FERMENTATION PROCESS EVENTS AFFECTING THE

BIOPHARMACEUTICAL QUALITY

INTRODUCTION
BIOPHARMACEUTICALS
Biopharmaceuticals are defined
Complex macromolecules derived from recombinant DNA technology,
cell fusion, or processes involving genetic manipulation
Biologically significant compounds like hormones and proteins useful for treatment of
variety of human health disorders, usually called as Biopharmaceuticals or
Biotherapeutics or Biologicals. They are usually obtained from biological source and
produced through industrial biotechnology.
Quality of these biopharmaceuticals depends upon:
Choice of proper host cell to correctly synthesize and process the desired proteins,
upon fermentation process
Fermentation process development to maintain protein consistency from lot to lot.
Fermentation processes are designed and operated to prevent biological
contaminants of the protein and to optimize product safety.
It is very important to study various variables and factors which affect the quality of the
finished product to ensure the safety and efficacy of these biopharmaceuticals.
Unlike orally delivered small molecule drugs, biopharmaceuticals are usually
administered by subcutaneous, intravenous, or intramuscular injection.
Biopharmceuticals includes the following classes:
Recombinant proteins,
(monoclonal) antibodies,
vaccines,
Blood/plasma-derived products,
Non-recombinant culture-derived proteins, and
Cultured cells and tissues.
EXAMPLES
Blood factors (Factor VIII and Factor IX)
Thrombolytic agents (tissue plasminogen activator)
Hormones (insulin, glucagon, growth hormone, gonadotrophins)
Hematopoietic growth factors (Erythropoietin)
Interferons (Interferons-, -, -)
Vaccines (Hepatitis B surface antigen)
Monoclonal antibodies (Various)
Additional products (therapeutic enzymes)
FERMENTATION
Fermentation technology is the oldest of all Biotechnological process. It is actually
process of growing a culture of micro organisms in a nutrient media and converting feed
into desired end product. It is described as a biochemical reaction in which micro
organisms (bacteria or fungi) serve as biocatalyst.
MICROBIAL FERMENTATION
Microbial fermentation is the basis for the production of a wide range of pharmaceutical
products (Biopharmaceuticals), targeting any medical indication.
Examples of such medical indication range from cancer, viral infectious diseases, to
hormonal disorders and many other indications.
Microorganisms may be genetically modified (recombinant technology) or metabolically
engineered by substantial alteration of their endogenous routes. During their growth and
lifespan microorganisms build a wide range of different molecules types required for
Viability and multiplication;
Adaptation to changing environment;
Stressful conditions and
Defence against hostile, competitive microbial threats.
Microorganisms that are typically used within the pharmaceutical industry include:
Prokaryotes such as Bacteria (e.g. Escherichia coli, Staphylococcus aureus) and
Streptomycetes.
Eukaryotes such as Filamentous Fungi (e.g., Nigrospora spp, Aspergillus spp,) and
Yeast.
FERMENTER
Also termed as Bioreactor.
REQUIREMENTS OF BIOREACTORS
There is no universal bioreactor. However the general requirements of the bioreactor are
as follows:
The design and construction of bioreactors must keep sterility from the start point
to end of the process.
Optimal mixing with low, uniform shear.
Adequate mass transfer, oxygen.
Clearly defined flow conditions.
Feeding substrate with prevention of under or overdosing.
Suspension of solids.
Gentle heat transfer.
Compliance with design requirements such as: ability to be sterilized; simple
construction; simple measuring, control, regulating techniques; scale-up; flexibility; long
term stability; compatibility with up- downstream processes; antifoaming measures.


TYPES OF FERMENTATION PROCESS
Batch Fermentation
Continuous Fermentation
Fed batch
BATCH FERMENTATION PROCESS
Its a dynamic processes that are never in a steady state. Often, the critical parameter is
gas exchange or balance between respiration rate and oxygen transfer. Sterilized media
components are supplied at the beginning of the fermentation with no additional feed
after inoculation.
Cells are grown in a batch reactor, they go through a series of stages:
Lag phase
Exponential phase
Stationary phase
Death phase
Lag Phase
Microbial population remains constant as there is no growth. However it is the period of
intense metabolic activity.
Exponential Phase
Cell divides with increasing frequency till it reaches the maximum growth rate (max)
At this point logarithmic growth begins and cell numbers or cell biomass increase at a
constant rate.
Stationary Phase
Overall growth rate has declined to zero and there is no net change in cell numbers/
biomass ie. rate of cell division equals rate of cell death.
Microorganisms are still metabolically active, metabolizing intracellular storage
compounds, utilizing nutrients released from lysed cells and in certain cases produce
secondary metabolites.
Death Phase
Cells die at constant rate and often undergo lysis.
CONTINUOUS FLOW FERMENTER.
Here the raw materials are trickled in at the top of a column in which there are
immobilised micro-organisms or enzymes. The product flows out the bottom in a pure
state. It does not need to be separated from the catalyst. However this process can only be
used for reactions that are fast possibly taking 10 minutes.
FERMENTATION PROCESS EVENTS AFFECTING
BIOPHARMACEUTICAL QUALITY
We can divide these events in two groups
A. Biological Events
B. Physicochemical Events
A. BIOLOGICAL EVENTS
a) Protein synthesis process
b) Genetic construction
c) Cell Physiology
d) Medium contaminants
e) Biological contaminants
f) Pharmacologically active contaminants

a) Protein synthesis process
Genetic information chemically determined by DNA structure is transferred to daughter
cells by DNA replication and is expressed by Transcription (conversion of DNA into
RNA) followed by Translation (conversion of RNA into proteins)

DNA RNA Protein
Proteins are translated by ribosomes from mRNA which have been transcribed from
cellular genes (DNA)
Some important events that may impact product quality include:
Post translational covalent modifications
(glycosylation, - carboxylation, acetylation, disulfide formation)
Folding
Intracellular transport and secretion
Interaction with other proteins

b) Genetic construction
Portion of DNA needed for the transfer of a gene of interest in a cell also includes the
promoter and regulators essential to its expression and regulation in the receiving cell
Failure to utilize well-known genetic elements may lead to expression of gene sequences
of unknown function as well as structure and function of uncharacterized DNA sequences
Incorrect amino acids may be incorporated into the protein of interest due to mutation,
mistranscription, mistranslation of transfected gene results in product related molecules
might be immunogenic or toxic
The probability that at least one mutation will occur in at least one copy of the
transferred gene during fermentation is quite high.
c) Cell physiology
During cultivation cell physiology should be optimized to provide maximum amount of
recoverable qualitatively acceptable protiens.
Major physiological events that affects product quality are post-translational processing
events
Proteolysis
Processing of proenzymes and prohormones
Sulfation
Phsphorylation
Transcription Translation
Glycosylation
Posttranslational modification may interact with cell physiology, e.g. glycosylation
pattern of expressed proteins may change when mammalian cells are deprived of glucose.
d) Medium contaminants
Medium components can directly interact with protein of interest. One of such
interactions is enzymatic action. Interaction of protein of interest with other components
of the cell or medium (or buffer), if not considered, the final product may be
contaminated by altered molecules. For example
Proteases liberated from dead or dying host cells cleave the peptide backbone of
protein.
Other contaminants include microbes like bacteria fungi (and their toxic
byproducts) and Viruses.
Viral contaminants of plasma products (and organ donations and transfusion) have
killed thousands of recipients in US. Many people developed hepatitis and HIV
Safety is assured by four separate mechanisms, which include:
Use of characterized cell banks and certified raw materials
Process validation to remove or inactvate potential contaminants
Use of appropriate assays to screen for viral contaminants
Procedural control on manufacturing process, equipment, and facilities

e) Pharmacologically active contaminants
Some biopharmaceuticals manifest toxicity but this is due to inherent pharmacology of
active ingredients and not due to the result of pharmacological action of unrelated
contaminants or degradation products of active ingredients.
B. PHYSICOCHEMICAL EVENTS
a) Agitation
b) Temperature
c) Mass Transfer
d) Transport of Gases
e) Formulation Fill-Finished Consideration
1) Bulk FreezeThaw
2) Handling Stresses During Mixing and Filling
3) Filtration
4) Lyophilization
5) Photostability
6) Cleaning

a) Agitation
Agitation should produce homogeneous conditions and promote
Nutrient transfer
Gas transfer
Heat transfer

b) Temperature
Automatic temperature control during the fermentation is accomplished by injecting
either cold or hot water or glycol into the outer jacket and/or internal coils.
Inefficient system of heat transfer control can result in higher fluctuations of temperature
of the bioreactor.
c) Mass Transfer
Like the gas transfer the transfer of nutrient can also be a potential source of
contamination. So pre-sterlized nutrient should be used. Further filters can be placed at
inlet and outlet for the mass transfer.
d) Transport of Gases
To prevent the risk of contamination, gases introduced into the fermenter should be
passed through a sterile filter. A similar filter on the air exhaust system avoids
environmental contamination. Sterile filtered air or oxygen normally enters the fermenter
through a sparger system.To promote aeration in stirred tanks, the sparger is usually
located directly below the agitator.
e) Formulation and FillFinish Considerations
1. Bulk FreezeThaw
Stability challenges during freezing
Cold denaturation may also lead to spontaneous unfolding at cold temperatures,
due to the weakening of the hydrophobic effect with decrease in temperature.
An increase in protein concentration also increases the possibility of molecular
collisions, thereby resulting in protein aggregation.
Smaller freezing path lengths and higher heat transfer uxes are recommended to
minimize such freeze concentration effects
Stability challenges during thawing
Slow thawing rates can result in ice recrystallization with small ice crystals
growing into larger ones. Proteins may get sheared at iceliquid interfaces and
lose their activity
Faster thawing rates are usually preferred for protein stability and are likewise
achieved through improved heat transfer uxes and smaller path-lengths
2. Handling Stresses During Mixing and Filling
The mixing step during the formulation process may impact protein stability due
to mechanical stresses created through stirring and shaking in presence of different
contact surfaces.
Mixing and pumping can contribute to introduction of foreign particles that can
further trigger protein aggregation.
Scaled down Mnufacturing process or at very small scale rheometers can be used
to generate high shear under controlled conditions to evaluate the impact of
mixing and lling shear on product.
3. Filtration
An aspect of ltration that may impact product quality is adsorption of surfactants
and proteins on the membrane surface, resulting in either misfolding on the
membrane surface or protein loss. This loss could be signicant for low-
concentration products.
High transmembrane pressure could stress the protein while pushing it through the
lter pores, leading to protein denaturation.
So scale-down studies should be conducted prior to large-scale processing to
assess the impact of ltration on product quality and to recommend the optimum
lter size and the membrane type for the manufacturing process. Other process
parameters to be monitored include the temperature and the rate of liquid ow.
4. Lyophilization
The presence of water is needed for most covalent degradation phenomena, like
residue fragmen- tation and isomerization events, the process of lyophilization is
often an effective way of overcoming these instabilities.
The process of freeze drying, also called lyophilization, removes most of the water
in the drug product, except that presumably associated with the protein (which is
typically less than 1%).
However, the process of lyophilization, especially when not optimized, can also
introduce instability in proteins, typically manifested by either an irreversible
change in structure, or by greater levels of aggregation in the lyophilized product
5. Photostability
During ll and nish operations, including visual inspections, packaging and
during clinical usage the drug product is exposed to both natural and articial
light, of varying intensity and duration.
In proteins, tryptophan, tyrosine, phenylalanine, and cysteine are residues that are
more prone to modication due to light exposure than are other residues.
The peptide backbone may also be susceptible to photo-degradation.
ICH Guidelines recommends performing photostability testing in a controlled light
chamber with one of the following two options for light sources:
A single lamp source with wavelengths below 320 nm ltered out, with an output
similar to either D65 (international standard for outdoor light) or to ID65 (standard
for equivalent window-ltered indoor direct daylight)
Two lamp sources, one for uorescent lighting per ISO 10977 guideline and
another for UV in the 320400 nm range
6. Cleaning
In biotech industry, the cleaning process is designed to remove various process
soils, including media, buffer, process intermediates and puried bulk as well as
nal drug product.
A robust cleaning process, with its consistency and effectiveness at removing the
residual soils from the equipment surface, is critical to ensure product quality.