milk Powder in 0.05 % PBST 1.788 0.187
3. 1% BSA Powder in 0.1 % PBST 1.352 0.127
48

4. 1% BSA Powder in 0.05 % PBST 1.232 0.084
5. 3% Milk Powder in 0.1 % PBST 0.483 0.029
6. 3% Milk Powder in 0.05 % PBST 0.457 0.034
7. 3% BSA in 0.05 % PBST 0.387 0.020
8. 1% Milk Powder alone with WFI 1.744 0.152
9. 0.1% PBST 1.933 0.68
It is seen from Table-4 that 1% Milk powder in 0.1% PBST gave better delta absorbance value
and hence was selected as the assay buffer for further studies. The highest delta absorbance
value for assay buffer 0.1% PBST (without milk powder) was similar to 1% Milk Powder in
5 0.1 % PBST. However, 0.1% PBST showed high background noise.
The blocking buffer containing milk (5%) is used for blocking unbound sites in the wells.
Similarly, assay buffer containing milk may bind to any unbound site in the wells, which might
lead to background noise and false result. However, 1% Milk powder in 0.1% PBST showing
highest delta absorbance and larger gap between highest and lowest delta absorbance values
10 was found to be the most ideal for use as the assay buffer, as it displayed the least background
noise, indicating accurate results. The higher delta absorbance of 0.1% PBST may be because of higher binding of antibody to the unbound sites in the well.
Blocking buffer
Different blocking buffers were studied for determining potency of biological products in
15 accordance with the present disclosure. Details of the blocking buffers along with the delta
absorbance is provided in Table-5.

Table-5: Delta Absorbance values for different blocking buffers
Sr. No. Name of Blocking Solution Highest Delta Absorbance Lowest Delta Absorbance
1. 1 % Non-Fat Milk Powder in 0.1% PBST 1.057 0.114
2. 3% Non-Fat Milk Powder in 0.1% PBST 1.063 0.120
3. 5 % Non-Fat Milk Powder in 0.1% PBST 1.113 0.113
4. 1% BSA 0.405 0.069
5. 3% BSA 0.413 0.073
6. 5% BSA 0.445 0.062
It is seen from Table-5 that use of 5 % milk powder in 0.1 % PBST gave higher delta
absorbance and also provided clear background compared to other buffers and hence was
20 selected as blocking buffer for further studies.
49

Dilution for secondary antibody
Mouse anti- human HRP conjugate was used as secondary antibody. To select the optimum dilution fold, different dilution folds of 5, 10, 50, 100, 500, 1000, 10K, 15K, 25K, and 50K were studied as illustrated in Table-6.

Table-6: Study of dilution for secondary antibody
Sr. No. Dilution Fold of Secondary Antibody Highest Delta Absorbance
1 5 4.266
2 10 4.194
3 50 4.130
4 100 4.209
5 500 2.977
6 1000 3.305
7 10K 1.689
8 15K 1.133
9 25K 0.688
10 50K 0.297
5
Based on Table-6, 10K (10000) fold dilution gave highest signal: noise ratio compared to the other fold dilutions and hence was chosen for further ELISA studies.
In ELISA, balance between the antigen and antibody is crucial for accurate results. If there is
an excess of antibody compared to the antigen, it could lead to Hook effect. It occurs when the
10 concentration of an antibody or an antigen is extremely high, impairing the formation of
antigen-antibody complex. This can result in signal loss, and producing hook shape on a graph, leading to false negative or inaccurately low results.
Antigen present at high concentration can interfere with the antigen-antibody reaction and lead
to false low results. Therefore, it is important to maintain an optimal balance between antigen
15 and antibody concentrations to ensure accurate and reliable results. Hence, 10000 (10K) fold
dilution was chosen for further studies.
Wavelength for absorbance
Potency determination of the present disclosure uses HRP-TMB based iELISA. Different wavelengths were tested as illustrated in Table-7.

Table-7: Wavelength for absorbance
Wavelength (nm) 280 350 450 550
Highest Absorbance Value 1.269 1.744 2.023 1.269
50

It is seen from Table-7 that at 450 nm, the yellow substrate color showed maximum absorbance and hence was selected to detect absorbance for determining monoclonal antibody potency.
Example-2: Determining potency of biological products using indirect ELISA (iELISA)
Materials:
5 - 96 wells flat bottom NUNC Maxisorp plate (Sigma-Aldrich)
- Plate sealing tape (Thermo Fisher);
- Multi-channel pipettes (Eppendorf);
- Lint-free duster;
- Centrifuge tubes (1.5ml, 2mL, 15 mL and 50 mL Volume),
10 - Glass Bottles: 100mL, 1000mL and 2000mL (Borosil)
Antigen:
- Rabies Antigens (Rabies vaccines): Licensed rabies vaccine (RABIVAX-S) S (2.5
IU/mL) manufactured by Serum Institute of India Pvt. Ltd. (SIIPL).
Antibodies:
15 - Human anti Rabies monoclonal antibody: Rabies monoclonal antibodies and its charge
variant/s (SIIPL)
- Human anti Rabies monoclonal antibody reference standards: Rabies monoclonal
antibody In house reference standards (IHRS) SIIPL.
- Mouse anti human IgG antibody Fc specific HRP conjugate: Southern Biotech: Mouse
20 anti-human IgG Fc-HRP conjugate antibody
TMB substrate:
- TMB Micro well Peroxidase Substrate System was obtained from Sera Care. It is a 2
component system.
- Components: 3,3’,5,5’- tetra methyl benzidine (TMB) [C16H20N2] at a concentration
25 0.4 g/L in an organic base and KPL Peroxidase Substrate Solution B containing H2O2
at a concentration of 0.02 % in a Citric Acid Buffer. Equal volumes of both solutions were mixed before use.
2N Sulfuric acid (H2SO4): Make: Sigma
51

Equipment:
- ELISA plate reader (Bio Tek ELx) capable of measuring wavelengths at 450 and 630
nm;
- Plate washer (AquaMax 2000, 96 Well Wash Head Molecular Devices, ELISA Plate
5 washer (Fast));
- ELISA Plate Incubator capable of achieving and maintaining 37°C (Bio San PST-
100HL Thermo-Shaker ELISA Plate Incubator); Vortex (IKA), Magnetic Stirrer, Gen5
software
Chemicals:
10 - MQW/ WFI;
- Carbonate bi-carbonate buffer capsules (pH 9.6) (Sigma Aldrich: C3041 Lot#SLCG4253);
- Phosphate-Buffered Saline (PBS) tablets pH 7.4 (Make: Gibco, Cat. number: 18912 and 18912014, Lot Number: 2290955);
15 - Milk powder (Nestle dry milk powder or Nonfat Dry Milk 9999S, Cell Signaling
Technology);
- Tween-20: Mol. Formula [C26H50O10] BioXtra, viscous liquid Polyethylene glycol
sorbtan monhydrate, (from Sigma Life Science).
Reagents and Solutions:
20 - Coating buffer: Carbonate bi-carbonate buffer (pH 9.6): one capsule of carbonate
bicarbonate buffer (Make: bi-carbonate buffer capsules (pH 9.6) (Sigma Aldrich: C3041 Lot#SLCG4253); was dissolved in 100 mL of MQW/WFI.
- Phosphate buffer Saline (PBS) with pH 7.4: In 1000 mL of Milli-Q water, two PBS
tablets (pH 7.4 Make: Gibco, Cat. number: 18912 and 18912014, Lot Number:
25 2290955) were dissolved. It was prepared on the day of use as needed.
- Wash buffer (or 0.1% PBST or 0.1% Tween-20 PBS): Prepared on the day of use as
needed. 1 mL of Tween-20 was added to 1000 mL of PBS (pH 7.4) buffer. The pipette
was rinsed in the solution by pipetting up and down multiple times until all the Tween
20 in the pipette was removed. The solution was mixed properly by using magnetic
30 stirrer.
52

- 5% Milk powder solution: 5 gram of milk powder (Nestle dry milk powder) was
weighed and added to 80mL of 0.1% PBST buffer and mixed properly for uniform
solution. The final volume was made up to 100 mL with 0.1% PBST.
- Stop Solution: 2N Sulfuric acid (H2SO4): To make 100mL 2N H2SO4, 94.444 mL of
5 MQW was taken in 100mL glass bottle and 5.556 mL of concentrated sulfuric acid was
added, followed by gentle mixing.
Potency determination of rabies monoclonal antibody (RmAb) using iELISA was performed as per the protocol provided below:
i) Coating: The in house rabies antigen (RABIVAX-S 2.5 IU/mL rabies vaccine,
10 SIIPL) aliquot was thawed and diluted 100-fold (i.e. 0.025 IU/mL) in Carbonate
bicarbonate coating buffer. Maxisorp 96 well Nunc ELISA plates were coated with 100µL/well of 100F of this in-house rabies antigen as per the protocol given in Table-8. The plate was sealed using a plate sealer (Thermo Fisher Scientific) and incubated overnight (16±2Hrs) at 2-8°C.

Table-8: Coating Well Map
1
2
3
4
5
6
7
8
9 10 11 12 Batch No.07
Coating antigen in-house (Fold)
A B Primary antibody
Coating Antigen 100 F: 100µL/well,
Coating Buffer: Carbonate Bicarbonate
Buffer,
Blocking Buffer: 5% Milk
400000 400000 400000 ng/mL (or 400
µg/mL) & 4 fold serial
Dilution

100000 100000

25000 25000

6250 6250

1562.5 1562.5

390.63 390.63

97.66 97.66

24.41 24.41

6.10 6.10

1.53 1.53

0.38 0.38

Blank Blank

Secondary Antibody (Mouse Anti Human (Fc) HRP Conjugate Ab)
10K fold dilution
- A & B represent rows in the multiwell
average value t
- 100µL/well of 100-fold dilu
- S. No. 1-12 represent the serial 4-fold dil
- 10000-fold (10K) dilution of s plat aken tion o ution (400
econ e and the samples were tested in duplicate with for further calculation.
f antigen was coated onto each well. of primary antibody starting from 400000 ng/mL µg/mL). dary antibody was added to each well.
15
53

5
10
15

ii)
iii)
iv) v)

-

Washing: After incubation, the plates were washed five times with phosphate buffer saline (pH 7.2±0.2) with 0.1% tween-20 (0.1% PBST), and the plates were firmly tapped over a lint-free duster to remove residual buffer. Blocking: The plates were blocked with 200 µL/well 5% Milk powder solution prepared in 0.1% PBST, to prevent nonspecific binding and incubated at 37±1°C for 1 hr.
Washing: Post incubation, the washing step was repeated as mentioned above. Addition of Primary Antibody (Ab): The rabies monoclonal antibody samples from different batches were used as the primary antibody and in-house reference standard used as standard and positive control and assay buffer used as negative control. Also, charge variants of rabies mAb were used as primary antibody. The rabies mAb samples, standard, and positive control were diluted in assay buffer (1% Milk powder in 0.1% PBST). Rabies mAb sample was diluted up to 400 µg/mL concentration and then serially 4-fold diluted up to 0.39 µg/mL as per Table-9. The primary antibody was added according to the plate layout, 100μL/well and gradual 4-fold decrease in concentration across the plate. The plate was incubated at 37±1°C for 1 hr.

Table-9: Dilution chart for Primary Antibody
Sr. No. mAb (µg/mL) Fold Dilution

4 Fold

1 400.00 11.25
2 100.00 22.5
3 25.00 45
4 6.25 90
5 1.56 180
6 0.39 360
7 0.10 720
8 0.02 1440
9 0.01 2880
10 0.00 5760
11 0.00 11520
vi) Washing: Post incubation, the washing step was repeated as mentioned above.
20 vii) Addition of Secondary Antibody: The Mouse anti-Human IgG Fc-HRP conjugated
antibody was used as the secondary antibody to bind to the rabies mAb. The secondary antibody was diluted to 1:10000-fold using assay buffer. 100 µl of secondary antibody was added to each well, and the plate was incubated at 37±1°C for 1 hr.
54

viii) Washing: Post incubation, the washing step was repeated as mentioned above.
ix) Addition of TMB: 100 µL/well of TMB substrate was added to all the wells and
incubated for 30 minutes or up to color development at room temperature in dark
without disturbing the plate. This step was performed in dark.
5 x) Addition of Stop solution: The reaction was quenched using 50 µL/well of 2N
sulfuric acid. xi) Screening: The plate was screened in a plate reader, and the absorbance was recorded
at 450 and 630 nm wavelength and the result obtained is illustrated in Tables10-11.
Four parameter logistic (4PL) parameter standard curves were plotted on Gen 5
10 software by using delta value (450nm OD-630nm OD) and known concentrations.

Table-10: Absorbance at 450 nm, 630 nm and Delta absorbance
Absorbance at 450 nm
A B 1 2 3 4 5 6 7 8 9 10 11 12

1.417 1.402 1.329 1.088 1.049 0.783 0.535 0.344 0.183 0.134 0.12 0.107

1.572 1.526 1.394 1.338 1.136 0.881 0.586 0.34 0.188 0.137 0.123 0.114
Absorbance at 630 nm
A B 1 2 3 4 5 6 7 8 9 10 11 12

0.035 0.035 0.036 0.037 0.038 0.035 0.036 0.036 0.037 0.038 0.039 0.039

0.037 0.039 0.038 0.042 0.036 0.039 0.037 0.037 0.037 0.038 0.038 0.04
Delta absorbance
A B 1 2 3 4 5 6 7 8 9 10 11 12

1.382 1.367 1.293 1.051 1.011 0.748 0.499 0.308 0.146 0.096 0.081 0.068

1.535 1.487 1.356 1.296 1.1 0.842 0.549 0.303 0.151 0.099 0.085 0.074

Table-11: Delta Absorbance
Batch No. 7 MAb (ng/mL) 4F Fold Dilution OD1 OD2 Average SD % CV
1 400000.00 11.25 1.382 1.535 1.459 0.108 7.42
2 100000.00 45 1.367 1.487 1.427 0.085 5.95
3 25000.00 180 1.293 1.356 1.325 0.045 3.36
4 6250.00 720 1.051 1.296 1.174 0.173 14.76
5 1562.50 2880 1.011 1.100 1.056 0.063 5.96
6 390.63 11520 0.748 0.842 0.795 0.066 8.36
7 97.66 46080 0.499 0.549 0.524 0.035 6.75
8 24.41 184320 0.308 0.303 0.306 0.004 1.16
9 6.10 737280 0.146 0.151 0.149 0.004 2.38
55

10 1.53 2949120 0.096 0.099 0.098 0.002 2.18
11 0.38 11796480 0.081 0.085 0.083 0.003 3.41
12 Blank -- 0.068 0.074 0.071 0.004 5.98
Calculation of rabies monoclonal antibody potency:
The average delta absorbance values obtained as per Table-11 was entered in Graph Pad Prism
(Version:10). A set of data representing 11 points was obtained. The X-axis represents the
5 logarithm (usually base 10) of the rabies mAb concentration, and the y-axis represents the
response in term of absorbance.
The four-parameter logistic (4PL) curve was used to analyse the data and is illustrated in Figure-1.
The 4PL curve is expressed as:
10 Y = Min + Max-Min
Hill Coefficient
1 + (X/ IC50) where:
- (Y) is the response in the form of absorbance
- Min and Max are the lower and upper asymptotes of the curve.
15 - (X) is the log-transformed concentration.
- IC50 is the concentration at which the response is half-maximal.
- The Hill coefficient determines the slope of the curve
Figure-1 illustrates the graphical representation of delta absorbance versus log concentration
of the primary antibody, from which the potency of the rabies monoclonal antibody was
20 calculated to be IC50 2.525.
Example-3: Comparison of potency determination of monoclonal antibodies using iELISA of present disclosure and RFFIT
- Potency determination by iELISA: potency of rabies monoclonal antibody was
determined using iELISA as described in Example-2 and the result obtained is provided
25 in Table-12.

Table-12: RmAb potency using iELISA
Sample Name Run 1 (ng/mL) Run 2 (ng/mL) Run 3 (ng/mL) Average (ng/mL)

Log IC50
56

Acidic Variants 2.84 2.53 3.01 2.79
Main Variant 2.69 2.63 2.97 2.76
Basic Variant 2.85 2.52 2.89 2.75
Positive Control 2.78 2.69 2.89 2.79
- Potency determination by RFFIT Assay: potency of rabies monoclonal antibody was determined using RFFIT assay and result obtained is illustrated in Table-15.
Biological material for RFFIT assay:
5 - Reference Standard (National/ International/ Inhouse): Rabies Human Monoclonal
antibodies: In-house (Serum Institute of India Pvt. Ltd.)
- Dulbecco’s Modified Eagle Medium (DMES) containing 10% FBS (Foetus bovine Serum) [DMES+10% FBS]: Corning Inc., USA/ Mediatech Inc. USA
- Foetus bovine Serum: Moregate Biotech, Australia & New Zealand
10 - Commercially available Rabies antibodies conjugated to fluorescein isothiocyanate dye
(FITC): Bio-Rad, Millipore
- Mice Neuroblastoma (MNA) cells or cell culture: Diagnostic HYBRIDS, Inc. USA; MassBiologics, USA
- Rabies Challenge Virus Standard (CVS): In-house (Serum Institute of India Pvt. Ltd.)
15 Material/ Equipment:
- Test sample – Rabies Human Monoclonal antibodies
- Reference Standard (National/ International/ Inhouse)
- Water for Injection (WFI)
- Dulbecco’s Modified Eagle Medium (DMES) containing 10% FBS (Foetus bovine
20 Serum) [DMES+10% FBS]
- Disinfectant
- Inverted fluorescence microscope
- CO2 incubator
- Micropipettes
25 - Sterile Tips
- Sterile Lab – Tek Chamber slides
- Sterile Glass vials/disposable vials (10mL/5mL)
- Cyclomixer
57

- Commercially available Rabies antibodies conjugated to fluorescein isothiocyanate dye (FITC), Example Bio-Rad, Millipore etc.
- Phosphate Buffered Saline (PBS), 0.05M, pH 7.5
- Vortex Mixer
5 - Laboratory Biosafety Level-2 Cabinet
- Centrifuge
- Fixative – Chilled 80 % Acetone (80 mL acetone + 20mL WFI). Keep at 2-8°C or 20°C
- Mice Neuroblastoma (MNA) cells or cell culture
- Trypsine – Phosphate – Versene – Glucose (TPVG) Solution
10 - Sterile tissue culture flasks
- Cell Counting Chamber (Neubauer Chamber)
- Sterile 10 mL pipette
- Rabies Challenge Virus Standard (CVS)
- Glass or Plastic beaker
15 - Tissue paper role SS Bowl
- Sterile 60/125 mL Disposable bottles
Principle: Un-neutralized Rabies virus does not produce any cytopathic effect in MNA cells.
But when antibodies labelled with fluorescent dye are added, they bind to the rabies virus
infected MNA cells. When observed under fluorescence microscope, these cells fluoresce due
20 to the dye and indirectly confirm the presence of rabies virus.
Sample preparation:
- One chamber slide was taken and marked as sample slide. The wells were marked as
per Table-13. Added 75 µL of media (DMEM + 10 % FBS) to well number 1.
- Added 100 µL of media (DMEM + 10 % FBS) to wells 2-8 of the chamber slide.
25 - Added 50 µL of test sample to well 1 and mixed thoroughly (1:5) or (1: 2.5F).
- Transferred 25 µL from well 1 to well 2 and mixed thoroughly (1:25).
- Transferred 25 µL from well 2 to well 3 and mixed thoroughly (1:125).
- Transferred 25 µL from well 3 to well 4 and mixed thoroughly (1: 625).
- Transferred 25 µL from well 4 to well 5 and mixed thoroughly (1: 3125).
30 - Transferred 25 µL from well 5 to well 6 and mixed thoroughly (1: 15625).
- Transferred 25 µL from well 6 to well 7 and mixed thoroughly (1: 78125).
- Transferred 25 µL from well 7 to well 8 and mixed thoroughly (1: 390625).
58

-

Discarded 25 µL from well No. 8.

Table-13: Sample Slide Well Chart
4 5
3 6
2 7
1 8
Test Sample
The assay was performed in duplicate and the above-mentioned procedure was repeated.

5

Preparation of control:

10
15

-
-
-
-
-
-
-
-
-

One more chamber slide was taken and marked as control slide for the assay, the wells
were marked as per Table-14.
Added 75 µL of media (DMEM + 10 % FBS) to well number 1.
Added 100 µL of media (DMEM + 10 % FBS) to wells 2-7 of the control slide.
Added 200 µL of media (DMEM + 10 % FBS) to well 8 of the control slide.
Added 50 µL of 2 IU/mL reference standard to well 1 and mixed thoroughly (1:5) or
(1: 2.5F).
Transferred 25 µL from well 1 to well 2 and mixed thoroughly (1:25).
Transferred 25 µL from well 2 to well 3 and mixed thoroughly (1:125).
Transferred 25 µL from well 3 to well 4 and mixed thoroughly (1: 625).
Discarded 25 µL from well No. 4.

20

Table-14: Control Slide Well Chart
Ref Std. CVS
1:625 50 FFD50
1:125 10-1
1:25 10-2
1:5 Cells Only
Control Slide
One more Chamber slide was taken and the above mentioned procedure for control was repeated.
- Thawed Rabies Challenge Virus Standard (CVS) i.e. CVS-11 in water for injection kept at 20 to 25°C with gentle stirring for 3-5 minutes and immediately diluted it to 50 FFD50 in media (DMEM + 10 % FBS). Kept the rabies challenge Virus Standard (CVS) and

59

back dilution on ice/ice packs. The challenge virus standard was used within 10 minutes after thawing.
- Added 100 µL of diluted CVS rabies virus to wells 1-8 of all sample slides and to wells
1-5 of the control slide and mixed thoroughly.
5 - Taken two sterile glass/ disposable vials labelled – 1 and 2 and added 1.8 mL media
(DMEM + 10 % FBS) to both the vials.
- Added 200 µL of diluted virus (50 FFD50) to vial-1 and mixed thoroughly. Then
transferred 200 µL of diluted virus to vial-1 and mixed thoroughly.
- Added 100 µL of virus from vial-1 to well 6 of the control slide, and added 100 µL of
10 virus from vial-2 to well 7 of the control slide. Well 8 of the control slide contained
“cell control only”.
- Incubated the slides for 90 minutes at 36±1°C in humidified CO2 incubator.
Preparation of MNA cells Suspension:
- 205 days old confluent MNA cell monolayer flask (80/175 cm2 TCF) was used.
15 - Discarded the medium from the flask. Added approximately 2-5 mL TPVG solution.
From the TCF.
- Added fresh 10-15 mL TPVG solution so as to cover the cell monolayer completely
and kept the TCF as such for some time.
- Waited until the cells start rounding off. At this stage, poured off excess TPVG solution
20 leaving behind 1-2 mL in the flask. The flask was gently tapped to remove the cells
completely.
- Resuspended the cells in about 8 mL media (DMEM + 10 % FBS). Separated the cells
by repeated pipetting. The cell count was adjusted to 5-6 x 105 cells per mL using cell
counting chamber (Neubauer).
25 - After completion of incubation period, added 200 µL of the cell suspension to each well
of the chamber slides and mixed thoroughly. Incubated the chamber slides at 36±1°C for 20-24 hours in humidified CO2 incubator (CO2- 2.0 – 2.5 %).
Fixation or fluorescence staining: After completion of incubation period, the slide was removed
from CO2 incubator and fixed with 80 % acetone as follows:
30 - Two glass/ plastic beakers filled with 80 % chilled acetone was used.
60

- The slide cover was removed and decanted the media from the slide chambers in the SS bowl. Submerged the slide immediately in the first 80 % chilled acetone beaker, rinsed once and then filled the wells with 80% chilled acetone from second beaker.
- Kept the slides for 10-15 minutes at room temperature.
5 - Discarded the acetone in the SS bowl. The slides were allowed to dry at room
temperature or in an incubator.
Staining:
- Diluted the anti-rabies antibody conjugated to fluorescein Isothiocyanate (FITC) dye
with PBS to a predetermined dilution (1:40).
10 - Added 100-150 µL of conjugate to each well so that it covered the entire cell
monolayer.
- Incubated the slides at 36±1°C in humidified CO2 incubator 30-45 minutes.
Washing: After completion of incubation period, the slide was removed from CO2 incubator
and washed as follows:
15 - Removed the top chamber and discarded.
- Dip rinsed the slide twice in two beakers containing PBS.
- Then dip rinsed the slide in WFI.
Slide Observation:
The slide was observed on an inverted fluorescence microscope at a magnification of 160x to
20 200x. Observation of bright Gren intracellular granules indicated a positive result i.e., the MNA
cell was infected with rabies virus. Moving from left to right and top to bottom, counted 20 distinct fields of the chamber. The number of fields containing infected cells were noted.
Calculations:
ND50 (Neutralizing dose 50%) of the test and standard sample was calculated as per the Reed
25 and Muench method and the potency (neutralizing titre) was calculated as follows:
(Infectivity next above 50 %)
PD = x log of dilution factor
(infectivity next above 50 %) – (infectivity next below 50 %)
30 (PD= proportionate distance)
61

ND50 = Log of dilution showing next over 50 % infectivity – P.D. = A
Antilog of A = Neutralizing dose 50 %
Determine the potency (IU/mL) of the test sample as follows:
ND50 of the test sample
5 X potency of the reference standard
ND50 of the reference standard
The results related to potency was rounded up to 2 decimal places i.e. if the 3rd decimal point is 5 or more that 5, the 2nd decimal point was increased by 1. Rabies monoclonal potency obtained using RFFIT is shown in Table-15.
10 As the test was carried out in duplicate, the final potency of the sample is the mean of the two
potency results. Mean potency result was reported as per specification.

Table-15: RmAb potency using RFFIT
Sample Name Run 1 (IU/mL) Run 2 (IU/mL) Run 3 (IU/mL) Average (IU/mL)

ND 50
Acidic variant 55.41 48.84 44.55 49.6
Main variant 51.19 46.6 46.64 48.14
Basic variant 54.22 50.09 46.72 50.35
Positive Control 53.18 51.34 47.86 50.8
The RFFIT is rabies virus neutralization test performed in cell culture to determine the rabies virus neutralizing antibody level/potency in given sample. On other hand, ELISA tests measure
15 binding antibodies to viral antigens rather than functional neutralization measured in the
RFFIT. ND50, in RFFIT, indicates the antibody concentration correlating to 50% neutralization of the virus while in similar manner IC50, in ELISA, indicates antibody concentration correlating to 50% decrease in the highest observed absorbance. Thus, both ND50 and IC50 “qualitatively” denote and depict the activity/potency of the antibody under
20 test. However, RFFIT requires the use of biological materials like BHK-21 cells and virulent
rabies challenge virus, which may not be feasible in any virology laboratory, and a greater coefficient of variation limit criterion is required for this viral neutralization assay. Further, the usage of an international standard reference sample requirement for each sample run makes the assay costly. It requires the use of containment facilities (BSL 2/3), and skilled professionals.
62

On the contrary, the iELISA method of the present disclosure is relatively easy to perform, has
a short turnaround time, and is capable of determining the potency of monoclonal antibody
charge variants (acidic and basic). The present method uses vaccine strain (inactivated virus)
which is safe to handle and does not require BSL3 facilities as it can be performed in any
5 laboratory. Hence, the iELISA method of the present disclosure is an advantageous and a better
alternative to prior art methods such as RFFIT.
Example-4: Determination of potency of rabies monoclonal antibody (RmAb) charge variants using iELISA
(i) Identification of charge variants in rabies monoclonal antibody
10 Isoelectric focusing (IEF) was carried out for rabies monoclonal antibody (RmAb) from
upstream process (USP) and downstream process (DSP) and the result obtained is illustrated in Figures 2a and 2b. IEF Protocol:
Material:
15 - Gel: SERVA (Cat: 42866.00)
o SERVALYT PRECOTES Gel (pH range 3-10); PAG Layer 300 µm; size 125x125 mm; Lot P160374; (No. of gels used = 1)
- Sample Buffer: SERVA (Cat: 42537.01); 20 ml; IEF sample buffer (2x) sterile filtered;
Lot P170205
20 - Anode Fluid: SERVA (Cat: 42984.03); 50 ml; Anode fluid 3 For IEF; Lot P180004
- Cathode Fluid: SERVA (Cat: 42986.03); 50ml; Cathode fluid 10 For IEF; Lot P170386
- Cooling Fluid: SERVA (Cat: 14500.01); 100ml; Bayol F Research Grade; Lot 150584
- IEF Marker: SERVA (Cat: 39212.01); 500µl; IEF Maker 3-10, Liquid Mix; Lot P 170134
25 - Wicks: SERVA Cat:42987.03) 100 pieces Electrode wicks long size, 240x6x1mm; Lot
130645
- Sample applicator strips: SERVA (Cat: 42989.01) 3 pieces Applicator strips 7x1,2 24
slots, 263 mm long Lot 170062
- Fixing solution: 20 % Trichloroacetic Acid (TCA)
30 - Staining solution: 0.1% CBB G -250
- De-staining solution: Methanol: Acetic Acid: WFI [ 4:1:5]
- Samples: RmAb samples
63

o Sample Preparation:
▪ Diluted 2X Sample buffer (SERVA) to 1X with WFI. ▪ Diluted the samples to1µg/ µL with 1x sample buffer.
Method:

5
10
15

-
-
-
-
-
-
-
-
-

The chiller was switched on to 10°C and the plate was allowed to cool down.
(NOTE: the chiller was switched on after loading the samples on the gel).
Bayol F/ Cooling fluid was applied to the area where the gel is going to be placed.
Removed the plastic coating of the gel (Servalyte precoated: pH 3-10, 300µm, 125mm
x 125 mm) and placed it the on the plate.
The excess oil/ cooling fluid surrounding the gel was blotted dry using a tissue paper
The wicks were cut according to the size of the gel and were wet in anode and cathode
fluid respectively. Placed them according to the markings on the gel.
The sample applicator strip was placed ~1- 2cm from the anode-side wick.
Added 10uL of the sample (concentration/ well = 10ug/10µL) to respective wells.
Added 15uL of IEF marker (SERVA IEF Marker, Cat: 39211).
Placed plate cover over the respective anode and cathode ports.
Set the program for running the gel as illustrated in Table-16.

Table-16: Program for running gel
Step # Conditions Duration
Step 1 500V - 4mA - 10W 30min
Step 2 2000v - 8mA - 12W 130min
Step 3 2300V - 6mA - 10W 45min

20
25
30

-
-
-
-
-
-
-

The oil covered portion of the gel was blotted dry using a tissue paper.
Prepared 20% Trichloroacetic acid (TCA) to be used as a fixing solution.
The gel was given 2 washes X 5-10 minutes with WFI to make sure the oil was
completely removed.
Placed the gel in fixing solution for 30 minutes.
Post incubation, the gel was given 3 washes X 10 minutes with WFI so as to completely
remove 20% TCA.
Taken ~80-100mL of staining solution (0.1 % CBB G-250 solution) in a beaker and
heated in a microwave for ~30 seconds. Immediately poured the staining solution in the
gel container for 3-5 minutes.
Placed the gel in de-staining solution and gave 4 washes or until the background was

64

clear.
- Stored the gel in WFI and the gel image was taken using gel doc system.
It is seen from Figure-2a that the IEF of rabies monoclonal antibody (RmAb) from upstream
process (USP) and downstream process (DSP) revealed presence of two bands: a major mAb
5 form at pI of 8.4, and another band was observed at pI of ~8.3.
Further, Figure-2b illustrates a magnified IEF image of rabies monoclonal antibody showing
two distinct bands for the main form and the acidic variant (isoform). Basic variant is not
observed in IEF as a distinct band (Figure 1a and Figure 1b), as basic variant band being very
close to main form got merged with the main form. The basic and acidic variants can be clearly
10 viewed in chromatography and capillary isoelectric focusing (iCEF). In cation exchange
method, separated peaks of acidic variant, basic variants and main peak can be seen.
When Rabies mAb upstream process samples from 0 day and downstream process samples
were run on IEF Gel (pH range 3 to 10), two distinct bands were obtained: one major band on
8.4 pI and another band on 8.3 pI. These indicate the presence of charge variants from zero day
15 (before USP/ DSP processes) and that the charge variants are not process generated charge
variants.
(ii) For determining the potency of RmAb charge variants, first the charge variants were
separated using cation exchange, followed by potency determination of individual charge variants using iELISA.
20 Separation of RmAb charge variants: The charge variants in Rabies monoclonal antibody were
separated using salt gradient cation exchange chromatography (CEX) on the AKTA-HPLC Explorer chromatography system (AKTA, Cytiva).
- Clarified harvest was loaded onto the equilibrated Protein A chromatography column.
- The column was washed with protein A Wash-I buffer followed by Wash-II buffer at a 25 flow rate of less than 300 cm/hr.

- The column-bound protein was eluted using protein A elution buffer at a flow rate of less than 150 cm/hr.
- By monitoring at A280 nm, the elute was collected and further subjected to low pH treatment by adjusting pH to 3.5±0.1.
30 - After being subjected to one hour of low pH treatment, the protein A elute was loaded
onto XK 16 column (radius: 8mm, bed height: 15.5 cm, resin volume: 32 mL, and resin:
65

5
10

-
-
-
-
-
-

Fractogel) which was equilibrated with Equilibration Buffer with a flow rate of
˂300cm/hr and 6mL/min. Post-load wash was done using Equilibration buffer by
passing it at 90 mL by 6mL/min.
The column-bound antibodies were eluted by a linear gradient of Buffer B, elution took
place @ 3mL/min at 0 to 40% Buffer B with 500 mM NaCl.
A280 was monitored for absorbance and the entire peak was collected in fractions.
Linear gradient fractions of 1mL/vial were collected in 1.5 mL Eppendorf centrifuge
tubes.
The collected fraction was analyzed using the analytical cation exchange
chromatography method on HPLC.
Fractions containing the same variants were pooled together and re-analysed by CEX
and used for further analysis.
The column was cleaned with CIP Buffer and stored in Storage Buffer. Chromatograms
obtained were analyzed by using UNICORN software and is illustrated in Figure-3.

15 Following buffers were used for cation exchange chromatography:
a) Pre-Equilibration buffer (pH 5.0);
b) Wash buffer/ Buffer A (pH 6.0);
c) Neutralization buffer/ Buffer B (pH 6.0);
d) CIP buffer and 20 e) Storage buffer.
The composition of cation exchange chromatography buffers is provided in Table-17.

Table-17: Composition of cation exchange chromatography buffers
Sr. No. Buffer Name Chemical Name Quantity (g/L)
1 Pre-Equilibration (20 mM Sodium Acetate pH 5.0 ± 0.2) Sodium Acetate 1.64

Acetic Acid QS
2 Wash Buffer & Buffer A
(20 mM Citrate Buffer, pH 6.0
± 0.2 with 1 M NaOH/ 1 M
HCl) (Wash-I) Sodium Citrate dihydrate 4.76

Citric Acid 0.8
3 Buffer B/ Neutralization Buffer
(20 mM Citrate Buffer, 300 mM NaCl, pH 6.0 ± 0.2 with 1 M NaOH/ 1 M HCl) (Wash-II) Sodium Citrate dihydrate 4.76

Citric Acid 0.8

Sodium Chloride 17.53
4 CIP Buffer (0.5 M NaOH) Sodium Hydroxide 20
66

5

Storage Buffer (0.1 M NaOH)

Sodium Hydroxide

4

(iii) Determination of acidic charge variant and basic charge variant in rabies
monoclonal antibody
The charge variants were separated from the rabies monoclonal antibody using cation exchange
5 chromatography. Further, the potency of the charge variants was determined using iELISA as
described in Example-2. The result obtained are provided in Table-18 and Figure-4.

Table-18: Percentage of acidic and basic charge variant in RmAb
Rabies mAb
Acidic variant 21.11 %
Main form 66.12 %
Basic variant 11.77 %
It is seen from Table-18 and Figure-4 that the acidic charge variant and basic charge variant were 15-25 % and 7-15 %, respectively in Rabishield.
10 Example-5: Specificity of iELISA of the present disclosure
The present method was developed to estimate potency of the rabies monoclonal antibody against rabies antigen and estimated potency compared with rabies mAb standard.
Studies were carried out, where dengue monoclonal antibody was used in place of rabies
monoclonal antibody, and the result obtained is illustrated in Figure 5 and Tables 19-20. The
15 use of dengue mAb in place of rabies mAb did not result in binding with the rabies antigen
coated on to the multiwell plates and hence, it is seen that the rabies mAb exhibits specificity to rabies antigen. Dengue mAb did not show any binding with rabies antigen which means that this assay also shows the specificity of rabies mAb against rabies antigen.

Table-19: Relative potency of dengue and rabies mAb using iELISA with rabies antigen
Specificity Run 1 Run 2 Run 3 Run 4 Average SD %CV

IHRS 100.00 100.00 100.00 100.00 100.00 0 0

Sample A (RmAb) 98.14 104.39 87.78 96.67 96.74 6.85 7.08

Dengue MAb - - - - - - -

Bank - - - - - - -
Table-20: Potency of dengue and rabies mAb using iELISA with rabies antigen

Specificity
IHRS

Run 1
2.85

Run 2
2.76

Run 3
3.35

Run 4
2.94

Average
2.97

SD
0.26

%CV
8.72

67

Sample A (RmAb) 2.79 2.88 2.94 2.85 2.86 0.06 2.10

Dengue MAb - - - - - - -

Bank - - - - - - -
It is seen from Figure 4 and Tables 19-20 that rabies monoclonal antibody showed affinity to
rabies virus antigen, whereas dengue monoclonal antibody did not show any affinity towards
rabies virus antigen. Hence, the rabies mAb binding potency against rabies antigen was specific
5 and dengue mAb showed non-specific binding against rabies antigen when compared with in-
house reference standard (IHRS).
Example-6: iELISA of the present disclosure for determining potency of samples during stability studies
Samples were subjected to different temperatures for different time periods and potency of
10 these samples were determined using iELISA of the present disclosure. The temperature, time
duration and the potency obtained are illustrated in Tables 21-23.

Table-21: Temperature: 2-8 °C
Long-term stability Day 0 1
Mont
h 3 Months 6 Months 9 Months 12 Months Averag e SD %CV

IHRS 100.00 100.00 100.00 100.00 100.00 100.00 100.00 0 0

Sample A 92.60 100.08 95.43 95.43 95.14 84.42 93.85 3.10 3.30

Sample B 113.31 96.18 87.12 85.46 97.19 112.92 98.70 12.77 12.93

Sample C 104.78 94.09 109.39 90.95 103.10 98.91 100.20 8.71 8.70

Long-term stability Day 0 1
Mont
h 3 Months 6 Months 9 Months 12 Months Averag e SD %CV

IHRS 2.27 2.36 2.50 2.50 3.07 3.67 2.73 0.54 19.75

Sample A 2.10 2.36 2.38 2.38 2.92 3.10 2.54 0.38 14.98

Sample B 2.38 2.27 2.08 2.13 2.98 3.50 2.56 0.56 22.01

Sample C 2.50 2.13 2.27 2.27 3.16 3.46 2.63 0.55 20.73
Sample A, B and C refers to final bulk sample R & D batches 5, 6 and 7, respectively.
15
68

Table-22: Temperature: 25 °C
Stress stability 1
Mont
h 3 Months 6 Months Average SD %CV

IHRS 100.00 100.00 100.00 100.00 0 0

Sample A 105.32 102.79 104.24 104.12 1.27 1.22

Sample B 104.63 102.90 107.49 105.01 2.32 2.21

Sample C 102.14 99.76 106.53 102.81 3.44 3.34

Stress stability 1
Mont
h 3 Months 6 Months Average SD %CV

IHRS 2.894 2.899 2.710 2.834 0.108 3.800

Sample A 3.048 2.980 2.825 2.951 0.114 3.873

Sample B 3.028 2.983 2.913 2.975 0.058 1.948

Sample C 2.956 2.892 2.887 2.912 0.038 1.321
Sample A, B and C refers to final bulk sample R & D batches 5, 6 and 7, respectively.

Table-23: Temperature: 40 ° C
Stress stability 3rd Day 7th
Day 15th
Day 30th
Day Average SD %CV

IHRS 100.00 100.00 100.00 100.00 100.00 0 0

Sample A 107.04 97.07 97.67 95.73 99.38 5.17 5.21

Sample B 96.02 102.53 102.30 97.99 99.71 3.23 3.24

Sample C 106.79 99.84 85.63 94.37 96.66 8.94 9.25

Stress stability 3rd Day 7th
Day 15th
Day 30th
Day Average SD %CV

IHRS 3.27 3.86 3.69 3.16 3.50 0.34 9.59

Sample A 3.50 3.75 3.61 3.03 3.47 0.31 9.00

Sample B 3.36 3.85 3.69 2.97 3.46 0.39 11.23

Sample C 3.58 3.84 3.16 2.80 3.35 0.46 13.73
S ample A, B and C refers to final bulk sample R & D batches 5, 6 and 7, respectively.
It is seen from Tables 21-23 that different batches of rabies monoclonal antibody were stable
at 2-8 °C to up to 1 year, at 25 °C for up to 6 months and at 40 °C for up to 30 days. The
5 potency obtained using iELISA is comparable with the potency obtained using RFFIT.
However, do to various advantages such as short turnaround time, use of vaccine strain (inactivated virus as opposed to virulent strains) which is safe to handle and does not require BSL3 facilities as it can be performed in any laboratory, non-requirement of skilled
69

professionals, etc., the iELISA method of the present disclosure is an advantageous and a better alternative to RFFIT.
Example-7: Isoelectric point and charge distribution by capillary isoelectric focusing of charge variants of rabies monoclonal antibody
5 The Rabies Mab charge variant samples collected from AKTA Explore by salt gradient cation exchange chromatography and charge variants containing fraction confirmed using WCEXHPLC were desalted using EMD millipore Amicom Ultra-30 Centrifugal filter unit and buffer exchange to milli Q Water. To prepare a master mix Urea-cIEF Gel (3.75M), pharmalytes 3-10 carrier ampholytes, cathode stabilizer, anode stabilizer, pI marker A, B &C
10 mix in calculated quantity, were vortexed for 15 seconds to mix thoroughly. Freshly prepared 240 µL master mix was mixed with 10 µL charge variant samples (The protein sample solution should not contain more than 50mM of salt), positive and negative control of Rabies Monoclonal Antibody which contained 50 to 100 μg of protein in a volume not greater than 10 µL. This cIEF sample was vortexed for 30 seconds and 200 µL sample was transferred into the
15 micro vial and then centrifuged the vial for 20 sec at low speed to remove any air bubbles. Put the samples, added 1.5 mL reagent per vial, and filled the vials with 1.5 mL DDI water with a defined program. When sample preparation was completed, the cIEF sample was put on the inlet sample tray and run and the result obtained is illustrated in Figure-6. The 32- Karat software was used to calculate the experimental pI value of the samples. cIEF follows two steps
20 focusing and mobilization. First Capillary is filled with a solution containing carrier ampholytes and analytes. By applying a high voltage pH gradient established in capillary and proteins are focused until they reach their pI. At this point, the mobility of protein becomes zero due to their neutral charge.
It is seen from Figure-6 that use of cIEF results in proper separation of the charge variants, 25 which matches the positive control. cIEF represents one of the most appropriate methods (in terms of resolution) to separate the charge variants of proteins depending on their electric charge potential. During the process of cIEF separation, a continuous pH gradient is created by applying electric current through a capillary that is filled with carrier ampholytes. mAb tends to generate intricate profiles of charge variants when separated by cIEF.
30
70

TECHNICAL ADVANTAGES:
The method for determining potency of biological products (particularly potency of mAb charge variants) of the present disclosure described herein above has several technical advantages including, but not limited to, the realization of:
5 • a platform, reliable, rapid, safe and cost-effective method for determining the potency
of charge variants of monoclonal antibodies (as an alternative to existing RFFIT, FAVN potency testing methods);
• easy to perform method using specific monoclonal antibodies for capture or detection;
• use of inactivated strains of virus as antigens;
10 • optimization of antigen, buffers, dilutions, primary antibody, secondary antibody, and
color substrate;
• easy standardization of the method without using biological materials such as cell line,
virulent virus, and containment facilities; and
• avoiding use of BSL3 facility, sacrifice of mice, and live virus handling for potency
15 determination.
Additional embodiments and features of the present disclosure will be apparent to one of
ordinary skill in art based on the description provided herein. The embodiments herein provide
various features and advantageous details thereof in the description. Descriptions of well-
known/conventional methods and techniques are omitted so as to not unnecessarily obscure the
20 embodiments herein.
The foregoing description of the specific embodiments reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be
25 comprehended within the meaning and range of equivalents of the disclosed embodiments. It
is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments in this disclosure have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the
30 embodiments as described herein.
71

Throughout this specification, the term ‘combinations thereof’ or ‘any combination thereof’ or ‘any combinations thereof’ are used interchangeably and are intended to have the same meaning, as regularly known in the field of patents disclosures.
Numerical ranges stated in the form ‘from x to y’ include the values mentioned and those values
5 that lie within the range of the respective measurement accuracy as known to the skilled person.
If several preferred numerical ranges are stated in this form, of course, all the ranges formed by a combination of the different end points are also included.
Any discussion of documents, acts, materials, devices, articles and the like that has been
included in this specification is solely for the purpose of providing a context for the disclosure.
10 It is not to be taken as an admission that any or all of these matters form a part of the prior art
base or were common general knowledge in the field relevant to the disclosure as it existed anywhere before the priority date of this application.
All references, articles, publications, general disclosures etc. cited herein are incorporated by
reference in their entireties for all purposes. However, mention of any reference, article,
15 publication etc. cited herein is not, and should not be taken as, an acknowledgment or any form
of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.
While considerable emphasis has been placed herein on the particular features of this
disclosure, it will be appreciated that various modifications can be made, and that many
20 changes can be made in the preferred embodiments without departing from the principles of
the disclosure. These and other modifications in the nature of the disclosure or the preferred embodiments will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.
25
72

We Claim:
1) A method for determining potency of a biological product by ELISA comprising the
steps of:
- coating a protein or an antigen to an ELISA plate; 5 - blocking unbound sites in the ELISA plate;
- detecting the protein or the antigen by:
(i) adding a detection antibody-enzyme conjugate, or
(ii) adding a primary antibody and adding a secondary antibody-enzyme
conjugate; and
10 - adding a substrate for the enzyme for eliciting a measurable signal.
2) The method for determining potency of a biological product as claimed in claim 1,
wherein said method is indirect ELISA comprising the steps of:
- coating an antigen to an ELISA plate;
- blocking unbound sites in the ELISA plate;
15 - adding a primary antibody;
- adding a secondary antibody-enzyme conjugate; and
- adding a substrate for the enzyme for eliciting a measurable signal.
3) The method as claimed in claim 2, wherein the method for determining potency of the
biological product by indirect ELISA comprises the steps of:
20 - coating an antigen to an ELISA plate followed by incubating and washing;
- blocking unbound sites in the ELISA plate by employing a blocking buffer, followed by incubating and washing;
- adding a primary antibody in assay buffer, followed by incubating and washing;
- adding a secondary antibody-enzyme conjugate, followed by incubating and
25 washing; and
- adding a substrate for the enzyme for eliciting a measurable signal.
4) The method as claimed in any one of the claims 1 to 3, wherein the biological product
is a therapeutic protein or an antibody.
5) The method as claimed in any one of the claims 1 to 4, wherein the biological product
30 is an antibody selected from a group comprising monoclonal antibody, charge variants
of monoclonal antibody, humanized antibody, chimeric antibody human antibody,
73

bispecific antibody, multivalent antibody, multi-specific antibody, antigen binding
protein fragments, polyclonal, diabodies, nanobodies, monovalent, bispecific, hetero-
conjugate, multi-specific, autoantibodies, single chain antibodies, Fab fragments,
F(ab)'2, fragments, fragments produced by a Fab expression library, anti-idiotypic
5 (anti-Id) antibodies, epitope-binding fragments and CDR-containing fragments and
combinations thereof.
6) The method as claimed in claim 5, wherein, the isoelectric point (pI) of the monoclonal antibody is in the range of about 2 to 9, preferably in the range of about 7 to 9 and most preferably in the range of about 8 to 9.
10 7) The method as claimed in any of the claims 1 to 3, wherein the antigen is selected from
a group comprising rabies virus, dengue virus, respiratory syncytial virus (RSV),
influenza virus, zika virus, West Nile virus, yellow fever virus, chikungunya virus,
herpes simplex virus (HSV), cytomegalovirus (CMV), Middle East respiratory
syndrome (MERS), Severe acute respiratory syndrome (SARS), Ebola virus, Epstein-
15 Barr virus, Varicella-Zoster virus, mumps virus, measles virus, polio virus, rhino virus,
adenovirus, hepatitis A virus, Hepatitis B virus, hepatitis C virus, Norwalk virus,
Togavirus, alpha virus, rubella virus, human immunodeficiency virus (HIV) virus,
Marburg virus, Human papilloma virus (HPV), polyoma virus, metapneumovirus,
coronavirus, Vesicular stomatitis virus (VSV), Venezuelan equine encephalitis virus
20 (VEE), Staphylococcus aureus, Streptococcus pyogenes, Clostridia species,
Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus
pneumoniae, Diphtheria tetani, Bacillus anthracis, Campylobacter jejuni, and
Acinetobacter baumannii.
8) The method as claimed in any one of the claims 1 to 6, wherein the biological product
25 is rabies monoclonal antibody, charge variants of rabies monoclonal antibody, or a
combination thereof; or
wherein the biological product is rabies monoclonal antibody selected from a
group comprising Rabishield, SYN023, CL184, F11, GR1801, A2, B353,
Ormutivimab, 2B10, CR057, CR4098, RV08, RV3A5, NP-19-9, 11B6, Miromavimab,
30 Docaravimab, RVC20, RVC58, CTB011, CTB012, R16F7, R14D6, or combinations
thereof.
74

9) The method as claimed in claim 8, wherein the charge variants are separated by a
method selected from ion exchange chromatography, cation exchange chromatography,
anion exchange chromatography, isoelectric focusing, capillary electrophoresis,
capillary zone electrophoresis, capillary isoelectric focusing, electrophoresis, 2D gel
5 electrophoresis, hydrophobic interaction chromatography, mixed mode
chromatography, chromatofocusing, affinity chromatography, high-performance liquid chromatography, and ion pair HPLC.
10) The method as claimed in any one of the claims 1 to 3 or claim 7, wherein the antigen
is a rabies vaccine or any antigen thereof.
10 11) The method as claimed in any one of the claims 2 to 10, wherein the method for
determining potency of a therapeutic protein or a monoclonal antibody or charge variants of monoclonal antibody by indirect ELISA comprises steps of:
- coating the antigen to an ELISA plate;
- blocking unbound sites in the ELISA plate;
15 - adding the therapeutic protein or the monoclonal antibody or the charge variants
of monoclonal antibody, which is the primary antibody;
- adding the secondary antibody-enzyme conjugate; and
- adding the substrate for the enzyme for eliciting a measurable signal.
12) The method as claimed in any claim 11, wherein the method for determining potency
20 of a therapeutic protein or a monoclonal antibody or charge variants of monoclonal
antibody by indirect ELISA comprises steps of:
- coating the antigen to an ELISA plate followed by incubating and washing;
- blocking unbound sites in the ELISA plate by employing a blocking buffer, followed by incubating and washing;
25 - adding the therapeutic protein or the monoclonal antibody or the charge variants
of monoclonal antibody, which is the primary antibody in assay buffer, followed by incubating and washing;
- adding the secondary antibody-enzyme conjugate, followed by incubating and
washing; and
30 - adding the substrate for the enzyme for eliciting a measurable signal.
13) The method as claimed in any one of the claims 1 to 12, wherein the antigen is diluted
in a coating buffer prior to coating on to the ELISA plate;
75

wherein the antigen is diluted about 50 to 200-fold in the coating buffer;
wherein the coating buffer is selected from a group comprising carbonate bicarbonate buffer, phosphate buffered saline (PBS), tris hydrochloric acid buffer (Tris-HCl), and combinations thereof; or
5 wherein the coating buffer has pH ranging from about 8.0 to 10.0.
14) The method as claimed in any one of the claims 1 to 13, wherein the blocking is carried
out using a blocking buffer selected from a group comprising milk powder solution,
bovine serum albumin (BSA), ovalbumin, aprotinin, tween 20, casein, whole normal
serum, fish gelatin, and combinations thereof;
10 or wherein the blocking buffer is about 0.5 % to 7 % milk powder solution in
about 0.1 % phosphate-buffered saline with Tween 20 (PBST).
15) The method as claimed in any one of the claims 1 to 14, wherein the primary antibody
or the detection antibody is diluted in an assay buffer before being added to the ELISA
plate;
15 wherein the primary antibody or the detection antibody is diluted about 50 to
200-fold in the assay buffer;
wherein the assay buffer is selected from a group comprising milk powder solution, phosphate buffered saline (PBS), phosphate-buffered saline with Tween 20 (PBST), and combinations thereof;
20 wherein the assay buffer is about 1% milk powder in about 0.1% PBST; or
wherein the assay buffer has a pH ranging from about 6.5 to 8.0.
16) The method as claimed in any one of the claims 1 to 15, wherein the secondary antibody
is diluted about 1: 10000-fold in a buffer;
wherein the secondary antibody is selected from a group comprising mouse or
25 mice, goat, sheep, rabbit, donkey, monkey, horse, guinea pig, chicken, bovine and
human antibody; or
wherein the secondary antibody is mouse anti-human secondary antibody.
76

17) The method as claimed in any one of the claims 1 to 16, wherein the enzyme conjugated to the secondary antibody or detection antibody is selected from a group comprising horseradish peroxidase (HRP), urease, alkaline phosphatase (AP), and ß-D-galactosidase.
5 18) The method as claimed in any one of the claims 1 to 17, wherein the substrate is selected
from a group comprising 3,3',5,5'-tetramethylbenzidine (TMB), 2,2'-Azinobis [3-
ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS), o-phenylenediamine
dihydrochloride (OPD), p-Nitrophenyl Phosphate (PNPP), o-nitrophenyl-β-D-
galactopyranoside (ONPG), diethanolamine, chemiluminescent 1,2-dioxetane alkaline
10 phosphatase substrate (CSPD), QuantaBlu Fluorogenic Peroxidase Substrate, and
QuantaBlu NS/K Fluorogenic Substrate.
19) The method as claimed in any one of the claims 1 to 18, wherein the secondary
antibody-enzyme conjugate is mouse anti-human IgG Fc-HRP conjugate.
20) The method as claimed in any one of the claims 1 to 19, wherein measuring the signal
15 comprises measuring absorbance at wavelengths of about 450 nm and 630 nm.
21) The method as claimed in any one of the claims 3 to 20, wherein the washing is carried
out using a wash buffer selected from a group comprising Tween 20, phosphate
buffered saline (PBS), phosphate-buffered saline with Tween 20 (PBST), Polysorbate
80, and Tris;
20 wherein pH of the wash buffer ranges from about 6.5 to 8.0;
wherein the wash buffer is 0.1% Tween 20 or 0.1 % PBST; or
wherein the washing of the ELISA plates is carried out with wash buffer for about 5 times to 20 times.
22) The method as claimed in any one of the claims 3 to 21, wherein each incubation step
25 is carried out at a temperature in the range of about 2 ℃ to 40 ℃ and for a time period
of about 10 minutes to 18 hours.
23) The method as claimed in any one of the claims 1 to 22, wherein the method comprises
determining the potency of a monoclonal antibody or charge variants of the monoclonal
antibody comprising the steps of:

coating vaccine (or an antigen thereof) diluted in carbonate-bicarbonate coating buffer to an ELISA plate followed by incubating at about 2°C to 8°C for about 14 to 18 hours, and washing the ELISA plate about 5 times with PBST buffer; blocking unbound sites in the ELISA plate by employing about 5% milk powder solution prepared in about 0.1% PBST, followed by incubating at about 36°C to 38°C for about 1 hour and washing the ELISA plate about 5 times with PBST buffer; adding monoclonal antibody or charge variants of the monoclonal antibody as the primary antibody in about 1% milk powder in about 0.1% PBST, followed by incubating at about 36°C to 38°C for about 1 hour and washing the ELISA plate about 5 times with PBST buffer;
adding mouse anti-human IgG Fc-HRP conjugate as the secondary antibody-enzyme conjugate, wherein the secondary antibody is diluted to about 1: 10000¬fold in about 1% milk powder in about 0.1% PBST, followed by incubating at about 36°C to 38°C for about 1 hour and washing the ELISA plate about 5 times with PBST buffer;
adding TMB substrate for the HRP enzyme and incubating for about 30 minutes for eliciting a measurable signal;
adding a stop solution for quenching the enzyme-substrate reaction; and measuring absorbance at wavelengths of about 450 and 630 nm, to determine the potency of the monoclonal antibody or the charge variants of monoclonal antibody.
24) The method as claimed in any one of the claims 1 to 23, wherein the method comprises determining the potency of rabies monoclonal antibody or charge variants of rabies monoclonal antibody comprising the steps of:
- coating rabies vaccine (RABIVAX-S) diluted in carbonate-bicarbonate coating
25 buffer to an ELISA plate followed by incubating at about 2°C to 8°C for about 14
to 18 hours, and washing the ELISA plate about 5 times with PBST buffer;
- blocking unbound sites in the ELISA plate by employing about 5% milk powder
solution prepared in about 0.1% PBST, followed by incubating at about 36°C to 38°C
for about 1 hour and washing the ELISA plate about 5 times with PBST buffer;
30 - adding rabies monoclonal antibody or charge variants of rabies monoclonal
antibody as the primary antibody in about 1% milk powder in about 0.1% PBST, followed by incubating at about 36°C to 38°C for about 1 hour and washing the ELISA plate about 5 times with PBST buffer;

- adding mouse anti-human IgG Fc-HRP conjugate as the secondary antibody-
enzyme conjugate, wherein the secondary antibody is diluted to about 1: 10000¬
fold in about 1% milk powder in about 0.1% PBST, followed by incubating at
about 36°C to 38°C for about 1 hour and washing the ELISA plate about 5 times
5 with PBST buffer;
- adding TMB substrate for the HRP enzyme and incubating for about 30 minutes for eliciting a measurable signal;
- adding a stop solution for quenching the enzyme-substrate reaction; and
- measuring absorbance at wavelengths of about 450 and 630 nm, to determine the
10 potency of the rabies monoclonal antibody or the charge variants of rabies
monoclonal antibody.
25) A method for determining potency of charge variants of rabies monoclonal antibodies,
the method comprising:
a. identifying charge variants in rabies monoclonal antibody;
15 b. separating the charge variants; and
c. determining potency of an acid charge variant and potency of a basic charge
variant of the rabies monoclonal antibody by the method as claimed in any one
of the claims 1-24.
26) An ELISA assay kit to determine potency of rabies monoclonal antibodies, comprising:
20 a. rabies antigen,
b. rabies monoclonal antibodies,
c. horse radish peroxidase,
d. tetramethylbenzindine (TMB), and
e. optionally mouse anti human antibodies;
25 wherein the rabies monoclonal antibodies comprise one or more of main form, acid
charge variants and basic charge variants of the rabies monoclonal antibodies;
and wherein the potency is determined by the method as claimed in any one of the claims 1-25.

“METHOD FOR DETERMINING POTENCY OF BIOLOGICAL PRODUCTS”