Downstream Bioprocess Development of P. Kudriavzevii: Experiment Title

Experiment Title
Downstream bioprocess development of P. kudriavzevii
Instructor
Sevil Dinçer İşoğlu
Özkan Fidan
Fatih Ortakçı
Responsible Assistant
Seray ZORA TARHAN
Submission date
21.01.2022
Prepared by
Gamze Hatlar
140120022
1
Aim
This week's experiment was carried out in order to understand the principles of downstream
bioprocess development of fermentation using P. kudriavzevii. The main aim was to understand
the filtration technique which is a purification technique used in the downstream process.
Calculating the dry weight of biomass purified from in different operation modes these are fed-
batch and batch.
Introduction:
Fermentation, which is conducted in order to produce biopharmaceuticals, can be divided into

upstream and downstream (Kesik‐Brodacka, 2017). The upstream process is the stage where
necessary microbial growth occurs during fermentation for the production of biomolecules. In
the upstream process, the main purpose is to transfer the necessary substrates into the desired
metabolic products. For this to happen, large-scale bioreactors and well-controlled conditions
must be provided. The upstream process has steps such as selection of cell line, culture media,
growth parameters, process optimization (Kiesslich & Kamen, 2020). Process optimization is
very significant, in order to ensure optimal conditions for cell growth and the production of
biopharmaceuticals. Many different parameters affect the upstream process, the first one is the
selection of process types such as batch, fed-batch, and continuous. Temperature, pH, oxygen
supply control, equipment employed, and maintenance of the environment are other factors
affecting the upstream process. Maintenance of the environment should be done in order to
provide it is free of contaminating microorganisms. Downstream processing includes all
necessary steps to obtain a final purified product. It is a step that involves removing impurities in
order to obtain the desired biomolecule. These impurities can be host cell related such as proteins
or DNA, process related impurities such as buffers, leached ligands, antifoam, and product
related impurities such as aggregates, fragments, clipped species (Winkelnkemper &
Schembecker, 2010). One or more impurities can be removed at each purification step. In
2
general, a downstream process is divided into three main steps, initial recovery, polishing, and
drying, respectively. Initial recovery can be explained as the extraction or isolation, polishing can
be explained as the elimination of most contaminants and drying can be explained as elimination
of specialized contaminants and unwanted form of final product. In the initial recovery step, cells
and supernatant are separated from each other. Techniques such as centrifugation, sedimentation,
filtration, and flotation can be used to separate cells and supernatants. If the desired biomolecule
is extracellularly produced, ultrafiltration which is a concentrated method, then purification
should be carried out with such methods chromatography,precipitation, or chromatography. On
the other hand, for intracellular biomolecules, cells that are harvested should be exposed to the
lysis with such methods; high-pressure homogenizer, sonication, passing through mills.
Recovery and purification are very important in the production process of biopharmaceuticals.
Purification operation should- be powerful, trustworthy, certainly scaled-up, and skilled in
removal of both processes- and product-related impurities in order to provide product safety. The
obtained purity, the rate of process improving, entire recovery yield, and throughput are some of
the principal key factors that must be considered into significante along downstream process
development. In order to get the tightness of purity needed in the biopharmaceutical production,
which can be sometimes 99%, chromatography processes are generally carried out.
Chromatography can provide high resolution and has been the power for purification and
polishing of protein (Kosanović et al., 2017). Besides, chromatography is not a cost effective
method due to the media and time. In order to get purified product at the end fermentation there
are many steps that should be carried out. First one is removing insoluble particles using such
techniques; filtration, centrifugation , settling, sedimentation, decanting. In order to enhance
product concentration primary isolation can be conducted by solvent extraction, absorption,
precipitation and ultrafiltration. The third step is product purification. In order to get extremely
pure products the impurities must be taken out for upward product concentration for instance
chromatography and adsorption. Final product isolation and drying of the crystallized product
are the last steps.These steps can be done with drum drying, spray drying or freeze drying. These
steps should provide the wanted product in a form appropriate for final formulation,blending,
and for direct shipping.
3
Figure 1 :Upstream and Downstream Steps in Biopharmaceutical Production (Benyahia et al.,
2020).
Materials and Method
Sample Centrifuged at 12,000 rpm for 5 min, and it filtered the cell-free medium through a 0.2-
μm syringe filter. Dry weight of biomass was measured using a sensitive balance. Dry weight
was collected in 2 mL pre-dried, pre-weighed microcentrifuge tubes.Sample and was washed
twice with equal volume distilled water and dry at 100 °C.
Results
4
Figure 2 : The pellet of biomass for fed-batch fermentation / Group 1 and 2
Figure 3 : The pellet of biomass for batch fermentation / Group 1 and 2
For group 1; fed-batch biomass : 326.0 mg and batch biomass 3.2 mg

For group 2 ; fed batch biomass : 692,4 mg and batch biomass 18.8 mg
References:
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1) Kesik‐Brodacka, M. (2017). Progress in biopharmaceutical development. Biotechnology
and Applied Biochemistry, 65(3), 306–322. https://doi.org/10.1002/bab.1617
2) Kiesslich, S., & Kamen, A. A. (2020). Vero cell upstream bioprocess development for the
production of viral vectors and vaccines. Biotechnology Advances, 44, 107608.
https://doi.org/10.1016/j.biotechadv.2020.107608
3) Winkelnkemper, T., & Schembecker, G. (2010). Purification performance index and
separation cost indicator for experimentally based systematic downstream process
development. Separation and Purification Technology, 72(1), 34–39.
https://doi.org/10.1016/j.seppur.2009.12.025
4) Kosanović, M., Milutinović, B., Goč, S., Mitić, N., & Janković, M. (2017). Ion-exchange
chromatography purification of extracellular vesicles. BioTechniques, 63(2), 65–71.
https://doi.org/10.2144/000114575
5) Benyahia, B., Brumano, L. P., Pessoa, A., & Santos Da Silva, F. V. (2020).
Biopharmaceutical development, production, and quality. New and Future Developments
in Microbial Biotechnology and Bioengineering, 69–89. https://doi.org/10.1016/b978-0-
444-64301-8.00003-2

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Experiment Title

Downstream bioprocess development of P. kudriavzevii

Sevil Dinçer İşoğlu

Seray ZORA TARHAN

Fermentation, which is conducted in order to produce biopharmaceuticals, can be divided into

Materials and Method

Figure 3 : The pellet of biomass for batch fermentation / Group 1 and 2

For group 1; fed-batch biomass : 326.0 mg and batch biomass 3.2 mg

and Applied Biochemistry, 65(3), 306–322. https://doi.org/10.1002/bab.1617

production of viral vectors and vaccines. Biotechnology Advances, 44, 107608.

3) Winkelnkemper, T., & Schembecker, G. (2010). Purification performance index and

separation cost indicator for experimentally based systematic downstream process

development. Separation and Purification Technology, 72(1), 34–39.

chromatography purification of extracellular vesicles. BioTechniques, 63(2), 65–71.

Biopharmaceutical development, production, and quality. New and Future Developments

in Microbial Biotechnology and Bioengineering, 69–89. https://doi.org/10.1016/b978-0-

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