Recent Innovations in Therapeutic Recombinant Protein
Dr Madhusudan P Dabhole, Group Manager – Bioprocess, Richcore Life Sciences Ltd

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.

Therapeutic 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 percent 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 percent 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.

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 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 non-nutritive (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 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 O2- 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 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 HBs -antigens. (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.

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.

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.

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 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.

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.

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.

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.

There are three Competent Authorities involved in approval process namely:
  1. Review Committee on Genetic 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. Central Drugs Standard Control 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 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 specialised skills in Microbiology, 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 university-industry 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 providing required infrastructure 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".