Production Of Recombinant Therapeutics Research Articles

Application of CRISPR/Cas9 Genome Editing to Improve Recombinant Protein Production in CHO Cells.

Genome editing has become an important aspect of Chinese hamster ovary (CHO) cell line engineering for improving the production of recombinant protein therapeutics. Currently, the engineering focus is directed toward expanding product diversity while controlling and improving product quality and yields. In this chapter, we present our protocol for using the genome editing tool Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) to knock out engineering target genes in CHO cells. As an example, we describe how to knock out the glutamine synthetase (GS) gene, which increases the selection efficiency of the GS-mediated gene amplification system.

In-house CHO HCPs platform: A promising approach for HCPs ELISA monitoring

A key aspect that must be supervised during the development of recombinant therapeutic products is the potential presence of impurities.Residual host cell proteins (HCPs) are a major class of process-related impurities derived from the host organism that even in trace amount have the potential to affect product quality, safety, and efficacy. Therefore, the product purification processes must be optimized to consistently remove as many HCPs as feasible, with the goal of making the product as pure as possible. The workhorse of HCP monitoring and quantitation during bioprocessing manufacturing is sandwich ELISA (enzyme‐linked immunosorbent assay), which employs polyclonal anti-HCP antibodies for both capture and detection. Commercial ELISA kits developed from Chinese Hamster Ovary (CHO) cell lines are widely applied in early drug development stages (preclinical, phase I, and phase II), but are not specifically designed for a given manufacturer's proprietary cell line, and users do not have control over reagent availability and lot-to-lot consistency. For later development stages, the upstream process-specific method is preferred to guarantee an improved sensitivity and coverage. In agreement with the USP General Chapter 〈1132〉, a platform assay can be used in place of the commercial one through all stages of product development, if already available when product development starts.This proof-of-concept study was carried out to demonstrate the feasibility and the advantages of the development of a proprietary CHO HCPs platform ELISA. Different proprietary mock materials have been characterized and compared by orthogonal bidimensional electrophoresis techniques (SDS-PAGE coupled to SS/WB and 2D DIGE) with the scope of selecting the best antigen-antibody couple for setting up the in-house ELISA. A preliminary evaluation of the in-house method performance has been done in comparison with the commercial assay, demonstrating that the platform method is promising for an accurate and precise CHO HCPs quantification during the early phase product and process development.

Antitumor effect of plant-produced anti-CTLA-4 monoclonal antibody in a murine model of colon cancer.

Cytotoxic T lymphocyte-associated protein 4 (CTLA-4) is an immune checkpoint regulator exclusively expressed on T cells that obstructs the cell's effector functions. Ipilimumab (Yervoy®), a CTLA-4 blocking antibody, emerged as a notable breakthrough in modern cancer treatment, showing upfront clinical benefits in multiple carcinomas. However, the exhilarating cost of checkpoint blockade therapy is discouraging and even utmost prominent in developing countries. Thereby, affordability of cancer care has become a point of emphasis in drug development pipelines. Plant expression system blossomed as a cutting-edge platform for rapid, facile to scale-up, and economical production of recombinant therapeutics. Here, we describe the production of an anti-CTLA-4 2C8 antibody in Nicotiana benthamiana. ELISA and bio-layer interferometry were used to analyze antigen binding and binding kinetics. Anticancer responses in vivo were evaluated using knocked-in mice implanted with syngeneic colon tumor. At 4 days post-infiltration, the antibody was transiently expressed in plants with yields of up to 39.65 ± 8.42 μg/g fresh weight. Plant-produced 2C8 binds to both human and murine CTLA-4, and the plant-produced IgG1 also binds to human FcγRIIIa (V158). In addition, the plant-produced 2C8 monoclonal antibody is as effective as Yervoy® in inhibiting tumor growth in vivo. In conclusion, our study underlines the applicability of plant platform to produce functional therapeutic antibodies with promising potential in cancer immunotherapy.

Screening of CHO-K1 endogenous promoters for expressing recombinant proteins in mammalian cell cultures

For the production of recombinant protein therapeutics in mammalian cells, a high rate of gene expression is desired and hence strong viral-derived promoters are commonly used. However, they usually induce cellular stress and can be susceptible to epigenetic silencing. Endogenous promoters, which coordinates their activity with cellular and bioprocess dynamics while at the same time they maintain high expression levels, may help to avoid such drawbacks. In this work, new endogenous promoters were discovered based on high expression levels in RNA-seq data of CHO-K1 cells cultured in high density. The promoters of Actb, Ctsz, Hmox1, Hspa5, Vim and Rps18 genes were selected for generating new expression vectors for the production of recombinant proteins in mammalian cells. The in silico-derived promoter regions were experimentally verified and the majority showed transcriptional activity comparable or higher than CMV. Also, stable expression following a reduction of culture temperature was investigated. The characterized endogenous promoters (excluding Rps18) constitute a promising alternative to CMV promoter due to their high strength, long-term expression stability and integration into the regulatory network of the host cell. These promoters may also comprise an initial panel for designing cell engineering strategies and synthetic promoters, as well as for industrial cell line development.

Induction of parturition in a large number of pregnant dairy goats and its benefits as a management tool in a commercial scale goat operation

At LFB USA, Inc., transgenic goats are utilized for the production of recombinant human protein therapeutics in their milk through the rPRO™ Technology platform. This retrospective analysis and report describes the results of induced parturition and its use as a management tool in this large herd of dairy goats. Over a three-year period, 342 does received pronuclear microinjected (MI) embryos transferred into the oviductal lumen via midline laparotomy (day 1). To initiate the induction process, does were given intramuscular injections (IM) of 10 mg each of prostaglandin (Lutalyse®) and dexamethasone to induce parturition on days 144–148 of pregnancy. Mean and Standard Deviation (±SD) time to parturition was 36.7 (±6.5) hours. Does were given these injections at 4pm on Sundays with an expected kidding time of late Monday into Tuesday morning. Of the 342 does, 333 or 97% had kidded by 3pm the following Tuesday, and 313 or 91% kidded in the 18 h between 9pm Monday and 3pm on Tuesday or between 29 and 47 h post induction. By the end of Tuesday, most kids had received colostrum and were transferred to the nursery. The incidences of kid mortality and retained placenta were 2.5% and 1.5%, respectively, clearly achieving a priority at this commercial operation for generating a high percentage of live kids (97.5%) of marked value being produced. The use of induced parturition allowed this large dairy operation to designate two 9-h time blocks in which to concentrate parturition times within the herd. This facilitated strategic scheduling to optimize availability of staff, in order to assist with parturition, separate kids from the dam at birth, and ensure adequate and prompt feeding of colostrum. Predicting the time of kidding in this way can serve as an effective management tool, especially to help reduce kid mortality and prevent disease spread by restricting suckling of colostrum.

Enhancing transient protein expression in HEK-293 cells by briefly exposing the culture to DMSO

BackgroundTransient expression of proteins in mammalian cells is a key technique for many functional and structural studies of human and higher eukaryotic genes as well as for the production of recombinant protein therapeutics. Maximizing the expression efficiency to achieve a higher expression yield is desirable and may be even critical when, for instance, an expressed protein must be characterized at the single-cell level. New MethodsOur goal was to develop a simple method by which protein expression yield in human embryonic kidney (HEK)-293 cells could be enhanced with a brief treatment of dimethyl sulfoxide (DMSO) solution. ResultsBy expressing green fluorescent protein (GFP) as a reporter protein using the calcium phosphate transfection method and imaging a large population of cells, we found that a 5-min exposure of 10 % DMSO to HEK-293 cells, 4 h after transfection of the protein of interest, leads to ∼1.6-fold increase in the expression yield without causing any appreciable cytotoxicity. By expressing an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and separately a kainate receptor in HEK-293 cells and measuring glutamate-induced whole-cell current response, we also found that such a brief DMSO treatment did not affect channel activity. ConclusionThis method is simple, efficient and inexpensive to use for enhancing transient transfection yield in HEK-293 cells.

Establishment of fast-growing serum-free immortalised cells from Chinese hamster lung tissues for biopharmaceutical production

Chinese hamster (Cricetulus griseus) ovary-derived Chinese hamster ovary (CHO) cells are the most commonly used mammalian hosts for the industrial production of recombinant therapeutics because of their ability to fold, assemble, and perform post-translational modifications, such as glycosylation, on proteins. They are also valuable for their ability to grow in serum-free suspension cultures. In this study, we established a cell line derived from lung tissue of Chinese hamsters, named Chinese hamster lung (CHL)-YN cells. The biosafety of CHL-YN cells was confirmed by in vitro sterility testing, mycoplasma detection, and reverse transcriptase assays. One of the key characteristics of CHL-YN cells was their doubling time of 8.1 h in chemically defined culture medium; thus, they proliferate much faster than conventional CHO cells and general mammalian cells. Transgenes could be introduced into CHL-YN cells with high efficiency. Finally, between 50% to > 100% of the amount of glycosylated immunoglobulin G (IgG)1 produced by CHO-K1 cells was produced by CHL-YN cells over a shorter period of time. In summary, fast-growing CHL-YN cells are a unique cell line for producing recombinant proteins.

Generation of goats by nuclear transfer: a retrospective analysis of a commercial operation (1998-2010).

At LFB USA, Inc., the ultimate use for transgenic cloned goats is for the production of recombinant human protein therapeutics in their milk. This retrospective analysis of the Somatic Cell Nuclear Transfer (SCNT) program, spanning from 1998 to 2010, examined parameters potentially affecting the outcomes and efficiencies in this commercial operation. Over 37,000 + ova were utilized in the SCNT protocol producing a total of 203 cloned goats. Fifty one (51) clones were produced from non-transfected (transgenic and non-transgenic animal donor) cell lines and 152 clones were produced from transfected cell lines. Comparisons and summaries of (a) transfected versus non-transfected cell lines, (b) relationship of SCNT parameters to offspring produced, (c) skin versus fetal cells, (d) fresh versus cryopreserved cells, (e) parameters from all cell lines used versus those producing SCNT offspring, (f) variation among cell sources, (g) methods of SCNT parturition management and effects on live offspring, and lastly (h) SCNT variation by program are reported. Findings indicate that (a) non-transfected cell lines were more efficient versus transfected cell lines in generating viable cloned offspring on a per reconstructed embryo transferred basis, (b) transfected fetal fibroblasts had improved efficiency versus transfected skin fibroblasts, (c) the percentage of non-transfected cell lines that produced offspring was statistically higher than transfected cell lines, (d) and induction of parturition improved the percentage of viable offspring. In summary, this retrospective analysis on the SCNT process has identified certain parameters for improved efficiency in producing viable cloned goats in a commercial setting.

Microaerobic fermentation alters lactose metabolism in Escherichia coli

Microaerobic fermentation has been shown to improve lactose transport and recombinant protein production in Escherichia coli. Mechanistic correlation between lactose and dissolved oxygen has been studied and it has been demonstrated that E. coli can switch its genetic machinery upon fluctuations in dissolved oxygen levels and thereby impact lactose transport, resulting in product formation. Continuous induction of lactose in microaerobic fermentation led to a 3.3-fold improvement in product titre of rLTNF oligomer and a 1.8-fold improvement in product titre of rSymlin oligomer as compared with traditional aerobic fermentation. Transcriptome profiling indicated that ribosome synthesis, lactose transport and amino acid synthesis genes were upregulated during microaerobic fermentation. Besides, novel lactose transporter setB was examined and it was observed that lactose uptake rate was 1.4-fold higher in microaerobic fermentation. The results indicate that microaerobic fermentation can offer a superior alternative for industrial production of recombinant therapeutics, industrial enzymes and metabolites in E. coli. KEY POINTS: • Microaerobic fermentation results in significantly improved protein production • Lactose transport, ribosome synthesis and amino acid synthesis are enhanced • Product titre improves by 1.8-3.3-fold.

Expression vector cassette engineering for recombinant therapeutic production in mammalian cell systems.

Human tissue plasminogen activator was the first recombinant therapy protein that successfully produced in Chinese hamster ovary cells in 1986 and approved for clinical use. Since then, more and more therapeutic proteins are being manufactured in mammalian cells, and the technologies for recombinant protein production in this expression system have developed rapidly, with the optimization of both upstream and downstream processes. One of the most promising strategies is expression vector cassette optimization based on the expression vector cassette. In this review paper, these approaches and developments are summarized, and the future strategy on the utilizing of expression cassettes for the production of recombinant therapeutic proteins in mammalian cells is discussed.

Influence of cytoskeleton organization on recombinant protein expression by CHO cells.

In this study, we assessed the importance of cytoskeleton organization in the mammalian cells used to produce therapeutic proteins. Two cytoskeletal genes, Actin alpha cardiac muscle 1 (ACTC1) and a guanosine triphosphate GTPase‐activating protein (TAGAP), were found to be upregulated in highly productive therapeutic protein‐expressing Chinese hamster ovary (CHO) cells selected by the deprivation of vitamin B5. We report here that the overexpression of the ACTC1 protein was able to improve significantly recombinant therapeutic production, as well as to decrease the levels of toxic lactate metabolic by‐products. ACTC1 overexpression was accompanied by altered as well as decreased polymerized actin, which was associated with high protein production by CHO cell cultured in suspension. We suggest that the depolymerization of actin and the possible modulation of integrin signaling, as well as changes in basal metabolism, may be driving the increase of protein secretion by CHO cells.

Engineering mammalian cells to produce plant-specific N-glycosylation on proteins.

Protein N-glycosylation is an essential and highly conserved posttranslational modification found in all eukaryotic cells. Yeast, plants and mammalian cells, however, produce N-glycans with distinct structural features. These species-specific features not only pose challenges in selecting host cells for production of recombinant therapeutics for human medical use but also provide opportunities to explore and utilize species-specific glycosylation in design of vaccines. Here, we used reverse cross-species engineering to stably introduce plant core α3fucose (α3Fuc) and β2xylose (β2Xyl) N-glycosylation epitopes in the mammalian Chinese hamster ovary (CHO) cell line. We used directed knockin of plant core fucosylation and xylosylation genes (AtFucTA/AtFucTB and AtXylT) and targeted knockout of endogenous genes for core fucosylation (fut8) and elongation (B4galt1), for establishing CHO cells with plant N-glycosylation capacities. The engineering was evaluated through coexpression of two human therapeutic N-glycoproteins, erythropoietin (EPO) and an immunoglobulin G (IgG) antibody. Full conversion to the plant-type α3Fuc/β2Xyl bi-antennary agalactosylated N-glycosylation (G0FX) was demonstrated for the IgG1 produced in CHO cells. These results demonstrate that N-glycosylation in mammalian cells is amenable for extensive cross-kingdom engineering and that engineered CHO cells may be used to produce glycoproteins with plant glycosylation.

A Lab-Scale Fermentation Course with an Emphasis on Recombinant Protein Production

Fermentation is responsible for the production of myriad products across a variety of industrial sectors. In particular, the biomanufacturing industry requires a labor force proficient in fermentation and its associated technologies to drive the production of recombinant protein therapeutics. This paper describes the development of a course that introduces students to fermentation technologies for protein production at the laboratory scale. The course aims to familiarize students with the theory underlying the operation of bioreactors, the growth of microorganisms, and the expression and characterization of recombinant proteins. The eight-week course consists of a weekly lecture covering topics including properties of proteins, expression systems, microbial growth, gas-liquid mass transfer, sterilization, and process controls. In addition to lectures, students participate in weekly lab sessions involving the evaluation of growth kinetics, protein production, expression system optimization, and oxygenation and substrate feeding strategies. Student learning is assessed through bi-weekly quizzes and team lab reports, as well as a final group presentation on a technology relevant to the production of recombinant therapeutic proteins. Analysis of student responses on pre- and post-course surveys informed efficacy of course delivery. Overall, student responses on pre- and post-course surveys indicate achievement of learning objectives.

Microalgal Aquafeeds As Part of a Circular Bioeconomy.

Photosynthetic microalgae are unicellular plants, many of which are rich in protein, lipids, and bioactives and form an important part of the base of the natural aquatic food chain. Population growth, demand for high-quality protein, and depletion of wild fishstocks are forecast to increase aquacultural fish demand by 37% between 2016 and 2030. This review highlights the role of microalgae and recent advances that can support a sustainable 'circular' aquaculture industry. Microalgae-based feed supplements and recombinant therapeutic production offer significant opportunities to improve animal health, disease resistance, and yields. Critically, microalgae in biofloc, 'green water', nutrient remediation, and integrated multitrophic aquaculture technologies offer innovative solutions for economic and environmentally sustainable development in line with key UN Sustainability Goals.

Clinical Pharmacology Review of Plasma-derived and Recombinant Protein Products: CBER Experience and Perspectives on Model-Informed Drug Development.

Clinical pharmacology studies are one of the major types of regulatory data submitted for review of therapeutic proteins regulated by the Center for Biologics Evaluation and Research (CBER). The primary objective of the current study is to provide an overview of the role of clinical pharmacology including pharmacokinetics (PK), pharmacodynamics (PD) and exposure-response analysis at CBER. Furthermore, we aim to provide a baseline estimate for the use of quantitative clinical pharmacology studies prior to implementation of FDA's model-informed drug development (MIDD) pilot programme. We survey original Biologics License Applications (BLAs) for plasma-derived and related recombinant therapeutic protein products approved by CBER/FDA (2008-2017). There were 37 original BLAs that met our inclusion criteria, and 34 of these products (92%) contained human PK data as part of the biological licensing. The products were broadly classified as coagulation factors (54%), IgG and related proteins (24%), and other therapeutic proteins (22%). Coagulation factor VIII and IX products constitute 32% of the BLAs and indicated for treatment of haemophilia A and B, respectively. Twelve products (35%) used model-based approaches (population PK/PD and exposure-response). Over the past 5years (2013 to 2017), there is a trend for increased application of MIDD approaches as compared to the previous cohort years (2008 to 2012). In conclusion, clinical pharmacology has played a major role in regulatory review of plasma-derived products, and we expect that the application of quantitative methods will further evolve for these products under the FDA MIDD programme.

Methodological Approaches to Validation of Therapeutic Recombinant Proteins Production Based on the Quality by Design Concept

Validation of production processes based on the Quality by Design (QbD) principles calls for thorough scientific understanding of the processes and enhancement of their stability by implementation of new technologies. The aim of the study consisted in substantiating a QbD-based technological approach to validation of commercial production of dornase alfa. For this purpose a design space was established in a scale-down model, i.e. 2 L reactors; the model was shown to be representative in terms of all parameters except for the reactor size; the similarity of hydrodynamic conditions, design characteristics and operation modes of laboratory, pilot and commercial scale reactors was established; the process scalability was demonstrated by using the PCA (Principal Component Analysis) multivariate mathematical model including the volume range of 2–1000 L, input and output process parameters and product quality attributes for a number of recombinant therapeutic products derived from the same CHO cell line and expression construction as dornase alfa producer. The article demonstrates the applicability of engineering space, which includes bioreactor design features and production process parameters, to different production scales by implementing 3 processes at the pilot scale (100 L) and 2 processes at the commercial scale (1000 L) and building a PCA model based on the obtained data.

Generation of transgenic goats by pronuclear microinjection: a retrospective analysis of a commercial operation (1995-2012).

Production of transgenic founder goats involves introducing and stably integrating an engineered piece of DNA into the genome of the animal. At LFB USA, the ultimate use of these transgenic goats is for the production of recombinant human protein therapeutics in the milk of these dairy animals. The transgene or construct typically links a milk protein specific promoter sequence, the coding sequence for the gene of interest, and the necessary downstream regulatory sequences thereby directing expression of the recombinant protein in the milk during the lactation period. Over the time period indicated (1995-2012), pronuclear microinjection was used in a number of programs to insert transgenes into 18,120, 1- or 2- cell stage fertilized embryos. These embryos were transferred into 4180 synchronized recipient females with 1934 (47%) recipients becoming pregnant, 2594 offspring generated, and a 109 (4.2%) of those offspring determined to be transgenic. Even with new and improving genome editing tools now available, pronuclear microinjection is still the predominant and proven technology used in this commercial setting supporting regulatory filings and market authorizations when producing founder transgenic animals with large transgenes (>10kb) such as those necessary for directing monoclonal antibody production in milk.

Assessment of the structure of pegylated-recombinant protein therapeutics by the NMR fingerprint assay

A number of recombinant protein therapeutic products, such as filgrastim (methionyl granulocyte colony stimulating factor [Met-GCSF] used to boost the immune system in chemotherapy treated cancer patients), and interferon alpha-2 (used for the treatment of various viral infections), have been chemically modified with the addition of a polyethylene glycol (PEG) chain. This modification prolongs residency of the drug in the body and reduces metabolic degradation, which allows less frequent administration of the products. Here we show how NMR spectroscopy methods can assess the higher order structure (HOS) of pegylated-filgrastim (Neulasta®), pegylated interferon-α2a (Pegasys®) pegylated interferon-α2b (PEG-Intron®) purchased from the marketplace. The addition of the PEG moiety effectively doubles the molecular weight of the three products. This presents a significant challenge for the application of NMR techniques. Nevertheless, the results showed that high-resolution spectra could be recorded for two of the three products. Comparison of the spectra of the pegylated protein and the non-pegylated protein shows that the chemical modification did not alter the HOS of these proteins.

Recombinant therapeutic proteins produced in plants: towards engineering of human-type O-and N-glycosylation

Background and Purpose : Recombinant DNA technology has allowed expression of different heterologous proteins in many host systems, ranging from prokaryotic to eukaryotic organisms. Therapeutic properties of recombinant proteins are very often affected by the composition and heterogeneity of their glycans. Conventional expression systems for recombinant pharmaceuticals typically do not address this problem and result with products that contain a mixture of glycoforms that are neither identical to human glycans nor optimized for enhanced biological activity. Over the last decade plants have been developed as production platforms for recombinant proteins with pharmaceutical or industrial applications. Namely, plant expression systems contain very small differences in the post-translational modifications, mainly glycosylation, which can partly be overpowered by glycoengineering, whose goal is production of recombinant proteins with highly homogenous glycosylation that closely resembles the native system. This review attempts to present current accomplishments in the production of plant-derived glycoconjugates with humanized N- and O-glycans. Materials and Methods: Main goal of N-glycoengineering is to reduce or eliminate plant-specific N-glycans, and at the same time to introduce mammalian-specific N-glycans through the several approaches. The easiest way is to change intracellular targeting of plant-made recombinant proteins and to ensure their retention in the ER; next approach is to eliminate the addition of plant-specific glycans; while the final step is engineering the plant glycosylation pathway to introduce mammalian glycotransferases into plants with generation of biantennary and multi-antennary structures on complex N-glycans. Due to significant differences in O-glycosylation between humans and plants, different approaches to engineering of O-glycosylation have been taken. Besides having their typical O-glycoslyation on Hyp-residues, plants in general miss the machinery for production of mammalian-type O-glycosylation. Attempts have been made to mimic mammalian O-glycosylation in plants, specifically the mucin-type addition of GalNAc residues. Result: Efficient generation of bisected tetraantennary complex N-glycans without typical plant glycoepitopes on human erythropoietin (hEPO) and human transferrin (hTF) was obtained in Nicotiana benthamiana plants, thus demonstrating generation of recombinant proteins with human-type N-glycosylation at great uniformity. As for the O-glycosylation, attempts to produce mucin-type O-GalNAc and disialylated core 1 O-linked glycan structures on hEPO in N. benthamiana transgenic plants proved to be successful. Moreover, although small amounts of Hyp residues were found on recombinant EPO, no plant-specific O-glycans were detected, which demonstrates that plants are eligible candidates for production of recombinant therapeutics with fully humanized O- and N-glycans. Conclusion : Plants and methods of plant molecular farming offer a powerful expression platform for the production of a variety of recombinant proteins, which show similar, or even higher, biological activity then protein or native homologs in cultured mammalian cells currently used for large-scale production.

Status and Application of Recombinant DNA Therapeutic Products Used in Clinical Practices: An Indian Scenario

Recombinant DNA (r-DNA) technology or Genetic Engineering is an umbrella term for a set of experimental techniques that empower specific genes and DNA sequences to be manipulated resulting in genetically modified organisms (GMO) and therapeutic products. There have been many potential applications of r-DNA in medicine, agriculture, and industry. Conventionally proteins and other biological products, processed from human or animal serum or tissues, often are of low purity. Production of therapeutic products by the r-DNA technology has several advantages such as provision of drugs that could not be produced by conventional methods, manufacture of adequate quantities of drugs and provision for manufacture of safe drugs. In India, nine products have been approved for marketing – insulin (diabetes drug), alpha interferon (cancer drug), hepatitis B vaccine, Granulocyte-macrophage colony-stimulating factor (GM-CSF), Granulocyte-colony stimulating factor (G-CSF or GCSF),blood clotting factor 7, erythropoietin (drug used in kidney failure), streptokinase (drug administered in heart attacks), and human growth hormone. All these products except Hepatitis-B are being imported. The four major recombinant products with high market potential in India are human insulin, alpha interferon, and erythropoietin. In India, technology has been developed in different centers like International Center for Genetic Engineering and Biotechnology (ICGEB), (insulin), IMTech Chandigarh (streptokinase), and Bharat biotech. This review focuses on the scenario of Indian manufacturers, biotherapeutics approvals in India and important aspects that need to be precisely analyzed and resolved from the Indian perspective to facilitate implementation of r-DNA technology, bio-similar products, to best serve the public health needs of the developing countries. The study is beneficial to biotechnology scientists, medical institutions, pharmaceuticals, and doctors.