Recent Innovations in Therapeutic Recombinant Protein
Therapeutic recombinant proteins
are one of the most important
and rapidly growing segments of
the biopharmaceutical market.
The emerging business of
therauptic Proteins, since 1980,
have witnessed a paradigm
shift with enhanced efficacy,
greater safety, and reduced
immunogenicity comes from the
conjunction of clinical, scientific,
technological and commercial
drivers that have identified unmet
needs. Since the first protein
therapeutics were approved two
decades ago, the field has seen
a transition from the development
of naturally occurring proteins to
design of molecules engineered
for optimal target recognition,
pharmacokinetics, bio distribution,
and therapeutic function.
Dr Madhusudan P Dabhole
Group Manager – Bioprocess
Richcore Life Sciences Ltd
10 July 2014
T
herapeutic proteins can be grouped
into molecular types that include:
antibody-based drugs, anticoagulants,
blood factors, bone morphogenetic proteins,
engineered protein scaffolds, enzymes, Fc
fusion proteins, growth factors, hormones,
interferons, interleukins, and thrombolytic.
Recombinant proteins are produced in
bacteria, yeast, filamentous fungi, insect
cells, mammalian cells, transgenic animals,
and transgenic plants. Overall, 39 per cent of
recombinant proteins are made by E. coli, 35
per cent by CHO cells, 15 per cent by yeasts,
10 per cent by other mammalian systems.
The first human protein therapeutic
derived from recombinant DNA technology
was human insulin (Humulin) created
at Genentech , developed by Eli Lilly,
and approved by the US Food and Drug
Administration (FDA) in 1982. The first
therapeutic application of an r-protein
produced in mammalian cells was approved
in 1986 (human tissue plasminogen activator,
tPA, Genentech). Recombinant Protein
drugs have changed the landscape for the
treatment of many diseases, including many
types of cancer and rheumatic conditions.
Moreover, the recombinant protein market
has grown at an annual average rate of
35 per cent since 2001, indicating a financially
sound future for the biopharmaceutical
industry.
Antibody-based drugs are the largest and
fastest growing class of protein therapeutics
with 24 marketed antibody drugs in the USA
and over 240 more in clinical development.
Antibody-based drugs contributed USD 38
billion of USD 99 billion in world-wide sales
for protein biopharmaceuticals in 2009.
Moreover, 5 of the 10 top-selling protein
therapeutics in 2009 were antibodies,
namely, infliximab (Remicade), bevacizumab
(Avastin), rituximab (Rituxan and MabThera),
adalimumab (Humira) and trastuzumab
(Herceptin). Existing mAb therapies are
based on ‘cocktails’ of molecules containing
sin¬gle- or double-site cell binding fragments
allow the formation of dimers, chains
and cycles. Currently, mammalian cell
line development technologies used by
biopharmaceutical industries are based on
either the methotrexate (MTX) amplification
technology or the glutamine synthetase
(GS) system.
The main strategies that have been adopted
for the creation of second generation Protein
products include reformulation, pegylation
and other forms of modification, or the
creation of analogues with different amino
acid structures. Most of these drugs are
aimed at being longer lasting in the body
and having improved pharmacokinetics (PK),
while still having similar pharmacodynamics
(PD) as the first generation molecules.
Achieving this aim can lead to highly
advantageous properties such as a lower
frequency of administration and improved
patient compliance. Examples of highly
commercially successful products include
Amgen’s Aranesp (an analogue of EPO)
and Roche’s Pegasys (pegylated interferon
alpha). The major challenges facing
recombinant protein production are to reduce
the production cost, improve the productivity
both in upstream and downstream and obtain
high titer while maintaining the quality of
the products.
The global biopharmaceutical industry is
currently worth over USD 116 billion and
should exceed USD 167 billion in 2015,
says IMARC. Geographically, North America
represents the largest market, as majority
of the key players are domiciled in United
States and thus many new drugs first are
introduced in these regions.
In 2010, the Department of Pharmaceuticals
(DoP) of the Government of India (GOI)
set the nation’s biopharmaceutical
industry (BioPharma) a goal: to become
a leading global producer of affordable
“biopharmaceutical” products by 2020.
Industry experts estimate that it could be
worth US USD 319 billion by 2020.
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Global Therapeutic Protein Market
Recent Innovations
Scientists searched for proteins from 17
bodily organs, including the frontal cortex
of the brain, the retina in the eye, ovaries,
testes and more. In addition, the scientists
analysed six types of cells found in the blood
and seven samples taken from organs in
human fetuses. The team identified proteins
made by 17,294 genes. 2,535 of those
genes made proteins that have never been
described by science before. 193 of those
genes weren’t previously predicted to be
protein-making genes at all.
The efforts included 72 scientists from six
countries: the US, India, Canada, Chile, the
UK and Hong Kong. The second team, made
up of 22 scientists from different institutes in
Germany, found similar, although not exactly
the same, numbers. The German team found
proteins made from 18,097 genes. Those
genes made 86,771 different proteins.
Now that the proteins in humans are
mapped, it will create a platform for
understanding genetic links to human
diseases. Bioinformatics will bridge the gap
in evaluation of data between the protein
function and expression in human diseased
states. In the near future, protein therapy for
novel diseases will be created by combining
MAbs and genes. According to Garcia &
Calantone, the essence of innovation can
best be described as: “an iterative process
initiated by the perception of a new market
12 July 2014
opportunity for a technology-based invention
which leads to development, manufacturing,
and marketing tasks aspiring commercial
success of the invention”.
So the stage is set for scientists across the
world who has initiated the study to work on
the protein disorders by aligning the protein
function and characterisation from different
organs and tissues.
In Mammalian cells, the culture media has
an important impact on both the yield and
quality of recombinant proteins. Mammalian
cells require a combination of both nutritive
(eg, sugars and amino acids) and nonnutritive (eg, trace metals, vitamins and
co-factors) components to support cell
growth and protein production. Continous
feeding and intermittent feeding of carbon
source at a specific interval is critical for
protein synthesis. In addition, the media
environment has been shown to affect protein
glycosylation, which is an important aspect
of protein quality and influences its efficacy
as a therapeutic. The efficacy of recombinant
glycoproteins as a human therapeutic is
strongly dependent on their carbohydrate
structures, or glycosylation. Glycosylation
has been implicated in bioactivity, receptor
binding, and susceptibility to proteolysis,
immunogenicity and in vivo clearance rate.
It is known that two mammalian cell lines
cannot be considered similar as proteins are
virtually very difficult to be copied. Protein
Modifications changes such as acetylation,
methylation, glycosylation, hydroxylation,
phosphorylation and sulphation may reduce
biological activity and cause an intrinsic
molecular heterogeneity which is difficult
to control. Furthermore, the structural
complexity of protein concentrates the final
product influenced by many variables, such
as the use of an expression system such
as bacterial, yeast and mammalian cells,
growth conditions, purification processes,
formulation, storage and transportation. The
process related impurities may increase the
severity of an immune response to a protein
product. Bottom-Up mathematical modeling
approach provides cellular responses to
different stimuli, but has limitations due
to low peptide recovery. Top-down Mass
Spectrometry is becoming a powerful
technology for comprehensive analysis of
protein modifications. Top-down approach
enables identification and analyses of an
entire cellular network for characterisation
of the recombinant protein mutants and
is applied together with evolution and
mutagenesis experiments.
For mammalian cell culture, Process
improvement requires and integrated
approach that can be achieved through
optimisation of bioreactor physicochemical
environment. Optimisation of culture
conditions needs to balance cell growth
with antibody production. The most common
approach to developing a feed medium uses
concentrated basal media without salts (to
avoid high osmolality). Certain key feeding
components (eg, phosphate) have also been
identified. During medium preparation, pH
and temperature may need to be adjusted
to completely dissolve some low-solubility
components. To optimise a feeding strategy,
consideration should be given to nutrient
consumption, by-product accumulation, and
the balance between growth and production.
Studies indicated that byproducts, such as
lactate and ammonia, could be minimised
by maintaining low glucose and glutamine
concentrations through frequent feeding.
In the breakthrough that provided the
details of large scale proteomic analysis of
6164 grouped proteins complemented by
the genomic data of CHO cells, the codon
Pharma Bio World
bias of CHO cells, distinct from humans
has been deciphered. The accessibility
of methods in the analysis of metabolites
in CHO cells with combined data from
genomics, transcriptomics, proteomics and
metabolomics now can identify novel genes
that affect the growth and protein production
rate of CHO cells.
The success of novel protein therauptic and
rise of antibody based drugs have led to
research on engineering protein scaffolds
which are in the early stages of study and
clinical development.
High cell density fermentation is a major
bio-process engineering consideration for
enhancing the overall yield of recombinant
proteins in E. coli. The development
and design of fermentation process and
fermenter itself play a key role for achieving
productivity and robustness at scale up.
Increasing aeration rate, feeding O 2- rich
air, decreasing temperature, gas holdup in
media, tip speed, increasing partial pressure
of the culture vessel are some of the methods
employed to maintain aerobic condition
during cell growth. One of the significant
technologies developed for microbial growth
and expression of recombinant proteins is
the use of Tender Coconut Water (TCW) as
Animal Origin Free (AOF) growth media by
C-CAMP, DBT India.
The dynamics of scale for each protein
production will differ based on rate of the
synthesis of protein in the cell, oxygen
availability, feeding strategy, carbon source
utilisation, operating fermentation parameters
and metabolic load. Generally, proteins that
are larger than 100 kD are expressed in
a eukaryotic system while those smaller
than 30 kD are expressed in a prokaryotic
system. The future of single use bioreactors
for recombinant protein production will
witness a complete revolution with respect
to processing and application on a prodigious
platform which will involve production of
high volume, high value products with high
yields as compared to low volume high value
products. The major challenge in single use
bioreactor is the usage of Pharma grade
14 July 2014
polymers which needs validated analytical
methods for leachables and extractables.
It is important to bring the flexibility in
biopharma operations and analytics with
platform technologies. Platform analytical
technologies can simplify timely acquisition
and reproduction of technology from clients,
which otherwise can pose a risk of substantial
project delays. Cost is another factor which
will have to be optimised during recombinant
protein production from R&D scale level.
The set-up of a large number of libraries of
Clone from bacteria, yeast and CHO cells are
in developmental stage which will be available
for commercial production of recombinant
proteins. Hansenula Polymorpha is looked
upon as one of the promising candidates for
insulin and Hepatitis B Vaccine production.
In yeast cells, the formation of the disulphide
bonds is efficient; however, the proteolytic
digestion of proinsulin is not possible. In this
case, proinsulin is produced and the purified
proinsulin is digested in vitro with trypsin.
Hepatitis B virus cannot be propagated
in vitro; therefore HBs-Ag is produced
by heterologous expression in yeasts
(Saccharomyces cerevisiae, Hansenula
anomala, Pichia pastoris). According to
its genome sequence, 8 genotypes (A-H)
have been identified. The newly described
G genotype has been isolated in the USA
and France, while the H genotype originates
from South and Central-America. The active
immunisation is generally based on HBsantigens. (SHBs, MHBs, LHBs, HBc-Ag,
or combination of HBs and HBc antigens
are also used.) . With FDA approving the
first inhaling insulin powder which will be
available by 2015, the insulin business has
scaled new heights with new innovations
being introduced periodically.
Scientists are working on to identify the effect
of Host Cell Protein during recombinant protein
production. Identification and elimination of
toxic molecules due to contamination and
absence of prion proteins is another area
of concern which is being addressed on
a global platform. Case Western Reserve
University researchers published findings
that point to a promising discovery for the
treatment and prevention of prion diseases,
rare neurodegenerative disorders that are
always fatal. The researchers discovered
that recombinant human prion protein stops
the propagation of prions, the infectious
pathogens that cause the diseases.
Scientists across the world are in the process
of developing recombinant fusion protein
markers and phage proteins for serological
diagnosis of human infectious diseases and
contaminants. Recombinant phage protein
technique is a novel technology on Vidas as
phage proteins bind efficiently to the receptor
and exhibit an extraordinary stability. A tail
fibre phage protein is used for detection of
contaminants. New peptide phage display
libraries allows gathering all the information
regarding a patient’s antibodies for mapping
the human response to the recent diseases
and allergies in just one step.
Proteomics technology is being applied in the
field of protein identification and quantification
at all stages of the development from cell line
expression to protein production. The use
of Proteomics has led to development of
a robust process optimisation to achieve
recombinant protein for clinical phases so
as to obtain faster regulatory clearances.
Biosidus is involved in a groundbreaking
initiative in the field of biodiversity known
as the White Genome Project, which
aims at the isolation, identification and
characterisation of Antarctic bacterial strains
for further sequencing of the complete
genome. In collaboration with the Argentine
Dirección Nacional del Antártico, research
is being conducted at the isolation and
characterization of certain microorganisms
from the Antarctic territory that are particularly
adapted to extreme temperature and have
isolated and identified a novel species,
Bizionia argentinesis and have sequenced
its full genome.
Current investigation is focused on the
identification and characterisation of
genes coding for “cold active enzymes” for
industrial processes, particularly in the field
of food processing.
Pharma Bio World
Frost & Sullivan recognised MicroProtein
Technologies for Technology Innovation for
the development of its proprietary MPTxpress
technology that provides a cost-effective
production method for pharmaceutically and
diagnostically relevant recombinant proteins,
along with the highest percentage of yield in
the industry. MPTxpress has achieved these
objectives and has the potential to become
the standard production protocol for bacterial
expression in this USD 2 billion market. The
platform uses a solid media to produce higher
yields of the total soluble protein. In addition
to significantly reducing procedural steps
for obtaining and extracting purified protein,
the solid media platform is biodegradable.
This makes it the first completely green
recombinant biologics production process
in the industry and it leaves behind a
considerably lower carbon footprint.
by USFDA. Rixubis [Coagulation Factor
IX (Recombinant) Fusion protein] is for
use in people with hemophilia B who are
16 years of age and older for the control
and prevention of bleeding episodes,
perioperative (period extending from the time
of hospitalisation for surgery to the time of
discharge) management, and routine use to
prevent or reduce the frequency of bleeding
episodes (prophylaxis). An inherited blood
clotting disorder mainly affecting males,
Hemophilia B is caused by mutations in the
Factor IX gene and leads to deficiency of
Factor IX.
Recombinant CBD (cellulose binding
domains) produced by E coli and Pichia
Pastoris are gaining importance as affinity
tags, scaffolds and for purification of
Hematopoietic stem cells. Hematopoietic
stem cells (HSCs) are the only type of stem
cells that have been routinely used to treat
patients with blood cancers and disorders
of the blood and immune systems by HSC
transplantation (HSCT). The difficulty to the
development of novel treatments using HSCs
is the lack of HSC source of sufficient purity
and yield. As a result, effort has been devoted
on HSC purification using recombinant CBD
and ex vivo expansion which will create
stem cell bio factories for the treatment of
human diseases.
Synthetic biology is a new emerging
field bringing engineers and biologist
to design and construct biomolecular
network pathways for pharmaceutical
applications. Artemisinic acid, as a precursor
towards the biosynthesis of artemisinin, an
important antimalarial drug, was successfully
transferred into E. coli and S. cerevisiae.
The plant derived kaempferol and quercetin
were heterologously synthesised in E. coli.
Synthetic biology is being applied to create
several artificial biological systems like
synthetic gene network – the gene toggle
switch for producing cheaper drugs. The
challenges are clear and range from host
design to producing non - natural chimeric
recombinant proteins.
Genetically engineered protein polymers with
specific monomer sequence and polymer
length has provided opportunities for the
utility of these polymers in drug delivery.
The development of elastin-like, silk-like,
and silk-elastin like protein polymers has led
to the study in gene delivery systems and
tissue engineering.
The first recombinant protein coagulation
factor IX that is specifically indicated
for routine use in preventing bleeding
episodes (prophylaxis) has been approved
16 July 2014
HIV associated Tuberculosis vaccine is in the
mid stage of clinical trials as we move into
2020. There is an urgent need to develop
an innovative tuberculosis vaccine with
immunogenicity that can be administered to
HIV patients.
Quality by Design (QbD) is a new system
towards the progress of recombinant
therapeutic protein that upholds a better
understanding of the product and its
manufacturing process. Quality features
observed in biopharmaceutical proteins
include product-related impurities and
substances, process-related impurities and
contaminants are evaluated each for their
impact on biological activity, immunogenicity,
and toxicity. The impact of structural
characteristics on the therapeutic proteins
is to reduce immunogenicity by controlling
critical quality attributes of proteins.
Worldwide, there are more than 500 biologic
products in various stages of clinical trials
and pre-registration. New Biotech clusters
may soon emerge that are able to reap the
benefits afforded by the development of
several types of recombinant proteins. The
success in future therapeutic protein markets
will require tractability, vision and the ability
to develop affordable biologics for patients
and physicians.
Regulatory Framework
The similar biologic drug products are usually
referred to as similar biotherapeutic product
(SBPs) by WHO, biosimilars by European
Medicines Agency (EMA) of the European
Union (EU), follow-on biologics (FOBs) by
the US FDA, and subsequent-entry biologics
(SEBs) by Health Canada. In some cases,
the term “biosimilar” has been used in
an inappropriate way, and therefore it is
important to review differences in definitions
of biosimilar products in different regions.
WHO defines SBP as a bio therapeutic
product, which is similar in terms of quality,
safety and efficacy to an already licensed
reference bio therapeutic product.
India is creating a framework to introduce
single window clearances for project and
approvals. Government of India is on the
path to abridge the procedures for import and
export biologics. The Regional Committee
on Genetic Manipulation (RCGM) committee
along with Department of Biotechnology
(DBT) meets once every month to evaluate
projects on biologics who should approve
the projects within specified timeframe by
planning to meet twice monthly. The RCGM
and DBT have the preeminent biosafety and
pollution control norms which the biotech
sectors needs to follow stringently.
In India, the Guidelines on Similar Biologics
were prepared by CDSCO and DBT laid
down the regulatory pathway for similar
biologic claiming to be similar to an already
authorised reference biologic. CDSCO is
the national regulatory authority in India that
evaluates safety, efficacy and quality of drugs
in the country.
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There are three Competent Authorities
involved in approval process namely:
1) R e v i e w C o m m i t t e e o n G e n e t i c
Manipulation (RCGM)/IBSC under
Department of Biotechnology (DBT),
Ministry of Science and Technology.
2) Genetic Engineering Appraisal
Committee (GEAC) under the Ministry
of Environment and Forests (MoEF) and
3)C e n t r a l D r u g s S t a n d a r d C o n t r o l
Organisation (CDSCO) under Ministry
of Health & Family Welfare.
The Biotech industries manufacturing
recombinant products will have to provide
1) Gene sequence, vectors and promotor
of the selected strain.
2) Three batches of reproducible
fermentation data at pilot scale.
3) Consistent Specific protein yield.
4) Overall productivity is reproducible and
scalable.
5) Steps involved in purification of protein.
6) Batch size for protein purification.
7) Consistency of recovery in 3 consecutive
batches of purification from 3 independent
batches of fermentation.
8) Determination of primary and higher
order structure of the product.
9) The target amino acid sequence of the
similar biologic should be confirmed and is
expected to be the same as for the
reference biologic.
10) In cases, where post translational
modifications are taking place, these
modifications need to be identified and
quantified.
11) In case any significant differences
are found, these should be scientifically
justified and critically examined in
preclinical studies and clinical trials.
12) Biological assays will be required to
characterise the activity and establish the
products mechanism of action and clinical
effects (in units of activity).
13) Assays should be calibrated against
an international or national reference
standard, where available and appropriate.
If no such standards are available, an
internal reference standard must be
established as per the ICH guidelines. If
18 July 2014
the methods of bioassay(s) are documented
in the specification, test(s) can be
conducted accordingly.
14) Evaluation by characterisation (antibody
or antibody-derived product); comparison to
reference biologic with respect to specificity,
affinity, binding strength and Fc function;
and evaluation by animal studies.
The clinical development of recombinant
proteins is commonly divided into three
phases (ie, phases I, II, and III). Each
phase is more complex, time-consuming,
and resource-intensive than the preceding
one. In India, Clinical drug trials including
biologics, post new rules in 2014, have
medical ethics committee to be registered
with DCGI which reviews the clinical
phases stringently. With new virus strains
being introduced for recombinant vaccines
production, DBT will have to review the
Biosafety norms and frame stringent
guidelines for manufacturing and purification
of the product.
Industry–Academia Partnership
A large number of students work towards
s p e c i a l i s e d s k i l l s i n M i c r o b i o l o g y,
Biotechnology, Biochemistry and Genetic
Engineering in and around the world.
Continous developments are seen across
in research journals of which less than
1 per cent gets translated to commercial
success. The economic significance
demonstrates the importance of universityindustry partnerships to biotech. It has been
observed that the Biotech courses taught
in the academic orientation are practically
diverse from the industrial requirement.
Few biotech industries like Biocon have
entered into agreement with Universities
to set up a course with industrial outlook
for aspiring candidates. Association of
Biotechnology Led Enterprises (ABLE)
facilitates strong industry - academia
interactions to explore opportunities for
collaborative research and technology
transfer as well as human resource
development through various platforms of
engagement. The biotech industries should
take the initiative to set up a research
projects in colleges across the country
which can transfer the technology from
the lab scale to the manufacturing scale.
The value addition created by the industry
academia interaction is unparalleled as
the industry will benefit from tangible
assessment created through insights and
industry ready employees.
Biotechnology Industry Research
Assistance Council (BIRAC) a Public
Sector Section 25 “Not-for-Profit Company”
of Government of India, registered under
India Companies Act 1956, has been set up
as Department of Biotechnology’s interface
agency, which serves as a single window
for the emerging biotech industries. It
was incorporated on 20th March, 2012.
.BIRAC has been set up as a separate
body for supporting product innovation
and prov iding required in fr a s tr u c tu r e
and services at different stages of the
value chain for promoting innovation and
product development.
Entrepreneurs are looking for a magic bug
which will initiate producing the product
and reach the market instantly. This has
created the gap in understanding for
manufacturing biological products which
needs incubation at various stages for
research, clinical trials, manufacturing
and validation. Further, these biological
products should be affordable to a common
man. Medicines have become an integral
part of human life and to combat the deadly
diseases, both existing and new arrivals,
scientists will have to develop a roadmap
for the next 50 years. The traditional
pharmaceutical manufacturers will have
to focus and invest now for these future
biologic blockbuster products. It will be a
challenge to bring the Innovation, instead
of renovation, to obtain pure, safe, high
efficacy, cost effective and high yielding
recombinant human therapeutic proteins
with quality to market. As Charles Darwin
rightly said “Survival of the fittest”.
Contact: [email protected]
Pharma Bio World