Castilho Leda R., Moraes Angela Maria (Ed.) Animal Cell Technology: From Biopharmaceuticals to Gene Therapy
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disadvantages of every change/innovation proposed for a process. More
extensive discussions on the topic presented in this chapter are available in
Janson and Ryde´n (1998), Ladisch (2001), Harrison et al. (2003), Pessoa
Junior and Kilikian (2005), Scopes (1994), and Harris and Angal (1989).
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Product purification processes 327
13
Quality control of
biotechnological products
Marina Etcheverrigaray and Ricardo Kratje
13.1 Introduction
Within the pharmaceutical industry, the term biotechnology, in its broad-
est definition, refers to the use of live organisms to produce biomedicines.
The general regulatory scheme for biotechnological products is the same
as for products made by traditional methods, with additional specific
requirements based on their biological origin. The aim of this chapter is to
consider the criteria for the production and quality control of products
obtained through biotechnological techniques. In Chapter 14 (Regulatory
aspects), complementary information is supplied about controls required
by governmental organizations for product registration and quality guar-
antees, during a product’s lifetime in the market. These controls are part of
a set of regulations and guidelines known as Good Manufacturing Practice
(GMP).
The main difference between traditional and biotechnological pharma-
ceutical products lies in the fact that the latter are produced from live
organisms that are often genetically modified. This category includes not
only proteins or polypeptides derived from recombinant DNA (rDNA)
but also monoclonal antibodies (mAbs). In the production stages, all the
pharmaceutical products, even those of biotechnological origin, share the
same basic requirements for process validation, environmental control,
aseptic production, and quality control systems. However, the biotechno-
logical systems usually present a greater degree of complexity.
The rDNA-derived products may contain potentially harmful contami-
nants that are normally not present in their equivalents prepared by
chemical methods, and which the purification process must be capable of
eliminating, such as the endotoxins expressed in bacterial cells, cellular
DNA, and viruses of animal origin. Contamination with nucleic acid from
transformed mammalian cells is of particular concern due to the possible
presence of potentially oncogenic DNA.
The selected production procedure affects the nature and level of
potential contaminants. The possible variability of the system during
production may result in modifications that favor the expression of
alternative genes in the host–vector system or produce lower product
efficiency, or quantitative and qualitative differences in the present impu-
rities. An example of such variability is a change in the profile of proteases
and this should be evaluated, as this may affect the quality of the
recombinant protein produced (Kratje et al., 1994). The production of
continuous cultures is subjected to similar considerations. Therefore, it is
important to rely on procedures that assure the uniformity of the condi-
tions of production and consequently the uniformity of the final product.
The development of biological materials as therapeutic agents must
involve guarantees about their safety, efficacy, and quality. Safety and
efficacy are established through well controlled toxicity studies and
clinical trials. Biological tests are important as a direct measure of bio-
logical activity and as safety indicators that may detect potential deviations
related to efficacy and safety. Quality should be confirmed using a variety
of analytical techniques, which can evaluate purity, biological potency,
stability, and consistency of production.
With the aim of establishing lot-to-lot consistency, the characterization
of a target protein should be performed during its development, to estab-
lish the required analytical criteria for production. This characterization
may be continued during its manufacture. The evaluation of a product is
based on its adequate characterization before and during process develop-
ment. This involves exhaustive research on the structure, physicochemical
properties, and biological activity, as well as its potential contaminants
(Jeffcoate et al., 1993). For a full overall evaluation, it is necessary to use a
combination of these procedures, as none of these can provide enough
information on their own.
Biotechnological production processes may include the following
stages.
(i) Stage of cell expansion (Upstream). This consists of obtaining suffi-
cient producer biomass or cells by bacterial fermentation or cell
culture. After this an impure biomolecule may be obtained under
conditions of pH, ionic strength, aggregation state, etc., which must
be optimized during subsequent processing.
(ii) Purification (Downstream). At this stage a molecule with the required
degree of purity is produced from the crude culture material while
fully maintaining its biological and therapeutic activity. This process
must ensure that the product is treated with criteria acceptable for its
eventual use as a pharmacological agent. The resulting material may
be called pure raw material (active pharmaceutical ingredient).
(iii) Galenic conditioning. The pure raw material must be conditioned to
achieve a final galenic formulation fulfilling all the requirements of a
pharmaceutical product, such as suitable pharmokinetics and safety.
The present chapter includes the following applications of cell cultures:
(i) Production of recombinant proteins (WHO, 1991),
– the initial materials, including the basic data of the host cell and
the origin, nature, and sequence of the gene used in production;
– its production process;
– the pure raw material;
330 Animal Cell Technology
(ii) Bioassays (Mire-Sluis et al., 1996; Mire-Sluis and Thorpe, 1998).
– Special emphasis on bioassays performed in cell culture.
rDNA-derived products and mAbs are considered to be similar to
biological substances produced by traditional methods, such as bacterial
and viral vaccines, in which adequate control of the initial materials and
the production procedure is as necessary as that of the product itself.
These considerations, therefore, put considerable emphasis on control
during the production, to insure the product’s safety and efficacy.
As detailed in Chapter 14 (Regulatory aspects), both the production
process validation and purification capacity to eliminate undesirable
materials are considered essential. The general criteria for quality control
of biotechnological products involve special attention to the quality of all
components used in production, including the components of fermenta-
tion and culture media. If additives of animal origin are used (e.g. fetal calf
serum), they should be proved to be free from adventitious agents. It is
not acceptable to use any agent with potential to provoke allergic reac-
tions, such as penicillin or other beta-lactam antibiotics.
Biotechnological products must satisfy the general criteria for the
quality control of biological products, such as tests of activity, abnormal
toxicity, pyrogenicity, stability, and sterility. The chemical identity of the
target molecule must also be assured, comparing it to a reference, and a
group of tests that verify its integrity, degree of aggregation, and correct
amino acid sequence.
As well as these general criteria there may be specific tests appropriate
to specific products with regard to quality control. Therefore, each
product’s production and quality control should be considered indepen-
dently, taking into account any special characteristic. The considerations
for each product must also relate to its clinical use. Therefore, a prepara-
tion to be administered repeatedly over a long period of time or in large
doses will probably need to be subjected to more stringent tests (particu-
larly for antigenicity) than a product that is applied only once (e.g. a
vaccine).
It is important to determine permissible maximum limits for impurities
and contaminants that may be present in these products, adapting the
limits according to technological advances for testing or from the contin-
uous assessment of the effects of clinical applications.
13.2 Production of recombinant proteins
13.2.1 Control of starting materials
Cloning and expression
As detailed in Chapter 14, regulatory organizations require information
on:
(i) Host cell characterization. This includes origin, phenotype and geno-
type, and the description of the culture medium, morphological
aspects, and growth rate. For eukaryotic cells used as host, the cell
Quality control of biotechnological produc ts 331
line history and general characteristics should be provided. These
must be stable from the master cell bank to the end of production. If
cells have a finite lifespan, the maximum number of population
doublings should be determined (CBER, 1993).
(ii) Characterization of host–vector system. Documentation concerning
the gene cloning strategy and characterization of the recombinant
vector is required (International Conference of Harmonization
Guidelines, 1995).
Cell banks
Once the cell line is selected as a host for production, a cell bank system
must be generated to guarantee the availability of an adequate source of
equivalent cells to use through the product’s lifetime in the market.
Usually, the cell bank system consists of two levels: a master cell bank
(MCB) and a manufacturer’s working cell bank (WCB).
Master cell bank (MCB)
This is a homogeneous suspension of the original cells, already trans-
formed with the expression vector and containing the desired gene, which
is distributed in equal volumes into individual containers (ampoules) for
storage (usually in liquid nitrogen). In some cases, it may be necessary to
establish a separate MCB for the expression vector and the host cell. The
localization, identification, and inventory of the individual ampoules must
be carefully documented.
All the necessary MCB identity tests should be performed to establish
the most significant cell characteristics and their stability over time. Data
must be provided to prove that the cells can be used for the desired aim, as
well as data showing the gene copy number and identity of the expression
system, and the quantity and quality of the protein being produced.
Normally, the nucleotide sequence of the cloned gene in the MCB should
be confirmed. However, in certain cases, as when multiple copies of the
cloned gene are introduced into the genome of the host cell, sequencing
can be inappropriate. In these circumstances, it might be useful to perform
either a Southern blot analysis of the total cellular DNA (Southern, 1975;
Chaplin and Kennedy, 1994) or a Northern blot from the mRNA
sequence (Alwine et al., 1977). In these cases, particular attention must be
paid to the characterization of the final product (see Section 13.4).
If a clone is selected from a population of host cells into which an
expression construct has been transferred, it will be necessary to create a
new MCB. From this MCB it will be important to guarantee the identity
of the clone and the produced protein in reference to the original.
Working cell bank (WCB)
This is derived from a homogeneous suspension of cells derived from the
MCB after a finite number of passages, distributed in equal volumes in
ampoules for storage. The localization, identification, and inventory of the
individual ampoule must be carefully documented.
For the production of a lot of biological product, one or more WCB
ampoules can be used. If cells from more than one ampoule are used, the
332 Animal Cell Technology
cellular suspensions must be mixed immediately after thawing. The popu-
lation doubling number of the cells used for production will be based on
previous criteria established by the manufacturer, assuring the integrity of
the cell product system. A WCB can be used for the production of cultures
with a finite number of passages or continuous cultures.
In both banks:
(i) All ampoules must be treated equally during storage, and once
removed from storage they must be discarded;
(ii) It is recommended that each MCB and WCB should be stored in two
or more sufficiently separated areas inside the production facilities to
avoid loss of the cell line.
13.2.2 Quality control of cell banks
The quality control of cell banks must include information on the follow-
ing.
(i) Stability by measurements of vector viability and retention.
(ii) Cellular identity through phenotypic characterization.
(iii) Evidence must be presented, proving that cell banks are free from
potential infectious or oncogenic agents (viruses, bacteria, fungi, or
mycoplasma). Special attention should be paid to viruses that com-
monly infect the species from which the cell line is derived. Certain
cell lines contain endogenous viruses, for example, retrovirus, whose
elimination might not be feasible. The expression of these endogenous
viruses should be tested in a variety of conditions that may cause their
induction. It is important to show that all lots of cells are free from
every adventitious contaminant.
(iv) In the case of eukaryotic cells derived from mammals, inform-
ation concerning the potential tumorigenicity of the cell bank is
necessary.
(v) Accurate records must be maintained of the composition and origin
of the culture medium used for the cell banks. As animal based sera
can produce allergic reactions in human beings, efforts must be made
to significantly reduce the level of serum required for the propagation
and production in cell cultures. The residual quantity of serum or
additives in the final product must be determined and it must not
exceed the established limits.
If porcine trypsin is used in the passage of cells, it should be free from
adventitious agents, including porcine parvovirus. The manufacturers of
biological products must provide information about the source(s) and
controls of any material derived from ovine or bovine species.
Penicillin or other beta-lactam antibiotics should not be used in produc-
tion cell cultures. Minimum concentrations of other antibiotics or inductor
agents in the culture may be acceptable; however, the presence of any
antibiotic or inductor agents in the final product is not acceptable.
Quality control of biotechnological products 333