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5.12 Summary
Researchers have advanced microbial nanoparticle manufacturing by looking for
particular properties in extremophiles and uncommon strains. For medication delivery and medical uses, biocompatible nanoparticles with minimal cytotoxicity are
created. A focus is on scaling up microbial nanoparticle synthesis for industrial
usage, particularly multimetallic nanoparticles. Microbial synthesis contributes to
environmental applications by eliminating heavy metals and contaminants in a sustainable manner. Tailoring characteristics, biomedical applications, nanoelectronics, and complex multimetallic architectures are all possibilities for microbial
nanoparticle manufacturing in the future. Plant-mediated nanoparticle synthesis is a
green approach that uses a variety of plant sources and has shown promise in medication delivery, catalysis, and energy applications. Current research is geared at
commercialization and industrial scale-up. The green technique of biopolymer and
biomolecule-mediated nanoparticle production yields versatile nanoparticles with
applications in medication administration, diagnostics, and environmental cleanup.
Tailor-made nanoparticle creation, breakthroughs in medicinal applications, environmental remediation, and investigation of developing biopolymers for sustainable
agriculture are all possibilities for the future. In conclusion, these approaches to
sustainable nanoparticle synthesis have a wide range of applications, ranging from
healthcare to environmental issues, with ongoing research focusing on large-scale
production and commercialization.
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Chapter 6
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Production ofBiopharmaceuticals
onGenetically Modied Organisms
SandipanJana, BarunDasGupta, AmitKar, AnirbitaGhosh,
MoniharPervin, AsisBala, MotlalepulaG.Matsabisa, RajeswarDas,
SomsubhraGhosh, PulokK.Mukherjee, andPallabK.Haldar
Abstract The production of biopharmaceuticals using genetically modied organ-
isms represents a transformative paradigm in the pharmaceutical industry. This
abstract explores the advancements, challenges, and implications of employing
genetically modied organisms for the synthesis of biopharmaceuticals. The genetic
modication of organisms, ranging from bacteria and yeast to mammalian cells,
enables the engineered expression of therapeutic proteins, antibodies, and vaccines.
This review delves into the molecular techniques employed in genetically modied
organisms to optimize protein production, the scalability of bioprocessing, and the
regulatory landscape governing the use of genetically engineered platforms.
Additionally, the chapter highlights the potential of genetically modied organisms
to revolutionize the production of complex biologics and discusses the ethical and
safety considerations associated with their widespread application in biopharmaceutical manufacturing. The synthesis of biopharmaceuticals on genetically modied organisms not only enhances production efciency but also contributes to the
expanding landscape of precision medicine, heralding a new era in therapeutic innovation and healthcare delivery.
S. Jana (*)
School of Pharmacy, The Neotia University, Sarisa, West Bengal, India
Department of Pharmaceutical Technology, Jadavpur University, Kolkata, West Bengal, India
B. D. Gupta
School of Natural Product Studies, Jadavpur University, Kolkata, West Bengal, India
A. Kar · P. K. Mukherjee
Institute of Bioresources and Sustainable Development, Autonomous Institute Under
Department of Biotechnology, Imphal, Manipur, India
e-mail: director.ibsd@nic.in
Ltd. 2024
S. Bose et al. (eds.), Concepts in Pharmaceutical Biotechnology and Drug
Development, Interdisciplinary Biotechnological Advances,
https://doi.org/10.1007/978-981-97-1148-2_6
91© The Author(s), under exclusive license to Springer Nature Singapore Pte

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S. Jana et al.
Keywords Genetically modied organisms · Biopharmaceuticals · Upstream
processing · Downstream processing
6.1 Biopharmaceuticals: AnIntroduction
In the past, the term “Biopharmaceuticals” referred to medications produced
through biotechnological procedures utilizing molecular biology techniques.
Today, these medications are widely used in various areas of medicine and are
considered to be highly effective therapy options for numerous illnesses including
cancer and metabolic disorders. Biopharmaceuticals encompass any benecial
medication produced using genetically modied organisms, microbes, or compounds derived from biological processing (Kesik-Brodacka 2018). The produc-
tion of these medications involves various biotechnology processes such as genetic
engineering, recombinant human technology, gene transfer, and antibody production (Walsh 2000). The rst therapeutic protein and anti-diphtheria serum was
introduced in 1894 by Farbwerke Hoechst to combat a catastrophic diphtheria epidemic in Europe (Strohl and Knight 2009). Later developments included the production of heterologous insulin rened from pigs and cows and sold under the
brand name “Iletin” and the cloning and expression of human insulin, somatostatin, and also growth hormone (HGH) in Escherichia coli in the late 1970s, which
led to the creation of the rst recombinant human insulin (Bhatia and Goli 2018;
Vecchio etal. 2018).
A. Ghosh · M. Pervin
Department of Pharmaceutical Technology, Jadavpur University, Kolkata, West Bengal, India
A. Bala
Division of Life Sciences, Institute of Advanced Study in Science and Technology (IASST),
An Autonomous Institute Under Department of Science and Technology (Govt. of India)
Vigyan Path, Guwahati, Assam, India
e-mail: asisbala_ju@yhaoo.co.in
M. G. Matsabisa
Department of Pharmacology, University of the Free State, Bloemfontein, South Africa
e-mail: motlalepula.matsabisa@mrc.ac.za
R. Das · S. Ghosh
School of Pharmacy, The Neotia University, Sarisa, West Bengal, India
e-mail: rajeswar.das@tnu.in; somsubhra.ghosh@tnu.in
P. K. Haldar
Department of Pharmaceutical Technology, Jadavpur University, Kolkata, West Bengal, India
School of Natural Product Studies, Jadavpur University, Kolkata, West Bengal, India

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6.2 Production Processes
There are two stages of biopharmaceutical production—upstream processing and
downstream processing.
6.2.1 Upstream Processing
Living cells such as those taken from patients that need to be genetically modied
are used to produce biopharmaceutical drug substances using viral vectors. To create an ideal environment for cell development, it is important to choose the best cell
line, culture medium, growth factors, and process optimization. Some crucial factors to take into account are the regulation of temperature, pH, oxygen supply, and
the sanitation of supplies and equipment utilized in the upkeep of an environment
free of microorganisms. The procedure involves identifying and isolating the gene
that produces the same protein expression in a healthy organism and selecting an
appropriate host cell type for genetic engineering. Strict control and cGMP compliance over the handling of these living cells are critical to the outcome of the upstream
production, which includes the amount of desired biopharmaceuticals produced, the
type and quantity of biomolecular structural variants formed, and the kind and also
the amount of specic impurities that need to be managed during purication
(Jozala etal. 2016; Gronemeyer etal. 2014).
6.2.2 Downstream Processing
Downstream processing is a crucial part of the purication of biological products. It
involves three steps: (1) removing most contaminants through purication, (2) eliminating particular impurities and undesirable target biomolecule forms that might
have developed during isolation and purication, and (3) polishing. Centrifugation,
ltration, sedimentation, and otation are the primary techniques used to separate
the products. However, continuous downstream processing has been known to
increase process complexity rather than efciency in purication. Nevertheless, the
benets of high output in continuous working units outweigh this drawback
(Mehta 2019).
The current and most commonly used purication methods include continuous
centrifugation, which involves retrieving the product from the extracellular media,
cleaning and recovering it from inclusion bodies, clarifying cell homogenates, and
harvesting precipitated protein. Filtration is another method used to eliminate
byproducts and concentrate culture media containing the desired expressed biomolecules. Cell lysis by physical, chemical, and enzymatic methods is yet another

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method used for the production of biopharmaceuticals like recombinant proteins
such as interferon-β, interleukin-2, protein C, and proinsulin, which are found to be
produced as inclusion bodies inside the cells (Mehta 2019).
S. Jana et al.
6.3 Bacteria inBiopharmaceuticals Production
Bacteria can grow quickly on low-cost substrates, and researchers have extensively
studied the method of producing recombinant proteins on a large scale in bioreactors. Microorganisms are a useful tool in producing recombinant proteins due to
their metabolic power, diversity, and ability to adapt to various environmental conditions. The most commonly used microorganism is Escherichia coli, which has
advanced genetics and a vast range of mutant host strains and cloning vectors.
E. coli’s high growth rate, basic media requirement, ease of handling, high yield,
and economic viability make it the most suitable for large-scale biopharmaceutical
production, and E. coli can also produce recombinant proteins up to 80% of its dry
matter (Sobolewska-Ruta and Zaleski 2019; Pham etal. 2019).
The incidence of diabetes is increasing globally, which is leading to an increase in
demand for recombinant insulin therapies. For many years, insulin derived from cows
and pigs was used to treat diabetes (Jozala etal. 2016). However, this was not the best
option because many people experienced adverse reactions such as serum sickness.
Genetic engineering made it possible to transfer genes across different biological species with ease and paved the way for efcient insulin manufacturing. Downstream
processing of recombinant human insulin/analogue manufacture from E. coli inclusion bodies has been carried out. Recombinant human insulin derived from DNA was
rst introduced by Eli Lilly & Company in 1982 and by Novo Nordisk in 1988 (Alyas
etal. 2021). Humulin, the rst of several recombinant insulin to be licensed for use in
general medicine, is made by Eli Lilly and Genentech. Recombinant insulin is still
mainly produced in E. coli, where inclusion bodies are often used to express human
insulin and its analogues. To generate sufcient amounts of proinsulin, recombinant
E. coli is used. E. coli is transformed with proinsulin producing plasmids to create this
recombinant protein. The plasmid includes proinsulin coding genes and a kanamycin
mono sulphate resistance gene, which prolongs its survival rate (Siew and Zhang
2021). Next, the transformed cells are cultured on tryptic soy broth supplemented with
kanamycin mono sulphate, an antibiotic. E. coli cells that have not undergone transformation are eliminated by kanamycin mono sulphate. To maximize yields during
product synthesis, the culture and fermentation conditions are closely monitored.
Biologically active human insulin is produced by post-translational processing and
thorough purication of the resultant medicinal product (Alyas etal. 2021).
The Pseudomonas genus is one of the most complex bacterial genera that
researchers have investigated, showing a great deal of metabolic and physiological
variety. A thorough investigation of Pseudomonas genomes and proteomics has led
to a greater understanding of the system and molecular level of Pseudomonas synthetic processes. Due to its property of a vast array of genetic manipulation, fast
growth rate, and the ability to secrete proteins, Pseudomonas uorescens has
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