Download 6. 7. 8. Brief Resume of the Intended Work

6. Brief Resume of the Intended Work:
6.1 Need For The Study:
With the trend towards the generation and production of increasing number
of complex biopharmaceutical (protein based) products, there is an increased need and
requirement to characterize both the product and production process in terms of
robustness and reproducibility. Characterization of the Drug substance/Drug product is
essential for the assessment of the quality of therapeutic substances intended for human
use according to regulatory bodies such as Food and Drug Administration and
European Medicines Agency. Such therapeutic substances may be broadly classified
into Pharmaceuticals and Bio-Pharmaceuticals. The Pharmaceuticals may be produced
by chemical synthesis while Bio-Pharmaceutical products may be produced by
expression through Recombinant DNA Technology. A one such biopharmaceutical
drug expressed by recombinant technology are Monoclonal Antibodies. Monoclonal
Antibodies represent a significant portion of sales in the biopharmaceutical market.
Ever-growing biotechnology pipelines have greatly increased demand for high
throughput, multi-product protein characterization methods. By taking a holistic
approach to improving the analytical process, one can identify strategies to increase
throughput and reduce development times and the costs of analytical methods.
Most of the drugs are analyzed by Mass Spectrometry1 (MS) method because of
several advantages like rapidity, specificity, accuracy, precision, reproducibility, ease of
automation and it eliminates tedious extraction and isolation procedures over other
analytical methods. Hence, in the present study an attempt will be made to characterize
protein therapeutics by Mass Spectrometry.
6.2 Introduction:
Biopharmaceuticals are the unique class of medicines due to their extreme
structural complexity.
Monoclonal antibodies2 are one such protein therapeutics
produced by a single clone of cells grown in culture that is pure and specific, capable of
proliferating indefinitely to produce unlimited quantities of identical antibodies. These
are used in the treatment of Cancer3, Tuberculosis4, Hepatitis5, Chronic Lymphocytic
Leukemia6, Transplant Rejection7, Crohn’s Disease8, Psoriasis9 and Melanoma10. The
structure of these therapeutic proteins is critically important for their efficacy and
safety. The process of characterizing these therapeutics at various levels is critical not
only at the quality control stage, but also throughout the discovery and design stages. A
physical characterization of monoclonal antibodies includes a determination of the
class, sub class, light chain composition (kappa and lambda chain) and the primary
structure of monoclonal antibody. The amino acid sequence of Monoclonal Antibodies
are deduced from DNA sequencing and also confirmed by adopting methods such as
peptide mapping, amino acid sequencing and Mass spectrometry analysis. Monoclonal
antibodies are characterized by following methods like Electrophoresis11, Ion Exchange
Chromatography12, Size Exclusion Chromatography13 and Mass Spectroscopy. Mass
Spectroscopy (MS) offers a variety of approaches to study the structure and behavior of
complex protein drug molecules and characterizing the covalent structure of protein
therapeutics, including identification of amino acid sequence and the post-translation
modification.
Mass Spectrometry has provided the means for accurately weighing atoms and
molecules. Recent development have transformed mass spectrometry to one capable of
weighing and identifying individual protein, under pining world wide effort to define
the individual protein components of living cells. High performance liquid
chromatography is basically improved form of column chromatography. Instead of a
solvent being allowed to drip through a column under gravity, it is forced through under
high pressure of up to 400 atmospheres that makes it much faster. It also allows a very
much smaller particle size for the column packing material which gives a much greater
surface area for interaction between the stationary phase and the molecules. The other
major improvements over column chromatography concern the detection methods
which can be used. These methods are highly automated and extremely sensitive.
Recently, Mass-based methods have also begun to enjoy a dramatic growth in
popularity as a mean to provide information on higher order structure and dynamic of
biotechnology products. In particular, Mass offers a convenient way to assess the
integrity of protein conformation.
MS-based
methods are
also applied to study
pharmacokinetics of Biopharmaceutical products, where they begin to rival traditional
immunoassays. Also MS provides valuable support at all stages of development of
biopharmaceuticals, from discovery to post-approval monitoring and its impact on the
fields of biopharmaceutical analysis continue to grow.
6.3 Review Of Literature:
David AB, Andrew AG, Gooley, Bernstein, George M, and Keith LW have
performed genetic diversity in cellular slime modls: allozyme electrophoresis and a
monoclonal antibody reveal cryptic species among Dictyostelium Discoideum
Strains11.
Marina Urman, Heniner G, Matthis Joehnck, Lother RJ and Christian Frech have
carried out cation-exchange chromatography of monoclonal antibodies: characterization
of a novel stationary phase designed for production-scale purification. In this method
the stationary phases has different base matrices and carry specific surface
modification. Three monoclonal antibodies were used as model protein to characterize
these chromatographic resins12.
Hongcheng Liu, Georgeen Gaza-Bulseco and Chris Chumase have performed
Analysis of reduced monoclonal antibodies using size exclusion chromatography
coupled with mass spectroscopy. This method was able to separate non reducible
cross-linked light chain and heavy chains species from free light chain and heavy chain.
A flow rate of 0.2mL/min was used in the current study and directly connected to mass
spectrometer13.
Marie-Luise Hagman, Christine Kionka, Martin Schreiner, Christine Schwer have
developed a method for characterization of the Fab fragment of a murine monoclonal
antibody using capillary isoelecteric focusing and electrospray ionization mass
spectrometry. They have characterized a Fab fragment obtained by pepsin cleavage from
a murine monoclonal IgG3 by mass of electrospray ionization (ESI) mass spectroscopy
(MS), capillary isoelectric focusing (clef) and LC-MS peptide mapping14.
Lintao Wang, Godfrey Amphlett, Walter AB, John M, Lambert and Wei Zhang
have performed structural characterization of the maytansinoid monoclonal antibody
immunoconjugate, huN901-DM1, by mass spectrometry; have studied the drug
distribution profile of huN901-DM1 by electrospray time-of-flight mass spectroscopy15.
Lobvi E, et al., have carried out a characterization of recombinant monoclonal
antibody by mass spectrometry combined with liquid chromatography. This method
includes direct analysis by matrix assisted laser desorption ionization mass spectroscopy
(MALDI-MS) of peptide mixtures and chromatographically isolated fraction allowed
identification of 94% and 85.4% of the amino acid sequence of light and heavy chains,
respectively.
MALDI-MS is used in the sequencing and analysis of peptides and
proteins, study of non covalent complex and immunological molecules and DNA
sequencing and the analysis of intact viruses16.
Lihua Huang, Jirong LU, Victor J Wroblewski, John M Beals, and Ralph M. Riggin
have developed
an In vivo deamidation
characterization of
monoclonal
antibody
by LC-MS/MS. This method was applied to deamidation half-life of amino acid residue
Asn55 in vivo and ratio of the deamidation derivatives has been determined. The
method is rapid and sensitive with low-nanogram quantities of protein detected in the
biological matrix17.
Ziad W Jaradat, Abrar M Rashdhan, Qotaiba O Ababneh, Saied A Jaradat and
Arun K Bhunia have carried out characterization of surface proteins of cronobacter
Muytjensii using monoclonal antibodies and MALADI-TOF mass spectrometry. In this
study they have concluded the immunological cross-reactivity among cronobacter
OMPs and their Enterobacteriaceae counterparts. Nevertheless, the identified protein
appeared to be different as inferred from the MALDI-TOF sequencing and
identification18.
Ping Lu, Brian W. Brooks, Ruth H Robertson, Klaus H Nielsen and Manuel M.
Garcia have developed a method for characterization of monoclonal antibodies for the
rapid detection of food borne Campylobacter. Immunoblot analysis revealed that MAbs
M337 and M357 reacted with a protein component with molecular mass of
approximately 62 kilo Daltons while M306 and M637 reacted with protein components
of approximately 31 kDa, respectively19.
Miroslav Pohanka, Oto Pavlis and Petr Skladal have developed a rapid
characterization of monoclonal antibodies using the piezoelectric Immunosensor.
Monoclonal antibodies with specificity against the Francisella Tularensis outer lipo
polysaccharide membrane were prepared and characterized using the piezoelectric
immune sensors with immobilized LPS antigen from F.tularensis. Signals obtained by
the immune sensor were compared with ELISA and similar sensitivity was noticed20.
Linda J Van Eldick, Beth Ehrenfried and Roy AJ have carried out a production and
Characterizations of monoclonal Antibodies With Specificity for the S1OO𝛽
Polypeptide of brain S100 fractions and reported that production and characterization of
2 mouse hybridomas that secret monoclonal antibodies that appear to be specific for the
S100𝛽 polypeptide of brain S100 preparations by ELISA, RIA and immunoblotting
analysis21.
Dietrich R, Fella C, Strich S and Martlbauer E have performed production and
characterization of monoclonal antibodies against the Hemolysin BL Enterotoxin
complex produced by Bacillus cereus22.
Wood PR. et al., have developed a production and characterization of monoclonal
antibodies specific for Mycobacterium bovis. In this method a series of monoclonal
antibodies (MAbs), specific for Mycobacterium bovis and BCG strains, were tested
extensively for cross-reactivity to wide range of Mycobacterium species using ELISA,
Western blotting and dot-blot analysis. The MAbs bound specifically to M. bovies and
BCG and showed limited cross- reactivity with some strains of M. tuberculosis23.
Zen K, Notarfrancesco K, Ooschot V, Slot JW, Fisher AB and Shuman H have
carried out a generation and characterization of monoclonal antibodies to alveolar type
II cell lamellar body membrane. This study provides evidence that the lbam180 protein
is a lung lamellar body and/or multivesicular body membrane protein and that its
antibody, MAb 3C9, will be a valuable reagent in further investigation of the biogenesis
and trafficking of type II cells organelles24.
Abel Baerga-Ortiz, Carrie A Hughes, Jeffrey G. Mandell, and Elizabeth A.
Komives have developed an epitope mapping of a monoclonal antibody against human
thrombin by H/D-Exchange. Mass spectroscopy reveals selection of a diverse sequence
in a highly conserved protein25.
Carola WN Damen, Hilde Rosing, Jan HM Schllens, Jos H Beijinen have
performed quantitative aspects of the analysis of the monoclonal antibody trastuzumab
using high-performance liquid chromatography coupled with electrospray mass
spectrometry26.
6.4 Main Objective Of The Study:
In the proposed work an attempt will be made:

To develop new analytical methods by MS for characterization of monoclonal
antibodies.

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 characterization of
monoclonal antibodies of marketed/RND samples.
7. Materials And Methods:
7.1 Source of Data:
The preliminary data required for the experimental study will be obtained from
1. Library, Govt. College of Pharmacy, Bangalore.
2. Library and Information Centre, Rajiv Gandhi University of Health Sciences,
Bangalore.
3. http//www.rguhs.ac.in/helinetHome.
4. JRD TATA Memorial Library IISc, Bangalore.
5. Journals and e-journals.
6.
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)
Bangalore.
7.3 Does the study require any investigations or interventions to be conducted on
Patients or other humans or animals? If so, Please describe briefly.
-NOT APPLICABLE.
7.4 Has ethical clearance been obtained from your Institution in case of 7.3?
-NOT APPLICABLE-
8. List of References:
1. Mike SL, Edward HK.
LC/MS Applications in Drug Development, Mass
Spectrometry Reviews. 1999;18:187-279.
2. Monoclonal Antibodies, Wikipedia, The free encyclopedia.
3. Ggillo- Lopez AJ, White CA, Varns C, et al., Overview of the Clinical
Developed of Rituximab: First Monoclonal Antibody Approved for the
Treatment of Lymphoma. Semin Oncol. 1999;26:66.
4. Chan CEZ, Chan AHY, Hanson BJ, Ooi EE. The Use of Antibodies in the
Treatment of Infectious Disease. Singapore Med J. 2009;50(7):663.
5. Morris Sherman. et al., Management of Chronic Hepatitis B: Consensus
Guidelines. Can J Gastroenterol C. 2007;21:5C-24C.
6. Karim A, Jennifer R B. Novel Agents for the Treatment of Chronic Lymphocytic
Leukemia. Clin Adv Hemat Oncol. 2010;8(12):7.
7. Clatworty MR, Watson CJE, Plotnek G. B-Cell Depleting Induction Therapy and
Acute Cellular Rejection. New Engl J Med. 2009;360(25):2683-88.
8. Van Deventer SJH. Anti-TNF Antibody Treatment of Crohn’s Disease. Ann
Rheum Dis. 1999;58(1):1114-20.
9. Prashant Singari, Dennis PW, Kenneth BGordon. Biological Therapy for
Psoriasis. ARCH Dermatol. 2002;138:657-9.
10. Rashid. et al., Potential of-109-Labeled Antimelanoma Monoclonal Antibody for
Tumor Therapy. J Nucl Med. 1984;25:796-9.
11. David AB, Andrew AG, Gooley, Bernstein, George M and Keith LW. Genetic
Diversity in Cellular Slime Modls: Allozyme Electrophoresis and a Monoclonal
Antibody Reveal Cryptic Species among Dictyostelium Discoideum Strains. Gen
Soc Am. 1987;117:213-20.
12. Marina U, Heniner G, Matthis J, Lother RJ and Christian Frech.
Cation-
Exchange Chromatography of Monoclonal Antibodies: Characterization of a
Novel Stationary Phase Designed for Production-Scale Purification. Landes Bio
Sci. 2010;2(4):395-404.
13. Hongcheng L, Georgeen Gaza-Bulseco, Chris Chumase. Analysis of Reduced
Monoclonal Antibodies using Size Exclusion Chromatography Coupled with
Mass Spectroscopy. J Am Soc Spectrom. 2009;20:2258-64.
14. Marie-Luise Hagmann, Christine K, Martin S, Christine S. Characterization of
the F(ab)2 Fragment of a Murine Monoclonal Antibody using Capillary
Isoelectric Focusing and Electrospray Ionization Mass Spectrometry.
J Chromatogr A. 1998;816:49-58.
15. Lintao W, Godfrey A, Walter AB, John ML, Wei Zhang. Structural
Characterization of the Maytansionoid Monoclonal Antibody Immunoconjugate,
huN901, by Mass Spectrometry. Protein Sci. 2005;14:2436-46.
16. Lobvi EMF, Dario EK, Loany C, Minerval Fernadez M, Antonio V, Peter
Roepstorff. Characterization of a Monoclonal Antibody by Mass Spectroscopy
Combined with Liquid Chromatography. J Chromatogr B. 2001;752:247-61.
17. Lihua H, Jirong L, Victor JW, John MB, Ralph MR. In vivo deamidation
characterization of monoclonal antibody by LC/MS/MS. Anal Chem.
2005;77(5):1432-9.
18. Ziad WJ, Abar MR, Qotaiba OA, Saied AJ, Arun KB. Characterization of
Surface Proteins of Cronobacter Muytjensii using Monoclonal Antibodies and
MALDI-TOF Mass Spectrometry. BMC Microbiol. 20;148(11):1471-2180.
19. Ping L, Brian WB, Ruth HR, Klaus HN, Manuel MG. Characterization of
Monoclonal Antibodies for the Rapid Detection of Foodborne Campylobacters.
Int J Food Microbiol. 1997;37(1):87-91.
20. Miroslav P, Oto Pavlis , Petr Skladal. Rapid Characterization of Monoclonal
Antibodies using the Piezoelectric Immunosensor. Sensors. 2007;7:341-53.
21. Linda JVE, Beth Ehrenfried, Roy AJ. Production and Characterizations of
Monoclonal Antibodies with Specificity for the S1OO𝛽 Polypeptide of Brain
S100 Fractions. Proc Natl Acad Sci. 1984;81:6034-38.
22. Dietrich R, Fella C, Strich S, Martlbauer E. Production and Characterization of
Monoclonal Antibodies against the Hemolysin BL Eenterotoxin Complex
Produced by Bacillus Cereus. Appl Environ Biotechnol. 1999;65(10):4470-74.
23. Wood PR. et al., Production and Characterization of Monoclonal Antibodies
Specific for Mycobacterium Bovis. J Gen Microbial. 1988;134:2599-2604.
24. Zen K, Notarfrancesco K, Ooschot V,
Slot JW, Fisher AB, Shuman H.
Generation and Characterization of Monoclonal Antibodies to Alveolar Type II
Cell Lamellar Body Membrane. Am J Physiol Lung Cell Mol Physiol.
1998;275:172-83.
25. Abel Baerga-Ortiz, Carrie AH, Jeffrey GM, Elizabeth AK. Epitope Mapping of a
Monoclonal Antibody against Human Thrombin by H/D- Exchange Mass
Spectroscopy reveals Selection of a Diverse Sequence in a Highly Conserved
Protein. Protein Sci. 2002;11:1300-08.
26. Carola WND, Hilde R, Jan HMS, Jos HB. Quantitative Aspects of the Analysis
of the Monoclonal Antibody Trastuzumab using High-Performance Liquid
Chromatography Coupled with Electrospray Mass Spectrometry. J Pharmaceutic
Biomed Anal. 2007;46:449-55.