Importance of Recombinant Protein Technology in Biopharmaceuticals

1 Introduction

Recombinant Protein Technology: A Cornerstone in Modern Biopharmaceuticals

In the past few decades, the field of biotechnology has seen unprecedented advancements, particularly in the development and application of recombinant protein technology. This technology, which involves the genetic manipulation of cells to produce desired proteins, has become a cornerstone in the biopharmaceutical industry. Recombinant protein technology is crucial for the production of therapeutic proteins, vaccines, and diagnostic tools that are essential for treating and managing various diseases.

Overview of Recombinant Protein Technology

Recombinant protein technology involves inserting a gene encoding a protein of interest into an expression system, such as bacteria, yeast, or mammalian cells. These genetically engineered cells are then cultured, allowing them to produce the recombinant protein. The process typically includes several stages: gene cloning, expression, purification, and characterization of the protein. This technology enables the production of proteins that are identical to their natural counterparts but can be produced in large quantities and with high purity and consistency.

Importance and Relevance in Modern Biopharmaceuticals

The importance of recombinant protein technology in biopharmaceuticals cannot be overstated. It has revolutionized the production of various therapeutic proteins, which were previously difficult or impossible to obtain in sufficient quantities from natural sources. For instance, human insulin, which is crucial for diabetes management, was the first recombinant protein to be approved for therapeutic use. Since then, numerous other therapeutic proteins, such as growth hormones, monoclonal antibodies, and cytokines, have been developed using recombinant technology.

One of the significant advantages of recombinant proteins is their ability to be produced in a controlled environment, ensuring consistency and reducing the risk of contamination. This is particularly important for biopharmaceuticals, where the purity and efficacy of therapeutic proteins are critical. Additionally, recombinant protein technology allows for the modification of proteins to improve their stability, efficacy, and safety. For example, engineered antibodies with enhanced binding affinity or reduced immunogenicity can provide better therapeutic outcomes.

The Aim and Scope of the Article

This article aims to provide an in-depth understanding of the importance of recombinant protein technology in the biopharmaceutical industry. It will cover the historical development of the technology, the mechanisms involved in recombinant protein production, and its diverse applications in biopharmaceuticals. Additionally, the article will highlight recent advancements and future prospects in the field, emphasizing how these developments continue to shape modern medicine.

Through this exploration, we hope to shed light on how recombinant protein technology has transformed the biopharmaceutical landscape, leading to more effective and innovative treatments for a wide range of diseases. By understanding the underlying principles and advancements in this technology, stakeholders in the biotechnology and pharmaceutical sectors can better appreciate its significance and potential for future applications.

2 Historical Background

The Evolution of Recombinant Protein Technology in Biopharmaceuticals: Key Milestones and Breakthroughs

Recombinant protein technology has transformed the landscape of biopharmaceuticals, enabling the production of vital therapeutic proteins, vaccines, and diagnostic tools. This section delves into the historical background of this technology, highlighting its evolution, significant milestones, and pioneering products that have shaped the industry.

Evolution of Recombinant Protein Technology

The journey of recombinant protein technology began with the discovery of the structure of DNA and the subsequent development of genetic engineering techniques. The concept of recombinant DNA (rDNA) technology emerged in the early 1970s, pioneered by scientists like Paul Berg, Herbert Boyer, and Stanley Cohen. This technology allowed the insertion of foreign genes into bacterial plasmids, leading to the expression of recombinant proteins. The first recombinant DNA molecule was created in 1972, marking the inception of a new era in biotechnology [3].

In the ensuing decades, advancements in molecular biology and genetic engineering refined these techniques, making them more efficient and versatile. The 1980s saw the commercialization of recombinant proteins, with insulin being the first therapeutic protein produced using recombinant DNA technology. The ability to produce human insulin in bacteria revolutionized diabetes treatment, replacing the less efficient and sometimes allergenic insulin derived from animal sources [4].

Milestones in the Development of Recombinant Biopharmaceuticals

Several key milestones have marked the progress of recombinant protein technology in biopharmaceuticals. One of the most notable achievements was the development of recombinant human growth hormone (rhGH) in the mid-1980s. This advancement addressed the limitations of pituitary-derived growth hormone, providing a safer and more reliable therapeutic option for patients with growth hormone deficiencies [5].

The introduction of monoclonal antibodies in the 1990s further expanded the applications of recombinant proteins. Monoclonal antibodies, produced through hybridoma technology combined with recombinant DNA methods, have become indispensable in the treatment of various cancers, autoimmune diseases, and infectious diseases. The approval of the first monoclonal antibody drug, muromonab-CD3, in 1986, marked a significant milestone in targeted therapy [6].

Key Breakthroughs and Pioneering Products

One of the groundbreaking breakthroughs in recombinant protein technology was the development of erythropoietin (EPO) for treating anemia in chronic kidney disease patients. EPO, a glycoprotein hormone that stimulates red blood cell production, was initially derived from human urine, making it scarce and expensive. Recombinant DNA technology enabled the mass production of EPO in Chinese hamster ovary (CHO) cells, revolutionizing the management of anemia and significantly improving patient outcomes [7].

Another pioneering product is the hepatitis B vaccine, the first recombinant vaccine approved for human use. Developed in the 1980s, this vaccine uses recombinant yeast cells to produce the hepatitis B surface antigen, providing a safe and effective means of preventing hepatitis B infection. This innovation paved the way for the development of other recombinant vaccines, contributing to global vaccination efforts [8].

The advent of fusion proteins, which combine two or more proteins to enhance therapeutic efficacy, represents another significant breakthrough. Fusion proteins like etanercept (Enbrel), used in the treatment of rheumatoid arthritis and other inflammatory conditions, demonstrate the versatility and potential of recombinant protein technology in creating innovative therapies [9].

3 Mechanism of Recombinant Protein Production

The Mechanism of Recombinant Protein Production: Principles and Systems

Recombinant protein production is a cornerstone of modern biopharmaceuticals, enabling the large-scale production of proteins that are essential for therapeutic and diagnostic applications. This section delves into the basic principles of recombinant DNA technology, the steps involved in producing recombinant proteins, and the common expression systems used in this process.

Basic Principles of Recombinant DNA Technology

Recombinant DNA technology, also known as genetic engineering, involves combining DNA molecules from different sources to create a new set of genes. This technology relies on the use of vectors, such as plasmids or viruses, to introduce foreign DNA into host cells. The key steps in recombinant DNA technology include:

1. Gene Cloning: The gene of interest is isolated and inserted into a cloning vector. This vector is typically a plasmid that can replicate within a bacterial host.
2. Transformation: The recombinant DNA is introduced into a host cell, usually a bacterium, through a process called transformation.
3. Expression: The host cell machinery is used to transcribe and translate the introduced gene, resulting in the production of the desired protein.
4. Selection: Host cells that have successfully incorporated the recombinant DNA are selected using markers, such as antibiotic resistance.
5. Purification: The recombinant protein is extracted and purified from the host cells.

This process has been refined over the years, making it possible to produce proteins with high purity and in large quantities [7].

Steps Involved in the Production of Recombinant Proteins

The production of recombinant proteins involves several critical steps, each requiring meticulous optimization to ensure high yield and functionality of the protein:

1. Vector Design: The gene of interest is cloned into an expression vector that includes regulatory elements, such as promoters and enhancers, to drive efficient transcription.
2. Host Cell Selection: The choice of host cell depends on the type of protein being produced. Common host cells include bacteria, yeast, mammalian cells, and plant cells.
3. Transformation/Transfection: The recombinant vector is introduced into the host cells through methods such as heat shock, electroporation, or chemical transfection.
4. Expression and Induction: The host cells are cultured under conditions that promote the expression of the recombinant protein. Inducers, such as IPTG in bacterial systems, may be used to initiate protein production.
5. Harvesting: The cells are harvested, and the recombinant protein is extracted. This step may involve cell lysis to release intracellular proteins.
6. Purification: The recombinant protein is purified using techniques such as affinity chromatography, ion-exchange chromatography, and size-exclusion chromatography.
7. Characterization: The purity, structure, and functionality of the recombinant protein are assessed using analytical techniques such as SDS-PAGE, mass spectrometry, and bioassays [8].

Common Expression Systems

The choice of expression system is crucial for the successful production of recombinant proteins. The most commonly used systems are:

1. Bacterial Systems: Bacteria, particularly Escherichia coli, are widely used for the production of recombinant proteins due to their rapid growth, ease of genetic manipulation, and cost-effectiveness. However, bacterial systems may not be suitable for producing proteins that require post-translational modifications [9].
2. Yeast Systems: Yeast, such as Saccharomyces cerevisiae, offers advantages over bacterial systems by providing eukaryotic post-translational modifications. Yeast systems are used for producing a variety of biopharmaceuticals, including insulin and hepatitis B vaccine [10].
3. Mammalian Cell Systems: Mammalian cells, such as Chinese Hamster Ovary (CHO) cells, are preferred for producing complex proteins that require precise folding and glycosylation. Mammalian systems are essential for producing monoclonal antibodies and other therapeutic proteins [11].
4. Plant Systems: Plants are emerging as a viable alternative for recombinant protein production due to their scalability and low production costs. Plant-based systems are used for producing vaccines and antibodies. They offer advantages in terms of safety, as plant pathogens do not infect humans [12].

4 Applications in Biopharmaceuticals

Harnessing Recombinant Protein Technology in Biopharmaceuticals: Therapeutics, Vaccines, and Diagnostics

Recombinant protein technology has revolutionized the biopharmaceutical industry, enabling the development of a wide range of applications that have significantly improved healthcare outcomes. This section explores the diverse applications of recombinant protein technology in biopharmaceuticals, including therapeutic proteins, vaccines, diagnostic tools, and emerging trends.

Therapeutic Proteins

Therapeutic proteins are among the most prominent applications of recombinant protein technology. These proteins, produced through genetic engineering, are used to treat a variety of diseases. Key examples include:

1. Insulin: The production of recombinant human insulin was a groundbreaking achievement that transformed diabetes management. Before recombinant technology, insulin was extracted from animal pancreases, which posed supply and purity challenges. Recombinant insulin is now produced in bacterial and yeast cells, ensuring high purity and consistent supply [10].
2. Growth Hormones: Recombinant human growth hormone (rhGH) is used to treat growth disorders in children and growth hormone deficiency in adults. The recombinant form replaced the limited and potentially contaminated pituitary-derived hormone, offering a safer and more reliable treatment option [11].
3. Monoclonal Antibodies: Monoclonal antibodies (mAbs) are engineered proteins that can specifically target and neutralize pathogens or diseased cells. They have become essential in the treatment of various cancers, autoimmune diseases, and infectious diseases. Examples include trastuzumab for breast cancer and adalimumab for rheumatoid arthritis [12].

Vaccines

Recombinant protein technology has also played a crucial role in vaccine development. Some of the most notable vaccines produced using this technology include:

1. Hepatitis B Vaccine: The recombinant hepatitis B vaccine was one of the first vaccines developed using genetic engineering. It utilizes the hepatitis B surface antigen produced in yeast cells, providing a highly effective and safe means of preventing hepatitis B infection [13].
2. Human Papillomavirus (HPV) Vaccine: Recombinant HPV vaccines, such as Gardasil and Cervarix, protect against cervical and other cancers caused by HPV. These vaccines use virus-like particles produced in yeast or insect cells, offering high immunogenicity and safety [14].
3. COVID-19 Vaccines: The COVID-19 pandemic accelerated the development of recombinant protein-based vaccines. Notably, the Novavax vaccine uses a recombinant spike protein produced in insect cells, highlighting the versatility and rapid scalability of recombinant protein technology in addressing emerging infectious diseases [15].

Diagnostic Tools and Enzymes

Recombinant proteins are widely used in diagnostic applications and as enzymes in various assays. These applications include:

1. Diagnostic Kits: Recombinant antigens and antibodies are used in diagnostic kits to detect diseases such as HIV, hepatitis, and COVID-19. These kits rely on the specificity and high affinity of recombinant proteins to provide accurate and rapid diagnostics [16].
2. Enzymes: Recombinant enzymes, such as Taq polymerase and restriction enzymes, are essential tools in molecular biology and clinical diagnostics. They are used in techniques like PCR (polymerase chain reaction) and DNA sequencing, which are fundamental in research and diagnostic laboratories [17].

Emerging Applications and Future Trends

The potential applications of recombinant protein technology continue to expand, driven by advancements in genetic engineering and synthetic biology. Emerging applications and future trends include:

1. Gene Therapy: Recombinant proteins are being developed to deliver therapeutic genes to patients with genetic disorders. This approach aims to correct or replace defective genes, offering the potential for long-term cures [18].
2. Personalized Medicine: Recombinant proteins are at the forefront of personalized medicine, where therapies are tailored to the genetic profile of individual patients. This approach aims to improve treatment efficacy and reduce adverse effects [19].
3. Synthetic Biology: Synthetic biology combines recombinant DNA technology with computational tools to design and construct new biological parts and systems. This field is expected to revolutionize the production of biopharmaceuticals, enabling the creation of novel therapeutics and vaccines [20].

5 Advantages of Recombinant Protein Technology

The Benefits of Recombinant Protein Technology in Biopharmaceuticals: Ensuring Quality and Efficiency

Recombinant protein technology has emerged as a transformative tool in the biopharmaceutical industry, offering numerous advantages that enhance the production and efficacy of therapeutic proteins. This section explores the key benefits of recombinant protein technology, including high purity and consistency, scalability and cost-effectiveness, customization and optimization for specific needs, and safety with reduced risk of contamination.

High Purity and Consistency of Products

One of the most significant advantages of recombinant protein technology is the ability to produce proteins with high purity and consistency. This is achieved through the use of well-defined genetic constructs and controlled expression systems. High purity is crucial for therapeutic applications to ensure safety and efficacy, minimizing the risk of adverse reactions in patients. Consistency in production batches is also essential for maintaining the therapeutic integrity of biopharmaceuticals over time [21].

For instance, recombinant insulin, one of the first therapeutic proteins produced using this technology, consistently meets the rigorous purity standards required for medical use. The precision in genetic engineering and production processes ensures that each batch of insulin is virtually identical, providing reliable and predictable outcomes for patients with diabetes [22].

Scalability and Cost-Effectiveness

Recombinant protein technology allows for the scalable production of therapeutic proteins, which is vital for meeting the growing global demand for biopharmaceuticals. Traditional methods of protein extraction from natural sources are often limited by the availability of raw materials and the complexity of purification processes. In contrast, recombinant technology enables large-scale production in bioreactors, significantly enhancing the scalability of biopharmaceutical manufacturing [23].

The cost-effectiveness of recombinant protein production is another key advantage. By optimizing fermentation and purification processes, manufacturers can reduce production costs while maintaining high quality. This is particularly important for making biopharmaceuticals more accessible and affordable to a broader patient population. For example, the production of monoclonal antibodies, which are used in cancer therapy, has benefited from recombinant technology's ability to produce large quantities at a lower cost [24].

Customization and Optimization of Proteins for Specific Needs

Recombinant protein technology offers unparalleled flexibility in customizing and optimizing proteins for specific therapeutic needs. Genetic engineering techniques allow for the modification of protein structures to enhance their stability, efficacy, and safety. This customization can include altering amino acid sequences to improve protein folding, stability, or to add functional groups that enhance therapeutic properties [25].

One notable example is the development of engineered antibodies with enhanced binding affinity and reduced immunogenicity. These modifications can improve the therapeutic outcomes of antibody-based treatments by increasing their ability to target specific cells or proteins within the body while minimizing immune reactions [26]. Additionally, recombinant technology enables the production of fusion proteins, which combine the functionalities of multiple proteins into a single therapeutic agent, offering innovative solutions for complex medical conditions [27].

Safety and Reduced Risk of Contamination

Safety is a paramount concern in biopharmaceutical production, and recombinant protein technology significantly reduces the risk of contamination compared to traditional methods. The use of well-characterized cell lines and defined growth media minimizes the risk of introducing pathogens or impurities into the final product. This is particularly important for therapeutic proteins, where contamination can have serious consequences for patient safety [28].

Furthermore, the controlled environment of bioreactors used in recombinant protein production allows for stringent quality control measures. This ensures that each batch of the product meets the required safety and efficacy standards. The ability to produce proteins free from animal-derived contaminants also addresses concerns related to zoonotic diseases and reduces the risk of immunogenic responses [29].

6 Future Prospects

The Future of Recombinant Protein Technology in Biopharmaceuticals: Innovations and Applications

The future of recombinant protein technology in biopharmaceuticals is poised for significant advancements, driven by innovations in gene editing, synthetic biology, and new expression systems. This section explores the future prospects of recombinant protein technology, highlighting advancements in gene editing and synthetic biology, emerging expression systems, potential applications in personalized medicine, and predictions for the next decade.

Advancements in Gene Editing and Synthetic Biology

The integration of gene editing technologies, such as CRISPR-Cas9, with recombinant protein production is expected to revolutionize the biopharmaceutical industry. Gene editing allows for precise modifications in the DNA of host cells, enhancing their ability to produce recombinant proteins with desired characteristics. CRISPR technology, in particular, has enabled targeted and efficient genetic modifications, paving the way for the development of novel therapeutic proteins and improved production strains [30].

Synthetic biology, which involves designing and constructing new biological parts and systems, is another promising area. This field aims to create customized biological systems that can produce recombinant proteins more efficiently and with greater control over their properties. Synthetic biology tools enable the creation of synthetic promoters, optimized gene circuits, and novel biosynthetic pathways, enhancing the overall productivity and functionality of recombinant proteins [31].

New Expression Systems and Technologies

Traditional expression systems, such as Escherichia coli, yeast, and mammalian cells, have their limitations. The development of new expression systems and technologies aims to overcome these challenges. One such advancement is the use of plant-based expression systems, which offer scalability, safety, and cost-effectiveness. Plants can produce complex proteins with proper post-translational modifications, making them suitable for producing biopharmaceuticals like vaccines and antibodies [32].

Another innovative technology is cell-free protein synthesis, which allows for the production of recombinant proteins without the need for living cells. This method involves using cellular extracts to perform transcription and translation reactions in vitro. Cell-free systems offer rapid production, ease of scalability, and the ability to produce toxic proteins or those that are difficult to express in living cells [33].

Potential Future Applications in Personalized Medicine

Personalized medicine, which tailors treatments to individual patients based on their genetic makeup, is set to benefit greatly from recombinant protein technology. Advances in genetic sequencing and bioinformatics allow for the identification of specific genetic mutations and the development of targeted therapies. Recombinant proteins can be engineered to interact with specific molecular targets, providing more effective and personalized treatments for diseases such as cancer and genetic disorders [34].

Additionally, the ability to produce patient-specific proteins, such as personalized vaccines or therapeutic antibodies, will enhance the efficacy of treatments. Recombinant technology can be used to develop custom-made biologics that match the unique genetic profile of each patient, reducing the risk of adverse reactions and improving therapeutic outcomes [35].

Predictions for the Next Decade in Biopharmaceuticals

The next decade is likely to witness several key trends and advancements in recombinant protein technology:

1. Increased Adoption of Gene Editing and Synthetic Biology: As gene editing and synthetic biology technologies mature, their integration into recombinant protein production will become more widespread, leading to more efficient and customizable biopharmaceuticals.
2. Expansion of Plant-Based and Cell-Free Expression Systems: These innovative expression systems will gain traction due to their scalability, safety, and cost advantages, opening new avenues for biopharmaceutical production.
3. Advancements in Personalized Medicine: Recombinant proteins will play a crucial role in the development of personalized therapies, offering targeted and effective treatments for a wide range of diseases.
4. Enhanced Production Efficiency and Yield: Continued research and technological advancements will focus on improving the efficiency and yield of recombinant protein production, reducing costs, and increasing accessibility to biopharmaceuticals.
5. Emergence of Novel Therapeutic Proteins: The discovery and development of new recombinant proteins with unique therapeutic properties will expand the arsenal of treatments available for various medical conditions.

Conclusion

The future of recombinant protein technology in biopharmaceuticals is bright, with numerous advancements and applications on the horizon. Innovations in gene editing, synthetic biology, new expression systems, and personalized medicine will drive the next generation of biopharmaceuticals, offering more effective and tailored treatments. As the technology continues to evolve, it holds the promise of transforming healthcare and improving the lives of patients worldwide.

FAQs

1. What is recombinant protein technology and why is it important in biopharmaceuticals?

Answer: Recombinant protein technology involves the genetic manipulation of cells to produce desired proteins. This is done by inserting a gene encoding the protein of interest into an expression system, such as bacteria, yeast, or mammalian cells. The cells are then cultured to produce the recombinant protein, which can be purified and used for therapeutic purposes. This technology is crucial in biopharmaceuticals because it enables the production of high-purity, consistent, and scalable proteins that are essential for treating a wide range of diseases, including diabetes, cancer, and genetic disorders.

2. How has recombinant protein technology evolved over the years?

Answer: Recombinant protein technology has significantly evolved since the early 1970s when the first recombinant DNA molecules were created. Major milestones include the commercialization of recombinant insulin in the 1980s, the development of recombinant human growth hormone, and the introduction of monoclonal antibodies in the 1990s. Recent advancements include the use of gene editing tools like CRISPR-Cas9, synthetic biology for designing new biological systems, and the development of plant-based and cell-free expression systems. These innovations have expanded the scope and efficiency of recombinant protein production, making it a cornerstone of modern biopharmaceuticals.

3. What are the advantages of using recombinant protein technology in drug production?

Answer: Recombinant protein technology offers several advantages in drug production:

• High Purity and Consistency: The controlled production environment ensures high purity and consistent quality of the proteins, which is essential for therapeutic use.
• Scalability and Cost-Effectiveness: Large-scale production in bioreactors allows for the efficient and cost-effective manufacture of therapeutic proteins.
• Customization and Optimization: Genetic engineering techniques enable the customization of proteins to enhance their stability, efficacy, and safety.
• Safety and Reduced Risk of Contamination: The use of well-characterized cell lines and defined growth media minimizes the risk of contamination, ensuring the safety of biopharmaceuticals.

4. What are some common applications of recombinant protein technology in biopharmaceuticals?

Answer: Recombinant protein technology is widely used in various biopharmaceutical applications, including:

• Therapeutic Proteins: Examples include recombinant insulin for diabetes, growth hormones for growth deficiencies, and monoclonal antibodies for cancer and autoimmune diseases.
• Vaccines: Recombinant vaccines, such as those for hepatitis B and HPV, as well as the recent COVID-19 vaccines, use recombinant proteins to induce immunity.
• Diagnostic Tools and Enzymes: Recombinant proteins are used in diagnostic kits to detect diseases and as enzymes in molecular biology techniques like PCR.

5. What are the future prospects of recombinant protein technology in biopharmaceuticals?

Answer: The future of recombinant protein technology in biopharmaceuticals looks promising, with several emerging trends and advancements:

• Gene Editing and Synthetic Biology: The integration of CRISPR and synthetic biology will enable more precise and efficient production of recombinant proteins.
• New Expression Systems: Innovations like plant-based and cell-free systems will enhance scalability and reduce production costs.
• Personalized Medicine: Recombinant proteins will play a key role in developing personalized therapies tailored to individual genetic profiles.
• Increased Production Efficiency: Ongoing research will focus on improving the yield and efficiency of recombinant protein production, making biopharmaceuticals more accessible.
• Novel Therapeutic Proteins: The discovery and development of new recombinant proteins with unique therapeutic properties will expand treatment options for various diseases.

References

[1] B. Shanmugaraj and S. Ramalingam, "Plant expression platform for the production of recombinant pharmaceutical proteins," Austin J. Biotechnol. Bioeng., vol. 1, no. 1, pp. 1-5, 2014. [Online]. Available: ResearchGate.

[2] Y. C. Chen and M. K. Yeh, "Introductory chapter: biopharmaceuticals," Biopharmaceuticals, 2018. [Online]. Available: IntechOpen.

 [3] M. Matejczyk, "Environmental and recombinant microorganisms for biopharmaceuticals production," Budownictwo i Inżynieria Środowiska, vol. 1, no. 1, pp. 1-5, 2014. [Online]. Available: https://bibliotekanauki.pl/articles/403071.pdf

[4] R. A. Rader and E. S. Langer, "Biopharmaceutical Manufacturing: Historical and Future Trends in Titers, Yields, and Efficiency in Commercial-Scale Bioprocessing," BioProcessing Journal, vol. 13, no. 4, pp. 1-8, 2014. [Online]. Available: https://www.biopharma.com/J134-Rader-Langer.pdf

[5] S. Berger, P. Lowe, and M. Tesar, "Fusion protein technologies for biopharmaceuticals: Applications and challenges," MAbs, vol. 7, no. 1, pp. 1-7, 2015. [Online]. Available: https://www.tandfonline.com/doi/pdf/10.1080/19420862.2015.1019788

[6] G. Jagschies, "Selected biotherapeutics overview," Biopharmaceutical Processing, pp. 1-15, 2018. [Online]. Available: https://www.sciencedirect.com/science/article/pii/B9780081006238000037

 [7] C. Altamirano, J. Berrios, and M. Vergara, "Advances in improving mammalian cells metabolism for recombinant protein production," Electronic Journal of Biotechnology, vol. 16, no. 3, pp. 1-10, 2013. [Online]. Available: https://www.scielo.cl/scielo.php?pid=S0717-34582013000300010&script=sci_arttext

[8] P. Habibi, G. S. Prado, P. B. Pelegrini, and K. L. Hefferon, "Optimization of inside and outside factors to improve recombinant protein yield in plant," Plant Cell, Tissue and Organ Culture, vol. 131, no. 2, pp. 1-12, 2017. [Online]. Available: https://www.alice.cnptia.embrapa.br/alice/bitstream/doc/1080464/1/Habibi2017ArticleOptimizationOfInsideAndOutside.pdf

[9] T. W. Overton, "Recombinant protein production in bacterial hosts," Drug Discovery Today, vol. 19, no. 5, pp. 590-601, 2014. [Online]. Available: https://www.sciencedirect.com/science/article/pii/S1359644613004029

[10] B. Shanmugaraj, C. J. I. Bulaon, and W. Phoolcharoen, "Plant molecular farming: A viable platform for recombinant biopharmaceutical production," Plants, vol. 9, no. 7, pp. 1-20, 2020. [Online]. Available: https://www.mdpi.com/2223-7747/9/7/842

[10] N. Tripathi and A. Shrivastava, "Recent developments in bioprocessing of recombinant proteins: expression hosts and process development," Frontiers in Bioengineering and Biotechnology, vol. 7, pp. 420, 2019. [Online]. Available: Frontiers

[11] M. Kesik‐Brodacka, "Progress in biopharmaceutical development," Biotechnology and Applied Biochemistry, vol. 65, no. 3, pp. 306-322, 2018. [Online]. Available: Wiley

[12] S. K. Gupta and P. Shukla, "Advanced technologies for improved expression of recombinant proteins in bacteria: perspectives and applications," Critical Reviews in Biotechnology, vol. 36, no. 1, pp. 108-120, 2016. [Online]. Available: Taylor & Francis

[13] B. Shanmugaraj, C. J. I. Bulaon, and W. Phoolcharoen, "Plant molecular farming: A viable platform for recombinant biopharmaceutical production," Plants, vol. 9, no. 7, pp. 842, 2020. [Online]. Available: MDPI

[14] N. K. Tripathi and A. Shrivastava, "Scale up of biopharmaceuticals production," in Bioprocessing, optimization, scale-up and biological aspects, 2018, pp. 49-69. [Online]. Available: ScienceDirect

[15] T. W. Overton, "Recombinant protein production in bacterial hosts," Drug Discovery Today, vol. 19, no. 5, pp. 590-601, 2014. [Online]. Available: ScienceDirect

[16] A. Lakowitz, T. Godard, R. Biedendieck, and R. Krull, "Mini review: recombinant production of tailored bio-pharmaceuticals in different Bacillus strains and future perspectives," European Journal of Pharmaceutics and Biopharmaceutics, vol. 118, pp. 25-38, 2017. [Online]. Available: ScienceDirect

[17] A. F. Jozala, D. C. Geraldes, L. L. Tundisi, et al., "Biopharmaceuticals from microorganisms: from production to purification," Brazilian Journal of Microbiology, vol. 47, no. 1, pp. 51-63, 2016. [Online]. Available: SciELO

[18] S. R. Schmidt, "Fusion protein technologies for biopharmaceuticals: applications and challenges," MAbs, vol. 7, no. 1, pp. 1-5, 2015. [Online]. Available: Taylor & Francis

[19] R. O'Flaherty, A. Bergin, E. Flampouri, et al., "Mammalian cell culture for production of recombinant proteins: A review of the critical steps in their biomanufacturing," Biotechnology Advances, vol. 40, pp. 107-120, 2020. [Online]. Available: ScienceDirect

[20] A. C. Silva, J. N. Moreira, J. M. S. Lobo, H. Almeida, and T. R. Abreu, "Current applications of pharmaceutical biotechnology," Springer, pp. 1-20, 2020. [Online]. Available: Springer

[21] M. Sabalza, P. Christou, and T. Capell, "Recombinant plant-derived pharmaceutical proteins: current technical and economic bottlenecks," Biotechnology Letters, vol. 36, no. 6, pp. 1231-1244, 2014. [Online]. Available: Springer

[22] B. Shanmugaraj and W. Phoolcharoen, "Addressing demand for recombinant biopharmaceuticals in the COVID-19 era," Asian Pacific Journal of Tropical Medicine, vol. 14, no. 2, pp. 61-67, 2021. [Online]. Available: LWW

[23] N. Tripathi and A. Shrivastava, "Recent developments in bioprocessing of recombinant proteins: expression hosts and process development," Frontiers in Bioengineering and Biotechnology, vol. 7, pp. 420, 2019. [Online]. Available: Frontiers

[24] R. O'Flaherty, A. Bergin, E. Flampouri, L. M. Mota, and P. J. Dunnill, "Mammalian cell culture for production of recombinant proteins: A review of the critical steps in their biomanufacturing," Biotechnology Advances, vol. 40, pp. 107-120, 2020. [Online]. Available: ScienceDirect

[25] A. Lakowitz, T. Godard, R. Biedendieck, and R. Krull, "Mini review: recombinant production of tailored bio-pharmaceuticals in different Bacillus strains and future perspectives," European Journal of Pharmaceutics and Biopharmaceutics, vol. 118, pp. 25-38, 2017. [Online]. Available: ScienceDirect

[26] S. K. Gupta and P. Shukla, "Advanced technologies for improved expression of recombinant proteins in bacteria: perspectives and applications," Critical Reviews in Biotechnology, vol. 36, no. 1, pp. 108-120, 2016. [Online]. Available: Taylor & Francis

[27] V. Gupta, M. Sengupta, J. Prakash, and B. C. Tripathy, "Production of recombinant pharmaceutical proteins," in Basic and Applied Aspects of Biotechnology, 2017, pp. 123-144. [Online]. Available: Springer

[28] L. Sanchez-Garcia, L. Martín, and R. Mangues, "Recombinant pharmaceuticals from microbial cells: a 2015 update," Microbial Cell Factories, vol. 15, no. 1, pp. 33, 2016. [Online]. Available: Springer

[29] B. Shanmugaraj, C. J. I. Bulaon, and W. Phoolcharoen, "Plant molecular farming: A viable platform for recombinant biopharmaceutical production," Plants, vol. 9, no. 7, pp. 842, 2020. [Online]. Available: MDPI

[30] M. Dabhole, "Recent innovations in therapeutic recombinant protein," Innov Recent Protein Ther Recomb Pharma Bio World, 2014. [Online]. Available: Academia.

[31] R. A. Rader and E. S. Langer, "Biopharmaceutical Manufacturing: Historical and Future Trends in Titers, Yields, and Efficiency in Commercial-Scale Bioprocessing," BioProcessing Journal, vol. 13, no. 4, pp. 1-8, 2014. [Online]. Available: BioPharma.

[32] A. Berlec and B. Štrukelj, "Current state and recent advances in biopharmaceutical production in Escherichia coli, yeasts and mammalian cells," Journal of Industrial Microbiology and Biotechnology, vol. 40, no. 3-4, pp. 257-274, 2013. [Online]. Available: OUP.

[33] N. K. Tripathi and A. Shrivastava, "Recent developments in bioprocessing of recombinant proteins: expression hosts and process development," Frontiers in Bioengineering and Biotechnology, vol. 7, pp. 420, 2019. [Online]. Available: Frontiers.

[34] L. Sanchez-Garcia, L. Martín, and R. Mangues, "Recombinant pharmaceuticals from microbial cells: a 2015 update," Microbial Cell Factories, vol. 15, no. 1, pp. 33, 2016. [Online]. Available: Springer.

[35] V. Picanço-Castro, R. T. Biaggio, et al., "Production of recombinant therapeutic proteins in human cells: current achievements and future perspectives," Protein and Peptide Letters, vol. 20, no. 12, pp. 1373-1381, 2013. [Online]. Available: IngentaConnect.