Download 6.0 BRIEF RESUME OF THE INTENDED WORK: 6.1

6.0 BRIEF RESUME OF THE INTENDED WORK:
6.1
INTRODUCTION:
Characterization of the Drug substance (DS)/Drug product (DP) is essential for
the assessment of the quality of therapeutic substances intended for human use according to
regulatory bodies Food And Drug Administration (FDA), European Medicines Agency
(EMEA). Such therapeutic substances may be broadly classified into pharmaceuticals and
bio-pharmaceuticals. The pharmaceuticals may be produced by chemical synthesis while biopharmaceutical products may be produced by expression through recombinant DNA
technology. Monoclonal antibodies (Mabs) are the most common biopharmaceutical drug
expressed by means of recombinant technology. During the process development of such
recombinant drugs, in addition to the main DS, other host cell proteins and structurally
related substance/proteins are co-expressed. Even in the presence of a robust downstream
processing of the (Mabs), few related substance may still be present in the final DS.
Therefore, there is a need to study and understand the type of the related substance/protein
and establishing their chemical identity and characterizing those impurities at all the stages
during manufacturing can be achieved by using MASS SPECTROMETRY (MS) etc.
Most of the drugs are analyzed by MS method since it has several advantages like
rapidity, specificity, accuracy, precision, reproducibility, ease of automation and it eliminates
tedious extraction and isolation procedures. The use of High Performance Liquid
Chromatography (HPLC) based methodologies involves analytical method development and
its validation which plays an important role in drug discovery and in the manufacture of biopharmaceuticals. Combination of both MS and HPLC methods are used to ensure the
identity, purity and quality of drug substances. The effort in majority of analytical method
development involves the validation of process related impurity identification.
Liquid chromatography –Mass Spectrometry1 (LC-MS) is a powerful technique
which has very high sensitivity and selectivity. In the present studies, we will discuss various
MS-based techniques for the structural characterization of Mabs. These techniques are
categorized as mass determination of intact antibodies, middle-up, bottom-up, top-down, and
middle-down structural characterization. Each of these techniques has its advantages and
disadvantages in terms of structural resolution, sequence coverage, sample consumption and
data analysis.
Mabs2 are an essential part of a molecular biologists toolkit. Antibodies are proteins
produced by the B-lymphocytes of the immune system in response to foreign substances,
called antigens. Antibodies function as markers, binding to the antigen and can be recognized
and destroyed by phagocytes as a part of defense mechanism. Antibodies have a short amino
acid sequence which recognizes the antigen and they are called as epitope. Epitope is a
binding site present in antibody. (Campbell NA, 1966).
The features of the antibody-epitope relationship are key to the use of monoclonal
antibodies as a molecular tool.

Specificity- the antigen binds only to its particular epitope

Sufficiency- the epitope can bind to the antibody on its own, i.e. the presence of the
whole antigen molecule is not necessary.
Structurally antibodies consist of four poly peptide chains. These four chains form a
quaternary structure resembling a Y - shape.
Each B cell in an organism synthesizes only one kind of antibody. In an organism, there
is an entire population of different types of B cells and their respective antibodies which are
produced upon exposure to various antigens. However to be used as a bio-tool, molecular
biologists need substantial amounts of Mabs for drug targeting. Therefore fermenters are used
to culture a population of B cells, which would allow us to harvest a single kind of antibody
that can be processed. This population of cells would be correctly described as monoclonal,
and the antibodies produced by this population of B cells are called monoclonal antibodies. In
contrast, antibodies obtained from the blood of an immunized animal are called polyclonal
antibodies.
6.2 ADVANTAGES OF USING Mabs:
Mabs have a variety of academic, medical and commercial uses. It would be impossible
to list all of these here. But the following list should indicate how ubiquitous Mabs
technology has become in biotechnology industry.

Antibodies are used in several diagnostic tests, used in pregnancy test kits. Another
diagnostic uses of antibodies is the diagnosis of AIDS by the ELISA test.

Antibodies are used in the radioimmunodetection and radioimmunotherapy of cancer
and Monoclonal antibodies can be used to treat viral disease

OKT3, an antibody to the T3 antigen of T cells is used to alleviate the problem of
organ rejection in patients who have had organ transplants.
6.3
IMPURITY PROFILE 3,4
“It is a description of identified and unidentified impurities present in DS and DP’’
Impurities are resulted from a chemical change in DS during its manufacture and storage by
the effect of light, temperature, pH, water or by reaction of excipient and\ or the immediate
container closure system etc.,
6.3.1 GOALS OF IMPURITY INVESTIGATIONS
Process related impurities
Understanding
the
process
Degradation related impurity
mediated To identify potential degradation product
impurities and to determine the origin of through stress testing and actual product
impurity.
through stability study.
External factor contributing to impurity To understand degradation pathway and
formation.
method
for
elimination
or
reduction
To establish a control system for impurity method to minimize degradation.
involving
I.
II.
I.
Processing / manufacture condition.
Suitable
analytical
method/
To
establish
manufacturing
conditions.
II.
specification
Suitable
analytical
method
/
specification
III.
Long
term
storage
condition
including package.
IV.
Formulation.
6.4 REVIEW OF LITERATURE

Bersinger.N.A. et al., have carried out the production and characterization of
monoclonal antibodies against pregnancy associated plasma protein A5.

Archelos.J.J. et al. have performed the production and characterization of monoclonal
antibodies to the extra cellular domain of P06.

Gururaj.V. Kavidal and Sotiros D. Chaparas have carried out the production
characterization
and species
mycobacterium tuberculosis7 .
specificity
of
five
monoclonal antibodies
to

Lobvi E. et al., have shown the potential of MALDI-MS in combination with
more
conventional
protein
chemistry
techniques for
the characterization of
monoclonal antibodies8.

Alain Beck. et al., have a characterization by liquid chromatography combined
with mass spectrometry of monoclonal anti-IGF-1 receptors antibodies produced
in CHO and NSO cells. The method includes 7H2M is a new humanized
monoclonal antibody (MA b) . Primary structure and disulfide bridge pairing were
analyzed by micro sequencing (Edmandegradation), Mass spectrometry (MALDITOF-ES-TOF) and peptide mapping after enzymatic digestion9.

Judy Glynn. et al., have developed an Application of a Monoclonal Antibody
Purification Platform. In this development the antibody purification platform was
used to reduce development time, while ensuring that product purity and virus
clearance are sufficient for clinical manufacture10.

Christa liedtke et al., have described the biochemical and immune cytochemical
characterization of monoclonal antibodies TOP 35 raised against purified tonoplast
from cress root11.

Alvarez M et al., have performed on line characterization of monoclonal antibody
variants by LC-MS operating in a two dimensional format12.

Lintao Wang et al., have
recombinant
monoclonal
carried
out
the
structural characterization
antibody by electro spray time of flight
of
a
mass
spectroscopy.13

Rasmus Hansen et al., have performed the rapid characterization of N- linked
Glycans from secreted and gel- purified monoclonal antibodies using MALDITOF mass spectrometry.14

John C. Le , Pavel V Bonda Renko have performed characterization of intact
monoclonal antibodies using reverse phase HPLC online with ion trap mass
spectrometry15.
6.5
MAIN OBJECTIVE OF THE STUDY
In the proposed project work on Mabs, the following things will be studied in detail:

To develop new analytical methods by HPLC and LC-MS for identification and
characterization of known as well as unknown impurities present in Mabs.

To characterize these identified impurities for their physicochemical properties.

Qualification/validation of these developed methods as per ICH and USP guidelines
for the intended analytical application.

To apply these proposed methods for routine analysis of known and unknown
impurities present in Mabs of marketed/RND samples.
7.0
7.1
MATERIALS AND METHODS:
SOURCE OF DATA:
The preliminary data required for the experimental study will be obtained from
1. Library, Govt. College of pharmacy, Bangalore.
2. http//www.rguhs.ac.in/helinetHome.
3. Journals and e-journals.
4.
Internet Sources:
www.scienencedirect.com ,
www.pubmed.com,
www.google.com,
www.ijp-online.com.
7.2 METHOD OF COLLECTION OF DATA:
Chemicals and other reagents required for the study will be collected from standard
companies. Data pertaining to the present study will be obtained from the experiments
performed at Govt. College of Pharmacy Department of Pharmaceutical Chemistry and
BIOCON LIMITED (Research and Development Department) Bengaluru.
7.3
Does the study require any investigations or interventions to be conducted on
Patients or animals? If so, Please describe briefly.
-NOT APPLICABLE.
7.4 Has ethical clearance been obtained from your Institution incase of 7.3?
-NOT APPLICABLE-
8.0 BIBILOGRAPHY
1. Snyder LR, Kirkland JJ.,Glajch JL. Practical HPLC Method Development. New
York: John Wiley and Sons;1997.
2. MonoclonalAntibodies.www.bio.davidson.edu/courses/molstudents/olrakik/mabhtml
9.30pm,29/11/2011.
3. Vasanti S, Sulabha S, Impurity Profiling. Drug invention. Today. 2009;1(2):81-88.
4. Impurities in New Drug Substance, International Conference on Harmonization, ICH
Harmonized Tripartite Guidelines. 2006.
5. Bersinger NA, Meisser A, et al., Production and Characterization of Monoclonal
Antibodies against Pregnancy associated Plasma Protein A. Molecular Human
Reproduction. 1999;5:675-81.
6. Archelos JJ, Roggenbuck K, et al. Production and Characterization of Monoclonal
Antibodies to the Extra Cellular Domain of P. J Neuro Sci Research. 1993;35:46-53.
7. Gururaj V, Kadival, Sotiros D Chaparas. Production, Characterization and Species
Specificity of Five Monoclonal Antibodies to M. Tuberculosis. J Clinical
Microbiology. 1987;25(1):76-80.
8. Lobvi E, Matamoros Fernandez, et al. Characterization of a Recombinant Monoclonal
Antibody by Mass Spectrometry combined with LC. J Chromatogr B. 2001;752:24761.
9. Alain Beck, Marie-Claire Bussat, et al. Characterization by LC- MS of Monoclonal
Anti- IGF-1 Receptor Antibodies produced in CHO and NS0 Cells. J Chromatogr B.
2005;819:203-18.
10. Judy Glynn, Timothy Hagerty, et al., The Development and Application of a
Monoclonal Antibody Purification Flat form Bio Pharm Int Suppl. 2009.
11. Christa Liedtke, Monika Polsakiewicz, et al., Biochemical and Immune Cytochemical
Characterization of Monoclonal Antibody TOP 35 Raised against Purified Tonoplast
from Cress Root. J Expt Botany. 1997;48(311):1215-21.
12. Alvarez M,Tremintin G, et al., On-line Characterization of Monoclonal Antibody
Variants by LC- MS. Operating in a Two Dimensional Project. Anal Bio Chem 2011;
419(1):17-25.
13. Lintao Wang, Godfery Amphlett, John M Lambert, Walter Blattler, Wei Zhang.
Structural Characterization of Recombinant Monoclonal Antibody by Electro Sprays
TOF Mass Spectrometry. Pharmaceut Research. 2005;22(8):1338.
14. Rasmus Hansen, Alan J Dickson, et al. Rapid Characterization of N- linked Glycans
from Secreted and Gel - Purified Monoclonal Antibodies using MALDI- ToF Mass
Spectrometry. Wiley online library. 2010 Jul 26; DOI 10.1002/bit.22879.
15. John C. Le, Pavel V Bonda Renko. Characterization of Intact Monoclonal Antibodies
using Reverse Phase HPLC online with Ion Trap Mass Spectrometry Elsevier 2005.