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About the Editors
paradigms and repurposing drugs, rapid DNA synthesis and DNA synthetic libraries. He earned his PhD from the University of Vermont and was a Post- doctoral
Fellow at the University of Virginia.
Stephen A. Kolodziej is an Associate Research Fellow in the Biotherapeutics
Pharmaceutical Sciences Division of Pzer at the Chestereld Missouri site. His
group develops downstream manufacturing processes for biotherapeutic drug and
vaccine candidates, specializing in protein conjugates. He made major contributions
to process and product understanding on late-stage bacterial vaccine projects,
including multivalent capsular polysaccharide conjugate vaccines against pneumococcus, staphylococcus and meningococcus. He started his professional career as a
medicinal chemistry in Searle, Pharmacia and then Pzer, developing novel smallmolecule candidates for the treatment of inammation, cancer and cardiovascular
disease. He applied the tools of combinatorial chemistry to produce small libraries
of candidates for evaluation in numerous preclinical programs and was a coinventor
for Xalkori®, an ALK inhibitor approved for the treatment of non- small cell lung
cancer. Steve has a BA and a PhD in Organic Chemistry from the University of
Missouri-St. Louis with Professor Rudolph E.K. Winter on the synthesis of natural
products.
Part I
Overview
Chapter 1
Bioprocessing, Bioengineering and Process Chemistry in the Biopharmaceutical Industry: Using Chemistry and Bioengineering to Improve the Performance of Biologics
KumarGadamasetti
Abstract The explosion and exploitation of biotechnology and bioengineering
toward the end of the twentieth century led to the invention of valuable biologic
drugs for the benet of patients around the world. Opportunities continue to expand
in dramatic and unforeseen ways through the present time. Advances in individual
elds as well as multidisciplinary integration across science, technology, and bioengineering drive this innovation, emphasizing the importance of reducing timelines
and cost for drug development and clinical studies, all under a regulatory and ethical
framework that is vital for delivering safe and effective therapies to patients.
Medical disorders with associated genetic components and the understanding of
single mutations in disorders like sickle-cell disease, cystic brosis, and progeria
have led to possible solutions by synthetic biologists for such diseases. The latest
advancements in synthetic biology and emerging new technologies manifested in
uncovering the expression of monoclonal antibodies, antibody drug conjugates
(ADCs), gene therapy and immunotherapies, cell therapies (e.g., CAR-T [Chimeric
Antigen Receptor-engineered T-cells]), gene editing (e.g., CRISPR [Clustered
Regularly Interspaced Short Palindromic Repeats]), and vaccines among other
applications. Efcient processes for generating monoclonal antibodies, gene therapy, and cell therapy products require signicant advances in upstream and downstream processes, analytical methods, as well as automation leading to process
efciencies and green processes.
The opening chapter discusses the differences and the relationship between small
molecules and biologic drugs and topics representing the latest advances of
K. Gadamasetti (*)
Certum Bio, San Francisco, CA, USA
e-mail: kumar@certumus.com
K. Gadamasetti, S. A. Kolodziej (eds.), Bioprocessing, Bioengineering
and Process Chemistry in the Biopharmaceutical Industry,
https://doi.org/10.1007/978-3-031-62007-2_1
3© The Author(s), under exclusive license to Springer Nature Switzerland AG 2024
4
K. Gadamasetti
bioprocessing, bioengineering, and process chemistry in biologics and biotechnology applications of interest to global readers from both industry and academia.
Keywords Building blocks of DNA · Mutations · Sickle cell disease (SCD) ·
Cystic brosis · Progeria · Crick’s adaptor hypothesis · Process chemistry
What is Life? The Physical Aspect of the Living Cell,” Erwin Schrödinger in his book
(1944) noted that “Organic chemistry, indeed, in investigating more and more complicated
molecules, has come very much nearer to that ‘aperiodic crystal’ which, in my opinion, is
the material carrier of life.” Schrödinger continues to mention in Chap. III of his book: “The
physicist would be inclined to call de Vries’s mutation theory (1902) guratively, the quan-
tum theory of biology… The mutations are actually due to quantum jumps in the gene
molecule” [1].
1.1 Introduction
The rst non-small molecule drug that made a major impact in alleviating pain and
suffering of patients over a hundred years ago was insulin, produced using
Escherichia coli (E. coli) cells. Canadians Frederick Banting and his medical graduate student Charles Best had successfully isolated the hormone insulin in 1921 from
dog pancreas [2], and Banting and John MacLeod were awarded the Nobel Prize in
medicine in 1923 for mass producing insulin for the treatment of diabetes.
Human insulin is composed of 51 amino acids (MW 5808). While insulin meets
the denition of a biologic or a biologic drug (a complex molecule made from
E. coli cells), it has been treated both as a drug and a biologic, being on the market
longer than the FDA rules for biologics were generated. Even though initially insulin was treated as a drug, during early 2020 it ofcially moved to biologic regulatory
framework by the FDA [3]. There is a special discussion and denition dedicated to
biologics in the related subheadings.
The premise in putting this volume together is not to show that either the biologics or small molecule drugs are superior to the other class of drugs. Typically, biologic drugs take longer lead times to develop, have stringent regulation requirements
for clinical trials, and need relatively high capital investments as compared to the
complementing small molecule drugs, but their aptness to certain drug targets and
the therapeutic applications makes biologics subjugate in treating autoimmune disorders and the rare diseases like sickle-cell disease (SCD), cystic brosis (CF), and
the genetic disease, progeria.
1 Bioprocessing, Bioengineering and Process Chemistry in the Biopharmaceutical…
5
1.1.1 The Synopsis oftheVolume
Biogenetic processes provide a pathway to biosynthesis of target molecules, leveraging the use and manipulation of the basic nucleotide building blocks, RNA, and
eventually modifying DNA for constructive purposes. Thus, the fundamental
aspects of genetic engineering allow the tweaking and maneuvering of building
blocks for the biochemical and organic chemical processes to create novel and synthetic oligonucleotides, including plasmids, and relying on cellular machinery for
applied functional outcomes. Extending the idea of utilizing the reconstruction processes of chemistry at the most fundamental level to generate signicant end products has unlimited potential. The utilization of these fundamental building block
maneuvering processes can facilitate the advancement of synthetic biology, in general, and the emerging new technologies and tools in biopharmaceuticals, including
expression of monoclonal, bispecic, and single-domain antibodies, antibody drug
conjugates (ADCs), fusion proteins, genetic alphabet engineering, gene therapy and
immunotherapy, gene editing (e.g., CRISPR [Clustered Regularly Interspaced Short
Palindromic Repeats]), cell therapies (e.g., CAR-T [chimeric antigen receptorengineered T-cells]), vaccines, and chemical processes to generate biologics with
desired properties, linking vaccines with enhanced immunogenicity and nanoparticles with targeting functions. Efcient processes for monoclonal antibodies (mAbs),
gene therapy, and cell therapy products require signicant advances in upstream and
downstream processes, analytical methods, as well as automation leading to process
efciency, cost containment, and green processes, all topics for invaluable discussions. Unlike small molecule generics, biologics (biosimilars, biobetters, and proteins) cannot be precisely replicated in manufacturing. They demand precise
processes to be put in place that meet regulatory constraints leading to signicant
manufacturing challenges. Certainly, the regulatory bodies for biologics are constantly challenged with continuous learning and evolution of regulatory perspectives and the ne-tuning of regulatory guidelines to support new technologies. The
International Conference of Harmonization (ICH) is specially challenged with
guiding the biopharmaceutical companies and allied CROs/CMOs to advance the
biological products into global markets.
In visualizing this futuristic volume to greater reach, a deliberate and conscious
attempt has been made by the editors to select the topics representing the latest
advances of bioprocessing, bioengineering, and process chemistry in biologics and
biotechnology applications of interest to global readers both from industry and academia. It is hoped that the readers will benet from learning directly from top
experts in the eld to improve and streamline processes, problem-solve the complex
challenges inherit in this effort, and further the vital and important work of advancing new drugs, vaccines, and therapies to alleviate the pain and suffering of
humankind.
6
K. Gadamasetti
1.2 Synthetic Biology: APerspective
As a chemist, it has always been intriguing and mindboggling to comprehend with
the fact how the chemistry and the array of atoms and molecules in nature, nucleotides specically, are essential to the storage, transmission, and evolution of
genetic information. Understanding the stereoregularity of the building blocks of
DNA (dA, dT, dG, dC; “d” refers to deoxy), and of RNA (A, U, G, C), the thermodynamic stability of the gene in the cell supporting the Darwinism of life is
vital for every discipline and the faculties in science and technology to appreciate.Protein metabolism [12], the manifestation of gene expression to proteins,and
functional proteins is a fascinating subject by itself. The captivating fact of mutations of DNA leading to evolution and the inevitable ugly truth of genetic disorders and diseases go hand in hand, so long as the existence of human beings and
living organismson the planet earth. The genetic mutations leading to medical
disorders opens a plethora of opportunities for the fraternity of medical, scientic,
bioengineers, and biotechnologists to look into nding the solutions to cure diseases and alleviate the human suffering. A selective group of the pharmaceutical
and biotechnology companies conceptually have the mission, based on genetic
modications, to discover, develop, and provide the most advanced innovative
therapies to patients and the medical community.An interesting description on
how six commercially-available products that are changing our worldbyVoight is
noteworthy [11].
1.2.1 Basic Alphabets andExpanded Alphabets ofGenetic
Building Blocks
The four natural deoxy nucleotides (dA, dT, dG, and dC) in DNA and nucleotides (A, U, G, and C) in RNA, respectively, as the genetic building blocks and
corresponding matching nucleotide base pairs, are the central foundation of creating 20 amino acids to build proteins through the 64 codons, the proteins, and
thus, the concept of the creation and the existence of the living organisms and
the human race. Benner [6a], Romesberg [6b], and Hirao and others [6c] have
been involved in experimenting with adding synthetic nucleotide-like base pairs
to natural deoxy nucleotides to create, potentially, several synthetic proteins and
unnatural mAbs.
This expanded genetic alphabet platform is expected to be a source for developing new class of synthetic proteins and possibly a differentiated therapeutic
pipeline.
1 Bioprocessing, Bioengineering and Process Chemistry in the Biopharmaceutical…
7
1.2.2 Mutations, Point Mutations, Chemistry,
andHealth Disorders
Mutation, in summary, is when a gene is altered through a modication in DNA
structure due to any chemical or physical change[5]. Point mutation[4c] is a type
of mutation in DNA or RNA in which a single nucleotide base is added, deleted, or
changed. A single base pair mutation in the genome can result in many congenital
disorders in humans and living organisms. The incidence of chemical and biochemical changes occurs that alters the genetic outcomes and amino acid sequences in
proteins by the cell, thereby manifesting in medical disorders during the mutations.
Point mutations contribute to about 62% of total genetic disorders. Base editing
precisely edits DNA sequences in a specic locus without inducing harmful doublestrand breaks (DSBs). Around 60% of pathogenic point mutations can potentially
be corrected by base editors and can provide a cure for many blood diseases [4f].
Readers interested in the discussion are encouraged to read the work in the citations
as it is beyond the scope of elaboration in this chapter. The content in Table1.1 [4,
4a, 4c] lists the class and the type of mutation, description, and the human diseases
associated with the specicmutation.
It is safe to say that nearly all medical disorders have a genetic component associated with the diseases. Some disorders like sickle-cell disease, cystic brosis, and
progeria are caused by single gene mutations. Going into some details calls forspecial attention in understanding the root cause of these diseases how the synthetic
biology and genetic engineering help with unraveling the intricacies of science
involved in dealing withdiseases like sickle-cell disease (SCD), cystic brosis (CF),
and progeria, also known as Hutchison–Gilford progeria syndrome (HGPS).
1.2.2.1 Sickle-Cell Disease (SCD) andPotential Treatment
Sickle-cell anemia [7a] is a disease caused by the alteration in a single nucleotide in
the gene (Fig.1.1) for the beta chain of hemoglobin (the oxygen-carrying protein
that makes blood red) that turns the normal hemoglobin gene into a sickle-cell
hemoglobin gene. This change in turn alters only one amino acid protein chain
reducing the ability to transport oxygen. The resulting protein with 147 amino acids
sequence contains valine instead of the glutamic acid as the sixth amino acid. The
impeded blood ow leads to recurrent pain and severe organ damage.
Recommended cure options include blood transfusions and stem cell transplantation. Clinical trials are ongoing to address stem cell transplantation in adults, and
gene therapies and CRISPR-Cas9-mediated gene editing methods are underway to
address the need to cure sickle-cell disease [7b]. Recent advances in the treatment
of SCD have been reviewed by Cisneros and Thein [7c] and Fortenberry and
co-authors[7d].
8
Table 1.1 Types of DNA mutations and the health disorder outcomes
Class of
mutation Type of mutation Description
Point mutation Substitution One base is incorrectly added during
replication and replaces the pair in the
corresponding position on the
complementary strand
Insertion One or more extra nucleotides are
inserted into replicating DNA, often
resulting in a frameshift
Deletion One or more nucleotides is “skipped”
during replication or otherwise
excised, often resulting in a frameshift
Chromosomal
mutation
Copy number
variation
Inversion One region of a chromosome is ipped
and reinserted
Deletion A region of a chromosome is lost,
resulting in the absence of all the
genes in that area
Duplication A region of a chromosome is repeated,
resulting in an increase in dosage from
the genes in that region
Translocation A region from one chromosome is
aberrantly attached to another
chromosome
Gene
amplication
Expanding
trinucleotide
repeat
The number of tandem copies of a
locus is increased
The normal number of repeated
trinucleotide sequences is expanded
K. Gadamasetti
Human disease(s)
linked to this
mutation
Sickle-cell anemia
One form of
beta-thalassemia
Cystic brosis
Opitz-Kaveggia
syndrome
Cri du chat
syndrome
Some cancers
One form of
leukemia
Some breast
cancers
Fragile X
syndrome,
Huntington’s
disease
Fig. 1.1 Molecular basis of sickle-cell disease
1 Bioprocessing, Bioengineering and Process Chemistry in the Biopharmaceutical…
9
1.2.2.2 Cystic Fibrosis
Cystic brosis (CF) is a progressive, genetic disease that causes persistent lung
infections and limits the ability to breathe over time. CF is caused by mutations in
the gene encoding the CF transmembrane conductance regulator (CFTR), a cyclic
AMP-activated chloride channel [8a]. Most of these mutations either substitute one
base pair (the building material for DNA) for another or delete a small number of
DNA bases. This mutation is caused by the deletion of three base pairs of the CFTR
gene leading to the loss of an amino acid, phenylalanine, in the CFTR protein.
Everyone receives a copy of the CFTR from each parent. Mutations in CFTR lead
to imbalanced water and ion movement across the airway epithelium, resulting in
thickened mucus, chronic bacterial infection, and inammation with progressive
loss of pulmonary function.
A detailed discussion is presented in the article “Molecular Basis of CF: From
Bench to Bedside” [8c]. The gene is located in chromosome 7 (a 27 exons structure), and the deletion of phenylalanine in position 508 of the polypeptide chain,
known as Phe508del or F508del, is the most common CFTR mutation affecting
from 50% to 90% of the chromosomes of CF patients along different geographical
areas. It is a chloride (and iodide in some cases) ion transporter localized at the apical membrane of several polarized epithelia, although other small molecules seem
to be transported by CFTR, including ATP.
There is no cure for cystic brosis, but treatment can ease symptoms, reduce
complications, and improve quality of life [8b]. Promising gene therapies and therapies involving CFTR mRNA and gene editing with CRISPR as well as new viral
and non-viral vector formulations are in development [8a].
1.2.2.3 Progeria
Progeria, also known as Hutchison–Gilford progeria syndrome (HGPS or HPS) [9],
is a genetic condition characterized by the dramatic, rapid appearance of aging
beginning in childhood. Mutations in the LMNA gene cause HGPS.The LMNA gene
expresses to generate the protein, lamin A [4b], which plays an important role in
determining the shape of the nucleus within the cells. The altered protein is accountable for progressively damaging the nucleus and the premature cell death.
There is no cure for progeria, but regular monitoring for heart and blood vessel
(cardiovascular) disease may help with managing your child’s condition [4d].
Recent studies by Fabrizio d’Adda di Fagagna and coworkers demonstrate an
important role for telomeric DNA damage response (DDR) activation in HGPS
progeroid detrimental phenotypes invitro and invivo [4e].