1. health and fitness
2. disease
3. cancer

GUIDANCE MANUAL FOR ASSURING QUALITY
OF TRASTUZUMAB
NATIONAL INSTITUTE OF BIOLOGICALS
(Ministry of Health & Family Welfare)
Government of India
A – 32, Sector 62,
NOIDA – 201309
Uttar Pradesh – INDIA.
E-mail: info@nib.gov.in
INTRODUCTION
Monoclonal Antibodies (mAbs) are immunoglobulin’s (novel protein therapeutics)
with a defined specificity derived from a monoclonal cell line. Their biological
activities are characterised by a specific binding characteristic to a ligand (commonly
known as antigen), and may also be dependent on immune effector function such as
antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent
cytotoxicity (CDC). mAbs may be generated by recombinant DNA (rDNA)
technology, hybridomas technology, B lymphocyte immortalisation or other
technologies (e.g. phage display technology, genetically engineered animals). As a
result of naturally-occurring molecular heterogeneity, imperfect cellular processing,
chemical and enzymatic changes during manufacturing and additional changes upon
storage, antibody drugs display a wide variety of minor chemical changes,
collectively termed microheterogeneity. Common examples include glycan structural
differences, deamidation, oxidation and glycation. Control of microheterogeneity
within predefined analytical specifications has been used in quality control
laboratories to guarantee consistent product quality during cGMP manufacturing.
The recent Quality by Design (QbD) initiative for therapeutic biotechnology products,
a joint pilot program between the regulators, academia and the biotechnology
industry, is providing new guidance and expectations on QbD approaches in
manufacturing for therapeutic proteins. As the name indicates, QbD encourages
developers to build quality into the drug from the start. This approach requires
significant knowledge of the drug’s mechanism of action and how the drug’s
attributes affect quality. Physical or chemical changes known to affect the safety or
efficacy of the drug are considered Critical Quality Attributes (CQAs). Manufacturing
is then designed to control the desired levels of CQAs within defined limits, providing
a consistent product quality. With this new QbD paradigm, the process is defined by
the target ranges for the CQAs which, in turn, provide assurance of consistent
product quality.
Of the various classes of antibodies, or immunoglobulin’s, IgG1 is the most common
immunoglobulin used for pharmaceutical and biomedical purposes, however, recent
developments have shown that other immunoglobulin types (e.g., IgG2, IgG4) and
mAb-related products (e.g., Fc-fusion proteins, Fabs, etc.) are also being used for
therapeutic purposes. The technological advancement in the evolution of therapeutic
antibodies is shown at Figure – 1.
Figure - 1: Various types of mAbs evolved with technological advancement.
The innovator product of trastuzumab is a humanized IgGl kappa monoclonal antibody
that selectively binds with high affinity to the extracellular domain of the human
epidermal growth factor receptor 2 protein, HER2 (Figure – 2). Trastuzumab is
produced by recombinant DNA technology in a mammalian cell (Chinese Hamster
Ovary) culture. It was the first targeted cancer drug, aimed at patients whose tumors
have specific genetic characteristics. In this case, the appropriate patients have
tumors that over-express the HER2 gene.
Antigen binding sites:
derived from mouse
Figure - 2Human IgG1 Antibody
Trastuzumab is mainly use for breast cancer treatment. Breast cancer is the most
common cancer among women worldwide. Each year, about 1.4 million new cases of
breast cancer are diagnosed worldwide, and over 450,000 women will die of the
disease annually.In India Breast cancer is the second most frequently diagnosed
cancer (22%) and is the second leading cause of death after cervix cancer (26%)
among women.
In October 20, 2010, the U. S. Food and Drug Administration granted approval for
trastuzumab (Herceptin®, Genentech, Inc.), in combination with cisplatin and a
fluoropyrimidine (capecitabine or 5-fluorouracil), for the treatment of patients with
HER2 overexpressing metastatic gastric or gastroesophageal (GE) junction
adenocarcinoma, who have not received prior treatment for metastatic disease.
In
HER2-positive breast cancer, increased quantities of the human epidermal growth
factor receptor 2 (HER2) are present on the surface of the tumor cells. This is known
as "HER2 positivity" and affects approximately 15% to 20% of women with breast
cancer. HER2-positive cancer is a particularly aggressive form of breast cancer.
2.
SCOPE
The guidance manual is aimed at emphasizing on the quality control testing
parameters particularly for trastuzumab intended for therapeutic use. Parallel to the
aforesaid, there is a need felt to meet the requirements of good manufacturing
practices, product-lot-testing to ensure product reproducibility, production process
verification and quality assurance.
3.
GLOBAL AND INDIAN MARKET DYNAMICS OF MAb PARTICULARLY
TRASTUZUMAB
The recombinant humanized anti-Her2 antibody trastuzumab (Herceptin) from
Genentech/Roche is among the world's commercially most successful antibodies
(2011 sales of over US$ 5.5 bn). Herceptin is approved and marketed for Her2 positive
breast and gastric cancer. The commercial attractiveness of trastuzumab has
stimulated many companies to create and develop next generation anti-Her2
monoclonal antibodies (mabs) with improved properties compared with the humanized
antibody trastuzumab. Accordingly, Genentech and Roche are still expanding the
indication label of trastuzumab and have developed 2nd and 3rd generation Her2
antibodies. These Biosuperior antibodies include antibody-drug conjugates, bispecific
antibodies and engineered Her2 antibodies. The forthcoming patent expiry for
Herceptin has boosted interest in and activities for developing biosimilar copies with
the most advanced projects already in phase III. At present, there are at least 7
Herceptin biosimilar antibodies in clinical development and further 15 in preclinical
R&D.
As the patents for biologics is expired or move closer to their expiration dates and the
challenging economic situation across the globe forces a decrease in healthcare
expenditures, biosimilars emerge as the cost-effective therapeutic alternative across
the globe. Market for biosimilar mAbs is on the constant rise. The average patent
expiry of $39.6 bn per annum between 2010 to 2015 as compared to just $14.2 bn in
last decade and 16.5 bn per annum between 2016-2020 (Table -1)
Table -1
Being an affordable yet equally-effective alternative for biologics, they can provide
savings that range from 25% to 85% of the original versions. Moreover, the patent
expiry of approximately 48 biologics (worth US $73 billion) in the next 10 years
will also fuel the demand for biosimilars. Analysts estimate that biosimilars could
quickly become a $10 billion category (Table – 2 & 3). Despite the resistance from the
innovator companies, and the regulatory hurdles involved in their production,
biosimilars have immense market potential for both developed, as well as growing
economies.
Table – 2: FDA approved therapeutic mAbs available in the world market.
Name:
Antibody
*OKT3:
Muronomab-CD3
Target:
Antibody Type
CD3:
Murine, IgG2a
Indication
Company
ReoPro:
Abciximab
PIIb/IIIa:
Chimeric, IgG1, Fab
Rituxan:
Rituximab
CD20:
Chimeric, IgG1
*Zenapax:
Daclizumab
CD25:
Humanized, IgG1
Autoimmune
Roche
Simulect:
Basiliximab
CD25:
Chimeric, IgG1
Autoimmune
Novartis
Synagis:
Palivizumab
RSV:
Humanized, IgG1
Infections
MedImmune
Remicade:
Infliximab
TNFa:
Chimeric, IgG1
Autoimmune
Johnson &
Johnson
Herceptin:
Trastuzumab
HER2:
Humanized, IgG1
Cancer
Genentech/
Roche
*Mylotarg:
Gemtuzumab
ozogamicin
CD33:
Humanized,
IgG4, immunotoxin
Cancer
Wyeth/
Pfizer
Campath:
Alemtuzumab
CD52:
Humanized, IgG1
Cancer
Genzyme
Zevalin:
Ibritumomab tiuxetan
CD20:
Murine, IgG1,
radiolabeled (Yttrium
90)
Cancer
Humira:
Adalimumab
TNFa:
Human, IgG1
Autoimmune
Abbott
Xolair:
Omalizumab
IgE:
Humanized, IgG1
Autoimmune
Genentech/
Roche
Approval Date
Autoimmune
Johnson &
Johnson
1986 (US)
Homeostasis
Johnson &
Johnson
1984 (US)
Cancer
Genentech
Patent
Expiry
Data Not
available
1997 (US)
Data Not
available
26.11.2017
1998 (EU)
02.06.2018
1997 (US)
1998 (US)
Data Not
available
Data Not
available
Data Not
available
Data Not
available
Data Not
available
Data Not
available
24.08.2018
1999 (EU)
13.08.2019
1999 (EU)
1998 (US)
1998 (EU)
1998 (US)
1999 (EU)
1998 (US)
2000 (EU)
25.09.2018
28.08.2018
2000 (US)
Data Not
available
2001 (US)
07.05.2021
06.07.2021
2001 (EU)
2002 (US)
Biogen Idec
2004 (EU)
2002 (US)
2003 (EU)
Data Not
available
Data Not
available
31.12.2022
08.09.2023
20.06.2023
2003 (US)
Bexxar:
Tositumomab-I-131
*Raptiva:
Efalizumab
CD20:
Murine, IgG2a,
radiolabeled (Iodine
131)
CD11a:
Humanized, IgG1
Cancer
Autoimmune
Corixa/GSK
2003 (US)
2003 (US)
Data Not
available
2004 (EU)
Data Not
available
Genentech/
Roche
2004 (US)
Erbitux:
Cetuximab
EGFR:
Chimeric, IgG1
Cancer
Imclone/
Lilly
Avastin:
Bevacizumab
VEGF:
Humanized, IgG1
Cancer
Genentech/
Roche
Tysabri:
Natalizumab
a4-Intergrin:
Humanized, IgG4
Autoimmune
Biogen Idec
2004 (US)
Actemra:
Tocilizumab
Anti-IL-6R:
Humanized, IgG1
Autoimmune
Chugai/
Roche
2005(JP)
2010 (US)
Vectibix:
Panitumumab
EGFR:
Human, IgG2
Cancer
Amgen
2006 (US)
Lucentis:Ranibizumab
VEGF: Humanized
IgG1 Fab
Macular
degeneration
Genentech/
Roche
Soliris:
Eculizumab
C5:
Humanized IgG2/4
Cimzia:
Certolizumab
pegol
TNFa:
Humanized, pegylated
Fab
Autoimmune
UCB
2008 (US)
Simponi:
Golimumab
TNFa: Human IgG1
Autoimmune
Johnson &
Johnson
2009
(US, EU, CAN)
Ilaris:
Canakinumab
IL1b:
Human IgG1
Infalmmatory
Novartis
2009 (US,EU)
Stelara:
Ustekinumab
IL-12/23: Human IgG1
Autoimmune
Johnson &
Johnson
Arzerra:
Ofatumumab
Prolia:
Denosumab
Numax:
Motavizumab
ABThrax:
Raxibacumab
Benlysta:
Belimumab
Yervoy:
Ipilimumab
Adcetris:
Brentuximab Vedotin
Perjeta:
Pertuzumab
Blood disorders
Alexion
2004 (EU)
2004 (US)
2005 (EU)
2006 (US)
2007 (US)
2009 (US)
2008 (EU)
CD20: Human IgG1
Cancer
Genmab
2009 (EU)
RANK ligand: Human
IgG2
Bone Loss
Amgen
2010 (US)
RSV: Humanized IgG1
Anti-infective
Meddimmune
Pending
B. anthrasisPA:
Human IgG1
Anti-infection
GSK
2012(US)
BLyS: Human IgG1
Autoimmune
Human
Genome
Sciences
2011 (US)
CTLA-4: Human IgG1
Cancer
BMS
2011 (US)
CD30: Chimeric, IgG1,
Drug-conjugate
Her2: Humanized,
IgG1
Cancer
Cancer
Seattle
Genetics
Genentech/
Roche
Data Not
available
2011 (US)
2012 (US)
12.02.2024
Data Not
available
26.02.2024
Data Not
available
Data Not
available
Data Not
available
Data Not
available
Data Not
available
Data Not
available
Data Not
available
Data Not
available
Data Not
available
Data Not
available
Data Not
available
Data Not
available
Data Not
available
Data Not
available
Data Not
available
Data Not
available
Data Not
available
Data Not
available
Data Not
available
Kadcyla:
Ado-trastuzumab
emtansine
Her2: Humanized,
IgG1, Drug-conjugate
Cancer
Genentech/
Roche
Data Not
available
2013 (US)
*Withdrawn by the sponsor
Table 3: List of biologics coming off patent from 2012 to 2015.
Product
Company
Therapeutic
Category
Patent Expiry
Enbrel
Amgen, Pfizer
Other anti-rheumatics
23-10-2012
Neupogen
Amgen
Immunostimulants
12-03-2013
Humalog
Eli Lilly
Anti-diabetics
07-05-2013
Avonex
Biogen Idec
MS Therapies
30-05-2013
Epogen
Amgen
Anti-anaemics
20-08-2013
Procrit/Eprex
Johnson & Johnson
Anti-anaemics
20-08-2013
Cerezyme
Genzyme
Other therapeutic
products
27-08-2013
Rebif
Merk KGaA
MS Therapies
21-12-2013
Novomix
Novo Nordisk
Anti-diabetics
06-06-2014
NovoRapid/NovoLog
Novo Nordisk
Anti-diabetics
07-12-2014
Rituxan
Roche
Antineoplastic Mabs
31-12-2014
Kogenate
Bayer
Anti-fibrinolytics
31-12-2014
Prevnar
Pfizer
Vaccines
01-01-2015
Lantus
Sanofi-Aventis
Anti-diabetics
12-02-2012
Actemra
Roche
Other Anti-rheumatics
07-06-2012
Gonal-F/Gonalef
Merk KGaA
Fertility agents
16-06-2015
Neulasta
Amgen
Immunostimulants
20-10-2015
Nimotuzumab
YM BioSciences
Anti-neoplastic Mabs
17-11-2015
Norditropin SimpleXx
Novo Nordisk
Growth Hormones
15-12-2015
Helixate
CSL
Anti-fibrinolytics
31-12-2015
3.1 Indian Market:
India, with a share of over 2 per cent in the global biotechnology industry, is set to
become one of the world's largest biotechnology hubs. Nearly 64 per cent of the
biotech companies operate in the bio-pharma sector in India. The biotechnology sector
in India is expected to achieve revenue of US$ 11.6 billion by 2017, growing at a
compound annual growth rate (CAGR) of 22 per cent. Revenue from biotech exports
reached US$ 2.2 billion in FY13, accounting for more than half (51 per cent) of total
industry revenues. Biotech industry is expected to grow to around Rs 440,000 crores
(US$ 70.84 billion) by the year 2020.The vaccines and recombinant therapeutics are
the leading sectors driving the biotechnology industry's growth in India. M/s Biocon,
other Indian pharma companies like M/s Intas, M/s Mabpharma and M/s Dr. Raddys
Lab are also set to tap the Rs.130-crore market for breast cancer drug Trastuzumab.
Just five top Indian companies with manufacturing capabilities in recombinant
therapeutic products in the year 2005 are shown in Table – 4.
Table 4:
3.2 Biosimilar Therapeutics - Indian Scenario:
The drug classes for biosimilars in India comprise of Human Insulin, Human Growth
Hormone, Granulocyte Colony Stimulating Factor (G-CSF), Erythropoietin, and
Streptokinase. Recent estimates indicate that 20 companies in India are already
developing biosimilars. There are around 25 recombinant therapeutic available in India
and 15 of them are manufactured within the country. About 50 brands are
commercialized in the country by leading companies such as Biocon, Shantha
Biotechnics, Reliance Life Sciences, Wockhardt and Intas Biopharmaceuticals among
others. About 72 recombinant drugs are currently undergoing different stages of
clinical trials and their launch may cause an increase of 25% in the Indian Biopharma
sector. In the biosimilar market India was holding 26th position in 2005 and will be
expected to hold 6th position by 2015 in the global biosimilar market (Figure - 3)
Figure – 3
4.
ICH DOCUMENTS FOR BIOLOGICS:
5.
MANUFACTURING PROCESS (UPSTREAM & DOWNSTREAM)
6. STAGES OF MASTER CELL BANK (MCB) AND WORKING CELL BANK (WCB).
7.
QUALITY CONTROL:
7.1
Reference Material
There is a need to have reference standard of Trastuzumab. In the meantime
innovator drug is regularly used as a comparator as and when required for quality
control testing.
7.2
Lot release testing of Trastuzumab
Once the trastuzumab is purified and formulated, the resulting drug substance must
be tested prior to lot release (Table – 5). The product specific specifications are not
available in Pharmacopoeia. Therefore lot release of drug is done based on the limits
approved for the drug during the marketing authorization by National Regulatory
Authority (NRA). A set of tests and acceptance criteria are established based on
trastuzumab characterization and prevalent regulatory requirements in order to ensure
drug quality. These tests typically include appearance, identity, purity, protein
concentration, potency of the drug, microbial limits or bio-burden, bacterial Endotoxins,
Host cell protein and residual DNA etc.
Ion exchange chromatography, SDS under reducing and non-reducing condition by
Capillary Electrophoresis and Size Exclusion Chromatography are the most frequently
used lot release methods for purity for Trastuzumab. Test for immunogenicity is
performed by the manufacturer. EMA has prepared a concept paper to revise the
guidelines on immunogenicity assessment on 20th February 2014. The aim of reform
is not to increase the number of studies on immunogenicity but to increase the quality
of studies and their clarity to the assessors. Stability studies may be performed on the
Trastuzumab drug substance (e.g. frozen bulk for storage) and drug product, if
required (e.g. final vial) according to prevalent regulatory guidelines. EMEA 2006 &
2007 guidelines are available for characterisation of similar biological medicinal
products containing biotechnology derived proteins as active substances. WHO
(2013) guidelines have been published on evaluation of similar biotherapeutic
products. Similarly, guidelines on similar biologics; regulatory requirements for
marketing authorization in India have been published in the year 2012 by the DBT &
CDSCO, Govt. of India. Indian pharmacopoeia commission is in the process of
finalisaiton of monograph on Rituximab and other recombinant drugs.
Table – 5. Commonly used tests in a Certificate of Analysis for lot release of mAbs
including trastuzumab.
QC Parameter
Test Name
Appearance
Color, Opalescence and Clarity
Identity
Peptide mapping by RP-HPLC (Reverse Phase HPLC), or
MALDI (Matrix – assisted deionisation) Mass spectroscopy, or
UV spectroscopy (2nd derivative)
Purity
Limulus Amebocyte Lysate (Endotoxin)
Size exclusion chromatography (SEC)
CE-SDS (Capillary electrophoresis-Sodium dodecyl sulphate)
IEC (Ion exchange chromatography) or icIEF (Imaged capillary
isoelectric focusing)
Glycosylation profile
Peptide mapping by RP-HPLC
Potency
Potency (ELISA/Cell-Based Assay)
Strength
UV Spectroscopy
General Tests
Osmolality
pH
Protein concentration
Surfactant Concentration (e.g. Polysorbate 20)
Safety
Sterility testing
Bacterial Endotoxin Test
Critical Quality tests such as Identity, Purity, Potency and Safety are performed head
to head with the “Reference” by using test methods suitably approved by a National
Control Authority.
7.3 General Test:
Appearance, Solubility, pH, Osmolality, Water content, Protein content, Subvisible particulate matter and Extractable volume should be assessed wherever
appropriate as per the prevalent regulatory guidelines / pharmacopoeia. The
results complies with the limits approved for the particular product during
marketing authorization.
7.4 Identification:
A. By Peptide Mapping: Positive identity is confirmed if the elution profiles of the
test solution and reference solution are similar by visual observation and there
are no new significant peaks. It complies with the limits approved for the
particular product during marketing authorization.
B. By Iso Electric Focussing (IEF) using capillary electrophoresis (CE): The
identity is established comparing the product with reference preparation. It
complies with the limits approved for the particular product during marketing
authorization.
C. CZE by capillary electrophoresis: The difference in the migration time (MT)
for the main peak of the first and second reference solution injections bracketing
each test solution must be < 0.1 minute. It complies with the limits approved for
the particular product during marketing authorization.
D. Bio-assay: Same as potency assay
7.5 Impurities:
A. Size Exclusion Chromatography by HPLC (SEC-HPLC): Impurities are
determine by Size Exclusion Chromatography and results complies with the
limits approved for the particular product during marketing authorization
B. Ion Exchange Chromatography by HPLC (IEC-HPLC): Impurities are
determined by Ion Exchange Chromatography. Run the sample with
appropriate gradient program to optimize the separation. It complies with the
limits approved for the particular product during marketing authorization.
C. SDS by Capillary Electrophoresis (CE-SDS): Impurities are determined by
Capillary Electrophoresis. This test performed in under reducing as well as nonreducing condition. Analyze the sample and reference against the molecular
weight marker using an capillary electrophresis method capable of giving
separation in the range 10- 250 kDa followed by UV detection. Results complies
with the limits approved for the particular product during marketing
authorization.
7.6. Safety Test:
A. Sterility: It complies with the test for sterility
B. Bacterial Endotoxin Test (BET): Not more than 1.0 EU per mg.
7.7. Assay:
1. Potency (Anti-proliferation): Determine the potency of Trastuzumab by using
BT-474 or similar cell line in an antiproliferation assay. It complies with the
limits approved for the particular product during marketing authorization.
2. Glycan Profilling by Capillary Electrophoresis or by HPLC: By any one of
validated methods. It complies with the limits approved for the particular product
during marketing authorization.
8.
REFERENCES:
I.
A Report by Association of Biotechnology Led Enterprises (ABLE) for the
Department of Biotechnology, Government of India – May 2012.
II.
Biospectrum – Volume 9, Issue 6, June 2011.
III.
EMEA guidelines on similar biologicals medicinal products containing
biotechnology derived proteins as active substance: non-clinical and clinical
issues, London – 2006 (CHMP/BMWP/42832).
IV.
EMEA guidelines on immunogenicity assessment of biotechnology derive
therapeutics proteins, London – 2007 (CHMP/BMWP/14327).
V.
Guideline on similar biologics – Regulatory requirements for marketing
authorisation in India (2012), Department of Biotechnology, Ministry of Science
& Technology, Government of India, CDSCO – Ministry of Health & Family
Welfare, Government of India (Page No’s: 1 to 42)
VI.
VII.
ICH document – Manufacturing process of Biologics - K. Ho Afssaps, France
(2011).
Monoclonal Antibody Development and Physicochemical Characterization
Jennifer C. Rea, Yajun Jennifer Wang, Tony G. Moreno, Rahul Parikh, Yun Lou
and Dell Farnan, Genentech, Inc., Protein Analytical Chemistry, South San
Francisco, CA-USA – 2012.
VIII.
WHO-TRS 814 (1991), Annex 3 – Guideline for assessing the quality of
pharmaceutical & biotech products prepared by rDNA technology.
IX.
WHO guideline – “Guidelines for national authorities on quality assurance for
biological products” – WHO TRS 822 (Annex 2) – 1992.
X.
WHO Guidelines on Biosimilars evaluation of Similar Biotherapeutic products
(SBPs) – 2009.
XI.
WHO informal consultations on the revision of guidelines on the Quality, Safety,
Efficacy of Biological & Medicinal products prepared by rDNA technology- June
2012.
XII.
Anti-malignant Tumor Agent with Anti-HER2 Humanized Monoclonal Antibody:
Presentation of Takahiro Yamaguchi, Herceptin Product Manager, Chugai
Pharmaceutical Co., Ltd. Oncology Unit, Breast Cancer Group.
XIII.
From pipeline to market R&D Directions 2009, 15(6)4-89.
XIV.
http://photos.prnewswire.com/prnh/20100302/LA63139LOGO).
XV.
http://www.researchandmarkets.com/research/jdh26g/competitor.
XVI.
http://www.businesswire.com/news/home/20130402005802/en/ResearchMarkets-Competitor-Analysis-Her2-Antibodies.
Disclaimer:
This guidance represents the current views of National Institute of Biologicals
(NIB's) on assuring Quality of Therapeutic (MAbs). It does not create or confer
any rights for or on any person and does not operate to bind NIB or the public.
An alternative approach may be used if such approach satisfies the
requirements of the applicable statutes and regulations.