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K. Gadamasetti
Fig. 1.2 Crick’s adaptor hypothesis. The amino acid is covalently bound at the 3′ end of a tRNA
molecule and that a specic nucleotide triplet elsewhere in the tRNA interacts with a particular
triplet codon in mRNA through hydrogen bonding of complementary bases [12]
1.3 Synergy Between Biologics andSmall Molecules
In nature, the small molecules interact with biological entities and vice versa to
produce outstanding outcomes, manifestations ofwhich make it a signicant event.
Among the following two examples, in the rst one, the small molecular amino acid
is selected via a covalent binding to the tRNA, a biological unit (Fig.1.2). In the
second example, the specic stereoselective chemical reaction takes place via binding of the substrate racemic mixture of 1 as the starting material to the enzymespecic strains, H. polymorpha SC 13865 and H. fabianii SC 13894, resulting in the
transformation to the optically active single diastereomer 2, which constitutes the
side chain of the cancer drug, Paclitaxel™ (Fig.1.3).
1 Bioprocessing, Bioengineering and Process Chemistry in the Biopharmaceutical…
Fig. 1.3 Enzymatic intervention in generating optically active small molecules. Synthesis of side
chain via enzymatic reaction in anticancer drug, Paclitaxel™
11
One can see, these manifestations (Fig.1.2) known as Crick’s adaptor hypothesis
are represented in the DNA expression of the proteins where the amino acid building block is selected via the covalent binding of the amino acid with the 3′-end of a
tRNA molecule and that a specic nucleotide triplet elsewhere in the tRNA interacts
with a particular triplet codon in mRNA through hydrogen bonding of complementary bases [12]. The particular amino acid sequence of a protein is constructed
through the translation of information encoded in mRNA.This process is carried
out by ribosomes.
In synthetic organic chemistry, myriads of examples are known to use enzymemediated reactions to create new chiral sites in the molecule. Although the synthetic
routes and processes developed to produce drug candidate compounds rely mainly
on conventional chemistry, the usage of enzyme-mediated intervention appeared
prudent for the production of chiral (optically active) intermediates [13]. Amino
acylation, hydrolysis, reductive amination, and reduction, all using enzymemediated reactions, were used at industrial scale for generating the optically active
chiral intermediate molecules. One such example is shown below for the semisynthesis (side chain of Paclitaxel™) of the anticancer drug Paclitaxel™ at BristolMyers Squibb (Fig.1.3).
1.3.1 Process Chemistry andBioprocessing
The process or the event of “making or breaking of a covalent bond” can be brought
forward between two atoms resulting in creating molecules or a molecule, respectively, using a traditional chemical reaction or an enzyme-mediated intervention.
Process chemistry is the term used for efciently carrying out the chemical transformations and the series of steps involved to reproduce the reaction or a series of
reactions.
Traditionally, process development chemistry involves devising efcient and
commercially viable synthetic routes in building small molecules. This process can
advance an anticipative drug candidate through the preclinical and clinical development timeline, establishing and transferring a commercial synthetic chemistry route
to the manufacturing division, and contributing to the CMC section of the new drug
12
application (NDA) submission. Biologics, on the other hand, are large molecules
such as therapeutic proteins, fusion proteins, monoclonal antibodies, and vaccines.
Most biologic medicines are developed using recombinant DNA technology.
Bioprocessing involves the processing of biologic molecules through genetically
engineering whole living cells to generate the protein. Unlike the chemical synthetic
process used to produce small molecules, bioprocessing involves upstream processing (upfront) starting with living cells and downstream processing involving larger
material handling and eventual purication of the nished biologic material.
The role of the process chemist has evolved not only to accommodate advancements in regulatory affairs and to improve business efciency but also to embrace at
times the technical challenges to learn processes involving and understanding of
synthetic biology and handling the biologics such as antibody drug conjugates
(ADCs). Interestingly, ADCs opened the door to productive interactions between
chemists and biologists. The FDA treats and regulates ADCs as biologics rather
than as conventional chemically synthesized drugs. ADCs, therefore, are approved
through a Biologics License Application (BLA).
K. Gadamasetti
1.3.2 Antibody Drug Conjugates (ADCs): AConjugation
ofSmall Molecules andBiomolecules
Antibody drug conjugates (ADCs) combine the cytotoxic potential of chemotherapeutic drugs (e.g., doxorubicin, auristatins, calicheamicins, maytansines, and other
drugs with sub-nano molar activities) with the specicity of monoclonal antibodies
(mAbs) bridged via the chemical linkers (Fig.1.4). The site-specic conjugation of
the cytotoxic drugs (e.g., doxorubicin, calicheamicin, auristatin, maytansine)
involve specic cancer antigen targets (e.g., CD33, CD30, Her2) linking to the antibody sites via cysteine S- or lysine N-bonding to the cytotoxic drug. The mechanism of action (MoA) encompasses internalization of ADC into cancer cell, the
release of the cytotoxic drug in the cell, and apoptosis (cell death). Some examples
of selected ADCs include Adcetris™ from Seattle Genetics; Kadcyla™ from
Genentech-Roche; and Mylotarg™ and Besponsa™ from Pzer. A detailed discussion on the development of ADCs is presented in this volume in Chap. 12 by David
Jackson and in Chap. 13 by the Pzer group of scientic teams that provide the
detailed work of discovery, development, manufacturing, and the regulatory challenges in launching Mylotarg™.
The author believes that the science and technology, as well as the development and manufacturing, involving process chemistry development tied to the
development of specic monoclonal antibody for a successful ADC creation and
the launching, embodies a genuine meaning of a “conjugation of small molecules
with biomolecules.” ADC technology and development challenges created an
1 Bioprocessing, Bioengineering and Process Chemistry in the Biopharmaceutical…
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Fig. 1.4 Site-specic homogenous ADCs
opportunity for the biologists, chemists, and bioengineers to come together to successfully launch the specic anticancer ADCs to alleviate the pain and suffering of
humankind.
1.3.2.1 ADC Drugs
Three of the marketed ADCs are shown in Fig.1.5.
1.4 Top 25 Best-Selling Drugs
Drug sales hit records with the COVID-19 Comirnaty vaccine jointly developed by
Pzer and BioNTech [10]. Table 1.2 lists the top 25 best-selling drugs based on
2021 sales.
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Fig. 1.5 Marketed ADCs, Adcetris™, Mylotarg™, and Kadcyla™. Cytotoxic warhead (in red) is
connected to the mAb through the linker (in purple) via cystine S-bond or lysine N-bond, covalently
K. Gadamasetti
1.5 Outline oftheContents oftheVolume
1.5.1 An Overview
The rst chapter of this volume opens with a general introduction, and the
author’s perspective on synthetic biology, genetic mutations, and associated diseases. It attempts to give a general idea about the plot of the book, the purpose, the
process, and the rational aspects behind putting the volume together. This chapter
lists the top best-selling 25 drugs and also summarizes the outline of contents of
the volume.
1.5.2 Synthetic Biology
In the overview, Gadamasetti in Chap. 1opens the discussion on the outline of synthetic biology, genetic alphabets and expanded alphabets, point mutations, and specic examples of diseases related to mutations. Remarkably, the cure of sickle-cell
therapy was through tweaking the specic genetic building block.
1 Bioprocessing, Bioengineering and Process Chemistry in the Biopharmaceutical…
Table 1.2 Top 25 best-selling drugs
2021 Sales
(millions
# Drug name Manufacturer(s)
1 Comirnaty
Pzer/BioNTech $59,109.00 Reduce risk of COVID-19
COVID-19 vaccine
2 Humira
AbbVie $20,694.00 Rheumatoid and psoriatic
(adalimumab)
USD) Indication(s)
infection
arthritis, ankylosing
spondylitis, Crohn’s disease,
ulcerative colitis
3 Spikevax
COVID- 19 vaccine
4 Keytruda
Moderna $17,675.00 Reduce risk of COVID-19
infection
Merck $17,186.00 Various cancers SM
(pembrolizumab)
5 Eliquis (apixaban) Bristol Myers
$16,732.00 Blood clots B
Squibb and Pzer
6 Revlimid
(lenalidomide)
Bristol Myers
Squibb (Celgene)
$12,821.00 Myelodysplastic syndrome,
multiple myeloma, and
mantle cell lymphoma
7 Imbruvica
(ibrutinib)
Pharmacyclics
(AbbVie) and
Janssen (Johnson
& Johnson)
$9777.00 Chronic lymphocytic
leukemia/small lymphocytic
lymphoma with 17p deletion,
Waldenström’s
macroglobulinemia
8 Eylea (aibercept) Regeneron
pharmaceuticals,
Bayer
$9244.00 Age-related macular
degeneration, macular
edema, and diabetic
retinopathy
9 Stelara
(ustekinumab)
10 Biktarvy
Janssen (Johnson
& Johnson)
$9134.00 Plaque psoriasis and
psoriatic arthritis
Gilead Sciences $8624.00 HIV SM
(bictegravir,
emtricitabine, and
tenofovir
alafenamide)
11 Xarelto
(rivaroxaban)
Janssen (Johnson
& Johnson)/Bayer
$8040.00 Reducing risk of stroke in
non-valvular atrial
brillation, deep vein
thrombosis, pulmonary
embolism, DVT prophylaxis
following knee or hip
replacement surgery
12 Opdivo (nivolumab) Bristol Myers
$7523.00 Various forms of cancer B
Squibb
13 Trulicity
Eli Lilly $6472.00 Type 2 diabetes B
(dulaglutide)
14 Dupixent
(dupilumab)
Sano Genzyme,
Regeneron
pharmaceuticals
$6210.00 Atopic dermatitis, asthma,
chronic rhinosinusitis with
nasal polyps
15
SM/
a
B
B
SM
SM
SM
B
B
SM
B
(continued)
16
K. Gadamasetti
Table 1.2
# Drug name Manufacturer(s)
15 Darzalex
16 REGEN-COV
17 Trikafta/Kaftrio Vertex
18 Gardasil/Gardasil 9
19 Veklury
20 Ibrance
21 Januvia/Janumet
22 Prevnar family
23 Tagrisso
24 Cosentyx
25 Ocrevus
Taken from Top 50 Best Selling Drugs [10]
B biologic drug, COVID-19 Coronavirus Disease-2019, DVT, HER2, HIV human immunode-
ciency virus, HR, SM small molecule drug
a
It is interesting to note the signicance of biologic drugs (13 out of 25) in the top 25 best-selling
drugs from 2021.
(continued)
(daratumumab)
(Casirivimab/
imdevimab)
(human
papillomavirus
9-valent vaccine)
(remdesivir)
(palbociclib)
(sitagliptin)
(pneumococcal
vaccine)
(osimertinib)
(secukinumab)
(ocrelizumab)
2021 Sales
(millions
USD) Indication(s)
(Janssen) Johnson
& Johnson
Regeneron
pharmaceuticals,
Bayer
pharmaceuticals
Merck $5673.00 Various cancers caused by
Gilead Sciences $5565.00 COVID-19 requiring
Pzer $5437.00
Merck & Co. $5288.00 Type 2 diabetes SM
Pzer $5272.00 Pneumococcal vaccine B
AstraZeneca $5015.00 Non-small-cell lung
Novartis $4718.00 Plaque psoriasis, psoriatic
Roche $4622.00 Relapsing or primary
$6023.00 Multiple myeloma B
$5828.00 Reduce risk of COVID-19
infection
$5697.00 Cystic brosis SM
human papillomavirus
hospitalization
HR-positive and HER2-
negative breast cancer
carcinomas
arthritis, ankylosing
spondylitis
progressive multiple
sclerosis
SM/
B
B
B
SM
SM
SM
B
B
a
1.5.2.1 Synthetic Biology inDrug Development andBeyond
In its simplest denition, Sato and Rife in Chap. 2 note that synthetic biology is the
creation of new biological entities for useful purposes. By manipulating an organism’s genome, synthetic biologists can produce novel proteins for a wide range of
applications, from the biosynthesis of industrial chemicals to the discovery and
optimization of biotherapeutics. The eld of synthetic biology has experienced a
renaissance in recent years as technological advances have lowered the barrier to
entry and increased the potential for innovation. Principal among these advances
has been the development of highly precise and large-scale DNA synthesis platforms.
1 Bioprocessing, Bioengineering and Process Chemistry in the Biopharmaceutical…
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1.5.3 Oligonucleotide Synthetic Chemistry toDNA Synthesis,
Bioprocessing, andManufacturing
Outlined in this section are three chapters. In its simplest denition, Aaron Sato and
Rebecca Nugent in Chap. 3 outline that creation of new biological entities dened
by synthetic biology facilitates in manipulation for several useful purposes. By
manipulating an organism’s genome, synthetic biologists can produce novel proteins for a wide range of applications, from the biosynthesis of industrial chemicals
to the discovery and optimization of biotherapeutics. The chapter outlines the evolution of gene synthesis starting with the humble beginnings of the oligonucleotide
synthesis leading to gene synthesis, antibody synthesis, and the library creation.
In Chap. 4, Greg Hiller outlines the recent innovations and advances in cell culture technology enabling process intensication in detailing the upstream process
development, the limitations of fed-batch processes, the recent resurgence of interest in traditional “classical steady-state” perfusion in which viable cell density is
held nearly constant, and the development of non-conventional “dynamic” perfusion processes in which viable cell density is allowed to peak and decline. Other
process intensication methodologies such as N-1 perfusion for high-density production reactor inoculation are also examined.
Timothy Iskra’s Chap. 5 summarizes the implication of bispecic and trispecic
antibodies on antibody process development paradigms. According to Iskra, in
recent years the bispecic and trispecic (or multispecic) antibodies have become
a large area of development for most companies, as the ability to bind two different
antigens at a time opens new and unique therapeutic areas.
1.5.4 Process Engineering, Gene Therapy, andVaccine
Under this title, three chapters are clustered. Sharfstein and coworkers, in Chap. 6,
review the basics of glycoprotein and glycosaminoglycans biosynthesis with particular focus on the current understanding of how glycan structures are controlled
invivo. The studies in which glycan structures for recombinant proteins and glycosaminoglycans have been optimized by cell line metabolic engineering and bioprocess manipulations are also reviewed. It is also noted that CRISPR has permitted
exquisite editing of host cells, allowing tailored production of glycan structures,
facilitating the production of biosimilars and bioengineered glycosaminoglycans as
well as setting the stage for “biobetters” in which improved functionality is obtained
by glycoengineering. Chapter 7 by Somanathan and coworkers at Pzer summarizes the host immune response activated by adeno-associated viral (AAV) gene
therapy vectors and the approaches currently being pursued to mitigate immune
recognition and activation. Kolodziej and coauthors, in Chap. 8, discuss the details
of Coronavirus Disease-2019 (COVID-19) vaccine manufacturing processes in
nding the solutions via making molecules to address the needs of eradicating the
pandemic.
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K. Gadamasetti
1.5.5 Special Topics: CAR-T andCRISPR Technologies
andApplications
CAR-T and CRISPR technologies and the associated applications are addressed in
Chaps. 9 and 10. In Chap. 9, the authors from Glaxo-Welcome Smith Kline (GSK)
present a high-level overview of the general process for production of chimeric
antigen receptor-engineered (CAR)-T cells. Also described are individual aspects of
the workow that ae utilized to generate and administer CAR-T cell products
including production of lentiviral vector, acquisition of lymphocyte starting material, cell activation, transduction and expansion, and formulation and product
release. The authors give some historical background about various technologies
and how they have been implemented in production, along with recent changes that
have been made in academia and industry to optimize CAR-T cell production.
Chapter 10 is a comprehensive guide to Clustered Regularly Interspaced Short
Palindromic Repeats (CRISPR) gene editing and its applications in biotherapeutics.
It begins with an introduction to CRISPR-Cas9 technology, examining its discovery
and adaptation, mode of action, advantages over previous tools, derivatives of the
original CRISPR-Cas9 technology, and gene editing methods. This is followed by a
review of the applications of CRISPR technology in biotherapeutics, including both
gene therapies and gene-edited cell therapies, for the treatment of three disease
categories: genetic disorders, infectious diseases, and cancer. The nal section
delves into the future of CRISPR therapeutics, including nascent applications, the
industrialization and projected growth of the industry, safety and ethical considerations, and its transformative potential for human health.
1.5.6 Fusion Proteins, Antibody Drug Conjugates,
andProcess Chemistry
Stefan Schmidt from BioAtrium AG, focuses in Chap. 11 on fusion proteins engineered from two or more genes that code for separate proteins joined by genetic
techniques. The result is a single polypeptide sharing functional properties of both
parent proteins. Fusion proteins belong to the class of next-generation therapeutics
as they are designed exclusively through human creativity and are not evolved from
a natural process. Very often they represent the so called “biobetters,” molecules
with improved functionalities.
The subsequent two chapters are dedicated to ADCs. In Chap. 12, David Jackson
summarizes the history of currently approved ADCs with emphasis on the challenges that were overcome during development and thenew technologies that likely
hadcontributed to their success. The safety and efcacy of each ADC are also discussed from a critical but honest perspective. The author’s intention in writing this
chapter is to encourage readers to educate themselves about the real benets and
risk of ADC therapeutics so that informed decisions can be made by cancer patients
1 Bioprocessing, Bioengineering and Process Chemistry in the Biopharmaceutical…
in collaboration with their doctors. And in Chap. 13, the team from Pzer discusses
the development and the challenges of the journey to FDA reapproval and broad
international approval process of the ADC, Mylotarg™.
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1.5.7 Biopharmaceutical Informatics andAnalytics
Chapter 14 from Sandeep Kumar and coworkers at Boehringer Ingelheim provides
a comprehensive overview of strategic vision of closer collaboration between drug
discovery and development functions of biopharmaceutical industry by integrating
the considerations of developability during early stages of drug discovery.
Computational tools already available to enable Biopharmaceutical Informatics are
reviewed in this work. While the focus is on monoclonal antibody-based biologics,
the concepts discussed in this work are also applicable to novel formats such as
multispecic biologics. Advanced data analytics (ADA) application in biomanufacturing processes is outlined in Chap. 15 by Jun Luo and coauthors from GenentechRoche. The authors believe that using ADA may elucidate previously undetected
relationships between process inputs and outputs, which holds promise as an additional tool to augment traditional design of experiments (DoE) as a means to gain
actionable insights, on process and product knowledge.
1.5.8 Biopharmaceutical Regulatory CMC
Two chapters are presented in this volume dedicated to regulatory chemistry, manufacturing, and control (CMC) of biopharmaceuticals. Richard Pelt from Pzer outlines the overview of complexities of global regulatory CMC aspects in Chap. 16.
Noted in the overview is the evolution of the technologies within the biopharmaceutical industry and relating to the global CMC regulatory environment for regulatory
approvals to benet the patients. In Chap. 17, Ding and Marino describe the monoclonal antibody (mAb) continuous bioprocess (CBP) from design, implementation
to manufacturing based on the scientic understanding of mAb physicochemical
properties, proven bioprocessing principles, available technologies, CMC considerations, current industrial practices, regulatory guidelines, challenges, potential solutions, and future perspectives.
1.5.9 Technology Transfer
Hughes and Brady from Bristol-Myers Squibb, in Chap. 18, describe in detail the
advancements in technical transfer of biologics. According to the authors, technology transfer of the manufacturing process and associated analytical methods is a