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Karlsruhe Institute of Technology

Institute of Engineering in Life Sciences

Section IV: Biomolecular Separation Engineering

Fritz-Haber-Weg 2

76131 Karlsruhe

Tel: +49 721 608 42557
Fax: +49 721 608 46240

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Experimental Process Development

 HTPD

The biopharmaceutical production industry is based on a vast number of complex process setups which all rely high experimental effort. A high amount of process knowledge and optimization helps meeting the requirements stated by the authorities such as the FDA, EMA, etc. High-throughput methodologies have become a central tool in experimental process development. Using high-throughput systems (HTS) including automated robotic platforms, development times and manufacturing costs can be reduced to a minimum due to miniaturization, parallelization and automation of experiments. In our institute HTS methods are used in cell cultivation and product formation studies (upstream processing) as well as downstream application (protein purification). Robotic screenings in high-throughput format include precipitation screenings, characterization experiments for aqueous two-phase systems, as well as chromatography in batch and column mode. 


Miniaturized Upstream Processing

 BioLector

The development of a successful biopharmaceutical upstream process is dependent on a large number of variables. The expression host, the expression vector, the inserted gene of interest as well as the cultivation conditions need to be scored and selected for maximizing the overall product concentration. For example the temperature, oxygen supply or the induction conditions can influence whether the product is formed at all or if it is produced in an insoluble form as inclusion bodies. To design upstream processes for the optimal product formation, high-throughput cultivations help exploring the experimental design space. We are using the BioLector system by m2p-Labs for 48-well cultivations in µL-Format for designing upstream processes for bacterial and yeast cells. Optimized cultivations using E. coli cells yielded up to 50 % of product of the total soluble protein fraction.

 

 

 

 

References

P. Baumann, T. Hahn, J. Hubbuch, High-throughput Micro-scale Cultivations and Chromatography Modeling: Powerful Tools for Integrated Process Development, Biotechnol. Bioeng. (2015), DOI: 10.1002/bit.25630.

C. Ladd Effio, P. Baumann, P. Vormittag, C. Weigel, A. Middelberg, J. Hubbuch, Establishment of a High-throughput Platform for the Production of Virus-like Particles in E. coli, J. Biotechnol. (2015), DOI: 10.1016/j.jbiotec.2015.12.018.

P. Baumann, N. Bluthardt, S. Renner, H. Burghardt, A. Osberghaus, J. Hubbuch, Integrated development of up- and downstream processes supported by the Cherry-Tag for real-time tracking of stability and solubility of proteins, J. Biotechnol. (2015), DOI: 10.1016/j.jbiotec.2015.02.024.


Aqueous Two Phase Systems (ATPS)

ATPS

ATPS represent a gentle way of biomolecule purification. Good scalablility, low cost and the possible integration of concentrating and purifying the target molecule within one process step are some of the advantages this technique can have over chromatography. Using a robotic platform, different ATPSs can be screened for their phase diagrams, phase volumes and distribution coefficient of biomolecules. Determining these values is critical for the evaluation of ATPS as a possible process step. The subsequent empirical process of multi parameter optimization can be done using a genetic algorithm. In studies with avidin from chicken egg white a purification factor of 5.7 with an avidin yield of 92% could be reached using an PEG/phosphate aqueous two-phase system, by using high-throughput techniques.

 

 

 

References

S. A. Oelmeier, F. Dismer, J. Hubbuch, Application of an aqueous two-phase systems high-throughput screening method to evaluate mAb HCP separation, Biotechnology and Bioengineering (2010), DOI: 10.1002/bit.22900.

P. Diederich, S. Amrhein, F. Hämmerling, J. Hubbuch, Evaluation of PEG/phosphate aqueous two-phase systems for the purification of the chicken egg white protein avidin by using high-throughput techniques, Chemical Engineering Science (2013), DOI: 10.1016/j.ces.2013.10.008.

C. Ladd Effio, L. Wenger, O. Ötes, S. A. Oelmeier, R. Kneusel, J. Hubbuch, Downstream processing of virus-like particles: Single-stage and multi-stage aqueous two-phase extraction, Journal of Chromatography A (2015), DOI: 10.1016/j.chroma.2015.01.007.


Precipitation

Loeslichkeit

Precipitation of biomolecules is an alternative widely used column based purification steps in the biopharmaceutical industry. The preparative purification of biomolecules by precipitation is widely acknowledged as simple, cost efficient and easily scalable process step. The precipitation of proteins could either be used as initial capture step in the beginning of the purification or in the end for polishing or concentration of the target product. For process development plenty of parameters, like type and concentration of the precipitant as well as kinetic parameters could be measured and optimized. For the purity and yield of the product the resolubilization condition of the precipitated product becomes important. We optimize these process parameters for proteins like antibodies, virus like particles or inactivated viruses with the help of robot-assisted high throughput experiments in 96-well scale.  For Antibodies an HCP reduction of 93% could and product recovery of 93% in the same time could be reached.