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Clinical Efficacy Assessment
• Target/ receptor(s) for each relevant activity/ function of the product
• B, dose/ concentration- response, and pattern of molecular signaling upon engagement of
the target/ receptor(s)
• Relationships between the product structure and target/ receptor interactions
• Location and expression of the target/ receptor(s).
• PK and biodistribution of the product in different patient populations (relevant PD measures
may also provide important information on the mechanism of action).
• Immunogenicity of the product in different patient populations.
• Differences in the expected toxicities in each condition of use and patient population (including
whether expected toxicities are related to the pharmacological activity of the product or offtarget activities).
• Any other factor that may affect the safety or efcacy of the product in each condition of use
and each patient population for which marketing authorization is sought.
• Differences between conditions of use concerning the factors described above do not necessarily preclude extrapolation. Scientic justication is needed to address the differences in the
totality- of- the- evidence context, supporting the demonstration of biosimilarity.
• When choosing which condition of use to study that would permit subsequent extrapolation
of clinical data to other conditions of use, it is recommended that the developers consider
choosing a condition of use that would be adequately sensitive to detect clinically meaningful
differences between the proposed biosimilar product and the reference product.
The developers of the proposed biosimilar product are required to obtain marketing authorization
for all conditions of use that have been previously approved for the reference product at the time of
applying. If the reference product receives marketing authorization for additional indications, then
the developers must add those indications before or after the marketing authorization. However, if
an indication is protected under intellectual property laws, then the developers may request fewer
indications and then add more indications as the intellectual property expiration allows them.
8.6.5 extRaPolation acRoss indications
The reference product may have more than one therapeutic indication. When biosimilarity in a
comparative study has been demonstrated for one indication, extrapolation of clinical data to other
indications of the reference product could be acceptable, but this needs to be scientically justied.
In case it is unclear whether the safety and efcacy conrmed in one indication would be relevant
for another indication, then additional data will be required for conrmation. Extrapolation should
be considered in the light of the totality of data, that is, quality, nonclinical, and clinical data. It is
expected that the safety and efcacy can be extrapolated when thorough physicochemical and structural analyses have demonstrated that the proposed biosimilar’s comparative and in vitro functional
tests complemented with clinical data (efcacy and safety and PK/ PD data) in one therapeutic indication. Additional data are required in certain situations, some of which are as follows:
• The active substance of the reference product interacts with several receptors, leading to a
possibly a different effect on the tested and nontested therapeutic indications.
• The active substance itself has more than one active site, and the sites may have a different
effect on different therapeutic indications.
• The studied therapeutic indication is not relevant for the others in terms of efcacy or safety,
that is, it is not sensitive to differences in all relevant aspects of efcacy and safety.
Immunogenicity is related to multiple factors, including the route of administration, dosing
regimen, patient- related factors, and disease- related factors (e.g., co- medication, disease type, and
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Clinical Efficacy Assessment
159
immune status). Thus, immunogenicity could differ among indications. Extrapolation of immunogenicity from the studied indication/ route of administration to other uses of the reference product
should be justied.
8.6.6 additional conditions of use
Agencies recognize that the application holder of a proposed biosimilar product may be interested
in seeking marketing authorization for an additional condition(s) of use after market authorization
of the product. While this option is generally available in many jurisdictions, regulatory agencies
allow a proposed biosimilar product to add any new indications allowed to the reference product in
the future, provided there are no changes to the reference product. If an indication is protected under
a patent, then it is the developers’ discretion to judge whether the patent is applicable in its region
and they are solely responsible for the litigation. Marketing authorization by the agencies does not
constitute an opinion regarding intellectual property associated with the reference product.
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Recombinant Manufacturing
9
System for
Biopharmaceuticals
9.1 OVERVIEW
A robust biopharmaceutical production process is governed by several elements: integrated process
design and critical process elements; the quality of starting materials; and the quality systems to
conrm that the manufacturing process is reproducible, consistent, and robust. The process design
has its own set of challenges both upstream and downstream. However, an upstream process and
an efcient purication process capable of providing maximal recovery can yield a highly pure
product, which are the most desirable features of any production process.
As biopharmaceuticals have complex chemical structures, it is impossible to synthesize them
with currently available technology. Furthermore, even if it becomes possible to synthesize these
macromolecules, it would be difcult to produce a molecule that does not have a xed structure.
Therefore, the production of biopharmaceuticals relies on recombinant systems where DNA produces
these molecules; DNA portions containing genes of interest are relatively easy to construct, allowing
their use as an engine to make the target molecules in living entities. Thus, the starting material for
manufacturing biopharmaceuticals is a genetically modied bacterial, yeast, insect, or mammalian
cell culture system expressing the target therapeutic protein of interest.
The technology for recombinant manufacturing is widely protected by patents, inclusive of the
critical steps and methods, such as isolation of the target gene sequence (coding for the desired
protein), its amplication, generation of recombinant DNA, transformation of host cells, screening,
and subsequent selection of the expression cell line. For example, the US Patent No. 4,237,224
(Cohen et al.), which is now expired, details the transformation process in microorganisms such as
Escherichia coli to generate recombinant plasmid DNA. The patent also describes the procedures
for the manufacture of the rst transformation vector. Cohen’s patent is now expired, and the technology is available in the public domain.
The recombinant expression is carried out in a variety of living systems such as
• Prokaryotes such as archaea and bacteria are unicellular organisms with an outer cell membrane but do not have any membrane- bound cellular components, including a nucleus or mitochondria. Thus, the transcription and translation processes are carried out simultaneously, and
mRNA translation occurs before the complete synthesis of a mature mRNA transcript.
• Eukaryotes such as animals and plants are multicellular organisms with a nucleus, mitochondria, and Golgi apparatus; these are also. The transcription and translation processes occur
sequentially. Transcription, that is, DNA to RNA, occurs in the nucleus, and translation, that
is, RNA to protein, occurs in the cytoplasm. Post- translational modications (PTMs), which
are chemical modications required for a protein’s functionality, occurs in the endoplasmic
reticulum and the Golgi apparatus following protein synthesis (at different stages, e.g., before
160
DOI: 10.1201/9781003392026-9
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protein folding, post- localization, etc.). The most common PTMs include glycosylation, phosphorylation, acetylation, proteolysis, specic conformation, and oligomerization reactions.
The degree and the complexity of these PTMs vary in eukaryotes. As such, depending on the
desired protein to be expressed, the choice of the expression system varies. For example,
• Glycosylation and phosphorylation are crucial PTMs occurring in simple eukaryotes such
as yeasts and fungi. However, yeasts produce mannose residues that have extensively
undergone N- glycosylation, and these residues can be highly immunogenic for humans. As
such, the choice of this expression organism is limited to the production of simple proteins.
• Complex proteins requiring a high degree of glycosylation or other PTMs are best produced
using eukaryotes such as mammalian cells (e.g., Chinese hamster ovary [CHO], human
cells, etc.), insect cells, and plant cells. Among them, mammalian cells, notably CHO cells,
have been the most extensively used expression systems for a wide range of complicated
proteins (monoclonal antibodies).
For more than 90% of the currently approved products, E. coli, CHO cells, and Saccharomyces
cerevisiae are the most utilized expression systems (hosts). Other cell lines include baby hamster
kidney (BHK) cells, mouse C127, African monkey kidney cells, lymphocyte activated, mouse myeloma, myeloma NS0, and prostate epithelium cells, in addition to these cell hosts.
Examples of approved biotherapeutic proteins and their corresponding host cell systems used for
protein production are summarized in Table 9.1.
DNA is responsible for producing proteins required by the cells for survival and physiological
functions. Recombinant protein expression differs widely— from in vivo use for structural studies to
large- scale development for biotherapeutic drugs.
Transcription and translation are the underlying mechanisms for gene regulation and protein
expression, respectively. The information contained in the genetic code (DNA) is transcribed into
mRNA. The mRNA- coded message is then translated into specic amino acid sequences, leading
to formation of the desired protein (Figure 9.1). R DNA is produced by a process wherein the gene
of interest is rst inserted into a plasmid (e.g., a virus) introduced into a living cell (such as a bacterium or mammalian cell). The plasmid modies the cellular DNA and forces it to start producing
the target protein.
In prokaryotes, transcription and translation occur simultaneously, whereas in eukaryotes, these
processes occur sequentially, followed by PTMs that further alter the protein structure or functionality, which are critical for complex therapeutic proteins. These processes are key to harnessing
living cells, including microbial systems, animal cells, and plant cells, to construct the desired protein of interest using recombinant DNA technology.
The DNA sequence according to the code of the desired protein is constructed and inserted into a
host system (also referred to as a production system or expression system). Chapter 3 discusses cell
line development in detail for microbial and mammalian expression systems.
The process of gene insertion starts from choosing a host plasmid that holds the gene of interest
in the entity designated to produce a recombinant protein (Figure 9.2). Cloning involves transferring
a gene of interest of a DNA fragment to an expression vector.
Expression vectors must possess the following four basic key features: (i) presence of the
gene- of- interest cassette, (ii) antibiotic selection cassette, (iii) bacterial origin of expression (ori),
(iv) multiple cloning site linkers, epitope tags, protease recognition sites, internal ribosome entry
sites, and secretion signals. However, not all elements are required.
In the gure, the arrows denote the direction in which the genes are transcribed. The ori denotes
the origin of DNA replication. The regions marked as “amp” and “tet” denote the antibiotic resistance genes ampicillin and tetracycline, respectively. The regions marked in blue at the specic
sequence (nucleotide) numbers represent the restriction sites for enzymes to recognize and cut out a
specic region in the DNA plasmid.
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TABLE 9.1
Recombinant Manufacturing System
Host Cells Used for Approved Biotherapeutic Products
Pichia pastoris Collagenase Santyl N/ A
Escherichia coli Interferon- Alpha- 2b Intron A 1986
Escherichia coli Epoetin Alfa Epogen/ Procrit 1989
Pichia pastoris Interferon Gamma- 1b Actimmune 1990
Escherichia coli Filgrastim Neupogen 1991
CHO cells Abciximab Reopro 1994
Pichia pastoris L- Asparaginase Oncaspar 1994
Escherichia coli Insulin Lispro Humalog 1996
Trichoplusia ni High Five cells Platelet- Derived Growth Factor Regranex 1997
CHO cells Rituximab Rituxan 1997
CHO cells Etanercept Enbrel 1998
CHO cells Iniximab Remicade 1998
CHO cells Palivizumab Synagis 1998
CHO cells Trastuzumab Herceptin 1998
Escherichia coli Insulin Aspart Novolog 2000
Escherichia coli Insulin Glargine Lantus 2000
CHO cells Alemtuzumab Campath 2001
CHO cells Darbepoetin Alfa Aranesp 2001
CHO cells Adalimumab Humira 2002
Sf9 cells Alpha- 1 Antitrypsin Prolastin- C 2002
Escherichia coli Peglgrastim Neulasta 2002
Escherichia coli Teriparatide Forteo 2002
Pichia pastoris Laronidase Aldurazyme 2003
CHO cells Omalizumab Xolair 2003
CHO cells Bevacizumab Avastin 2004
CHO cells Cetuximab Erbitux 2004
Escherichia coli Insulin Glulisine Apidra 2004
CHO cells Natalizumab Tysabri 2004
Sf9 cells Fibroblast Growth Factor Eperisone
Hydrochloride
CHO cells Ranibizumab Lucentis 2006
CHO cells Eculizumab Soliris 2007
CHO cells Golimumab Simponi 2009
Hansenula polymorpha Prucalopride Resolor 2009
CHO cells Ustekinumab Stelara 2009
HEK 293 cells Belimumab Benlysta 2011
CHO cells Brentuximab Vedotin Adcetris 2011
CHO cells Insulin Degludec Tresiba 2012
Nicotiana benthamiana tobacco plant Taliglucerase Alfa Elelyso 2012
CHO cells Vedolizumab Entyvio 2014
CHO cells Daratumumab Darzalex 2015
CHO cells Secukinumab Cosentyx 2015
HEK 293 cells Atezolizumab Tecentriq 2016
HEK cells Eftrenonacog Alfa Alprolix 2016
PER.C6 cells Follitropin Delta Rekovelle 2016
Sp2/ 0 cells Iniximab- Dyyb Inectra 2016
Escherichia coli Insulin Glargine/ Lixisenatide Soliqua 2016
CHO cells Ixekizumab Taltz 2016
BHK cells Octocog Alfa Kovaltry 2016
NS0 cells Olaratumab Lartruvo 2016
HEK 293 cells Avelumab Bavencio 2017
2005
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Recombinant Manufacturing System
TABLE 9.1 (Continued)
Host Cells Used for Approved Biotherapeutic Products
HEK 293 cells Axicabtagene Ciloleucel Yescarta 2017
CHO cells Brodalumab Siliq 2017
Escherichia coli Cenegermin- Bkbj Oxervate 2017
CHO cells Durvalumab Imnzi 2017
CHO cells Emicizumab Hemlibra 2017
CHO cells Guselkumab Tremfya 2017
HEK 293 cells Inotuzumab Ozogamicin Besponsa 2017
Saccharomyces cerevisiae Insulin Aspart Injection Fiasp 2017
CHO cells Non- Acog Beta Pegol Rexia 2017
CHO cells Rh Coagulation Factor IX Rebinyn 2017
CHO cells Sarilumab Kevzara 2017
HEK cells Simoctocog Alfa Vihuma 2017
HEK 293 cells Tisagenlecleucel Kymriah 2017
CHO cells Adynovi Rurioctocog Alfa Pegol 2018
CHO cells Aimovig Erenumab- Aooe 2018
CHO cells Benralizumab Fasenra 2018
CHO cells Coagulation Factor Xa Zhzo Andexxa 2018
NS0 cells Ibalizumab- Uiyk Trogarzo 2018
Pichia pastoris Insulin Glargine Semglee 2018
Escherichia coli Metreleptin Myalepta 2018
CHO cells Mogamulizumab Poteligeo 2018
Escherichia coli Pegvaliase- Pqpz Palynziq 2018
CHO cells Velmanase Alfa Lamzede 2018
CHO cells Aducanumab Aduhelm 2021
163
Source: www.acc essd ata.fda.gov/ scri pts/ cder/ daf/ 0
9.2 EXPRESSION SYSTEMS
The choice of the organism and the cell line type as the host organism are the primary and most
important aspects that govern a biotherapeutic production process. The starting material for manufacturing recombinant drugs, which is the primary focus of today’s biopharmaceuticals, is genetically modied prokaryotes (bacteria) or eukaryotes (e.g., mammalian cells such as CHO, yeast,
insects, or cells) expressing the target therapeutic protein product or monoclonal antibody of interest.
Biosimilar developers do not have to use the same expression system as that used for the reference
product, but it is not advisable, particularly if PTMs are involved.
Well- organized management for a systemic cell bank, with controlled monitoring and testing,
is key to assuring a cell line’s integrity and biological characteristics. A controlled environment
is likely to prevail with a reputable repository or commercial cell line developer. Upon acquiring
a new cell line, a collection center will generally characterize the cell line to establish and verify
its identity and ensure the purity of the culture— for animal cells, it is essential to demonstrate
that the cell line is free from microbial contamination and mycoplasma. The possibility of inadvertently introducing errors (e.g., cross- contamination, contamination with an adventitious virus,
etc.) in a cell line is innite and quite common. Therefore, care must be taken when handling all
material to ensure the safest possible working environment. This applies to both cells and to the
individuals handling this. Cell culture is considered a biohazard, and the degree of hazard depends
on the cells and experimental protocol. Primary cultures have a high risk of harboring undetected
viruses.
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Recombinant Manufacturing System
FIGURE 9.1 Flowchart of the development of expression and manufacturing system for recombinant
proteins
The robustness of the organism is critical to the success of the biomanufacturing process and
dictates commercial manufacturing scalability. It is essential to provide evidence of the organism’s
history, starting from the rst stage of creation to establishing a cell line for future production.
Given the high cost of biopharmaceuticals, there has been a signicantly continuous improvement
in product titer, cell densities, and even enhanced product quality. For example, in the early years,
antibody production using mammalian culture processes had relatively low yields of <0.8– 1.0 g/ L,
and in recent years, the productivity of commercial processes has increased to >3 g/ L.
9.3 BACTERIAL CELLS
However, bacterial expression systems (such as E. coli, Bacillus, Streptococcus) enable intracellular and extracellular protein expression without any PTMs. The key benets of using a bacterial
expression system for the production of simple target drugs are their relatively short doubling time,
easy manipulation, simplied handling, high productivity, and cost- effectiveness, particularly for
large- scale production.
The protein production process, which involve gene expression, cloning, and small- scale protein production, it can take approximately 5– 7 days to produce adequate protein (ranging from
a few hundred milligrams per liter to grams per liter). The nutritional and aeration demands for
bacterial production systems are simple, allowing relatively straightforward scale- up operations in
fermenters. Additionally, bacterial systems are robust in their ability to withstand the shear forces
occurring when increasing operational scales and volumes. These fermentation processes can be
operated in both batch and fed- batch modes.
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Recombinant Manufacturing System
FIGURE 9.2 Schematic representation of the pBR322 plasmid, one of the rst plasmids widely used as a
cloning vector
Source: By Ayacop (+ Yikrazuul)- Own work, Public Domain, commons.wikimedia.org/ w/ index.php?curid=
11840365
165
E. coli is the most widely used expression system since the approval of Humulin (insulin) in 1982,
specically for small proteins and peptides, and most recently, it has been used to produce antibody
fragments and derivatives such as Fab fragments single- chain variable fragment antibody that does
not require any PTMs. Some of the recombinant proteins that have been successfully manufactured
using E. coli are human growth hormone, interferon- alpha 2a, and 2b, interleukin, granulocyte–
colony- stimulating factor, parathyroid hormone, and somatostatin. Of the biopharmaceuticals
approved in Europe and the US, approximately 39% are manufactured using E. coli. Table 9.1 lists
examples of proteins produced using E. coli production systems.
A schematic detailing the process of gene expression, cloning, and generation of recombinant
bacterial cells is presented in Figure 9.3.
The rapid growth rates and basic nutrient requirements while not compromising titer are the
critical features of the E. coli expression system for the production of nonglycosylated molecules.
Therefore, optimal conditions including temperature, pH, dissolved oxygen, aeration, and agitation,
as well as convenient nutrient feeds, are essential parameters that determine the productivity of
fermentation.
The expression and production of recombinant proteins in E. coli occur intracellularly (e.g.,
cytoplasm, periplasm) and extracellularly (extracellular space). Typically, the protein expression
targeted in the cytoplasm or periplasm often leads to aggregates called inclusion bodies, which
harbor the protein. These inclusion bodies are isolated from the rest of the cellular components after
fermentation to extract and purify the protein. The protein present in the inclusion bodies exists in
the inactive state and requires conversion to the active state. This is typically accomplished through
solubilization and refolding to enable the movement of the protein into its native conformation. The
E. coli expression system lacks the ability and mechanism to carry out PTMs such as glycosylation,
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FIGURE 9.3 Bacterial expression system
Recombinant Manufacturing System
disulde bond formation, and phosphorylation, among others. Therefore, only relatively simple protein molecules that do not need any PTMs are often produced using bacterial cells. The formation of
inclusion bodies can be considered advantageous, particularly in cases where the protein produced
is susceptible to degradation or loss in its activity and function. The product, in its inactive state, is
safely harbored within these inclusion bodies. Inclusion bodies are generally recovered by centrifugation and are subjected to additional washing with various chemicals to obtain inclusion bodies of
high purity. To promote disulde bond formation, typically during protein refolding, an appropriate
redox pair (e.g., GSH/ GSSG) or reducing agents such as dithiothreitol or mercaptoethanol may be
added during and/ or after solubilization to reduce the formation of any undesirable intermolecular
and intramolecular disulde bridges. During refolding, controlling the product/ protein aggregation
is key to ensuring that aggregates are limited. This is typically accomplished by diluting the
medium to relatively low concentrations of the protein and a controlled rate. Additional processing
steps— solubilization and refolding, although it is crucial to regain the correct protein conguration
and activity during these steps— are considered disadvantageous, adding to the increase in liquid
handling volumes (due to refolding at low protein concentrations) which increases overall costs and
likely decreases the yield.
Another signicant drawback often encountered with bacterial production systems is the generation/ presence of cell- substrate impurities including endotoxins and host cell proteins (HCPs).
This requires various process controls to be monitored to ensure that endotoxins and HCPs are
within acceptable limits and meet regulatory requirements for permissible amounts in doses. Some
of the common processing steps that may require additional in- process controls or monitoring
include the cell lysis steps, body solubilization, and refolding. Thus, downstream operations must
be highly robust and capable of minimizing these impurities. Other alternatives such as in vitro
folding and N- terminal cleavage can be considered as overall process improvements. Despite
these limitations, E. coli cells are still popular and are the preferred choice for producing simple
protein molecules.
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Recombinant Manufacturing System
TABLE 9.2
167
Key Characteristics of Commonly Used Escherichia coli Expression Strains
Parent E. coli
Strain Strain Resistance Key Features Protein Expression
B BL21 (DE3) Ampicillin IPTG- inducible strain containing
T7 RNAP (DE3)
BL21 (DE3)
pLysS
BL21 (DE3)
pLysE
BL21 Star
(DE3)
BL21- AI Tetracycline/
BLR (DE3) Tetracycline Rec- A- decient derivative
Tuner (DE3) Chloramphenicol Contains mutated lac permease
Rosetta 2 (DE3) Chloramphenicol
T7 express Ampicillin,
K12 HMS174 (DE3) Rifampicin Rec- A decient Suitable for the expression of
Origami2
(DE3)
M15 pREP4 Kanamycin
Chloramphenicol
(pLysS)
Chloramphenicol
(pLysE)
Ampicillin T7 based, has high mRNA
kanamycin
(pRARE)
kanamycin
Streptomycin,
tetracycline
(pREP4)
pLysS expresses T7 lysozyme
Has lower basal expression levels
pLysE has higher T7 lysozyme
expression than pLysS
stability, capable of high yields
Arabinose- induced T7 expression Best suited for general protein
of BL21
Suitable for stabilizing plasmids
with repetitive sequences
(lac ZY deleted)
Derivative of BL21
Derivative of BL21
Contains seven additional tRNAs
for rare codons that can support
the expression of eukaryotic
proteins
IPTG- inducible T7 RNAP
expression
Protease decient
Mutated K12 strain for
thioredoxin reductase and
glutathione reductase genes
Cis- repression of the E. coli T5
promoter
Best suited for general protein
expression
Used for the expression of toxic
proteins
Most suitable for the expression
of toxic proteins
Best suited for a high level
of expression and for the
expression of nontoxic
proteins
expression but can also be
used for the expression of
toxic proteins
Suitable for the expression of
unstable proteins
Suitable for the expression of
toxic or insoluble proteins
Can be used for low levels of
expression
Expression of eukaryotic
proteins
Best suited for general protein
expression
unstable proteins
Best suited for the expression of
proteins that require disulde
bond formation for proper
protein folding
Suitable for the expression of
toxic proteins
The two most commonly used E. coli strains are K12 and B. Table 9.2 details the commonly used
strains derived from the E. coli B strain and K12 strain. The growth characteristics of the B and K12
strains are completely different. Therefore, the features and characteristics of these strains must be
carefully considered and evaluated for determining the appropriate strain for use as the expression
system for the desired protein. Additionally, the strains vary in terms of their coding of the disulde
bonds in the protein or even encoding toxic proteins.
In recent years, various new strains with signicant improvements are being used to manufacture
biopharmaceuticals (e.g., bacilli, Ralstonia eutropha, Staphylococcus carnosus).
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