Bioprocessing Strategies for Cell-Factory Systems

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Far-reaching Effects

Bioprocessing strategies are essential to the effective translation of basic biotechnology discoveries into practical applications, and impact significantly on the economic feasibility of biomanufacturing and bioremediation operations, and the efficacy of biomedical treatments and agricultural practices. While this is not a new realization,1 rapid, ongoing advances in the life sciences — especially molecular biology and genetics — are creating a renewed interest in bioprocessing research for the development of new and improved cell-factory systems for biomanufacturing.2,3

In this brief overview, we review the current state of research dealing with the major bioprocessing concerns in biomanufacturing. These concerns are illustrated with examples from research activities carried out at the Canadian Cell-Factory Bioprocessing Research Network at the University of Waterloo (UW) (Waterloo, ON).4
Figure 1 illustrates the three essential stages of a typical biomanufacturing process: upstream, midstream and downstream bioprocessing operations. In turn, these reflect the relevance of three corresponding systems for cellular expression, bioreactor and product purification as discussed below. The multidisciplinary tools are based on genetics, molecular biology, immunology, microbiology, biochemistry, chemical engineering, mechanical engineering.

Upstream Bioprocessing: Expression Systems
The Escherichia coli (E. coli) bacterial host system for recombinant protein production is currently acknowledged as the cell-factory workhorse. Using penicillin acylase (PAC) with a unique protein formation mechanism (Figure 2) as a model protein, genetic strategies for the construction of E. coli strains for high-level gene expression can be targeted on the basis of enhancing transcriptional/translational efficiency and “balancing” protein synthesis flux throughout the protein formation pathway.5, 6 An important area of concern is in vivo protein misfolding by which the aggregated periplasmic PAC precursors tend to hinder the formation of active PAC and even cause physiological stresses upon gene overexpression, and the problems can be alleviated by protease and/or chaperone functions.7, 8

Recently, there has been a growing interest in the yeast Pichia pastoris as a host system. Compared to E coli, this yeast has the potential advantages of extracellularly secreting gene products and having a post-translational processing mechanism.9 In our case, we are exploring its potential for producing the enzyme lipase for the production of biodiesel from oil feedstocks. Compared to current chemical technology, this transesterification route offers benefits for more specificity and relatively mild reaction conditions. These studies demonstrate that developing proper host/vector systems via genetic manipulation plays a critical role for effective biomanufacturing.

Midstream Bioprocessing: Bioreactor Systems
Although bacteria and mammalian cells are the hosts of choice for cell factories, fungal systems still account for a significant portion of economic returns in the biomanufacturing marketplace. These systems are the best antibiotic and bulk enzyme producers.1 Recently, our team has developed a bioprocessing strategy for improved simulation of the physico-chemical characteristics of fungal filamentous fermentation broths that allow the convenient evaluation of oxygen transfer requirements in the aeration-agitation operation of bioreactor systems.

These requirements are often of overriding importance to biomanufacturing economics. This strategy has been successfully tested for the production of the drug cyclosporin, which must overcome the complex rheological constraints of the fermentation broth.10, 11
A major advance in bioprocessing was made with the application of fed-batch protocols for bioreactor operations. These generic strategies can enhance biomanufacturing productivity several fold. We have developed protocols based on readily manageable operations such as dissolved oxygen monitoring and automatic control.12, 13 Medium composition optimization is also of increasing concern, driven by the current regulations related to restrictions on the use of components from animal sources. Recipes for replacements with plant-derived sources can be effectively developed with the aid of factorial designs and statistical analyses.14

Downstream Bioprocessing: Purification Systems
The high levels of product purity required for therapeutic products warrants innovative downstream bioprocessing strategies. For example, in one case study of a pre-clinical trial product, our team developed a series of steps to ensure a stable low-endotoxin purified product as illustrated in Figure 3. The protein is expressed as a GST fusion. Release and purification of the target protein moiety are simultaneously conducted when the fusion protein adsorbs on the GST affinity chromatography column. The high-quality protein product that was produced has recently been verified to be active in several in vitro and in vivo biological assays. The study not only demonstrates the impact of host/vector construction on subsequent midstream and downstream processing, but also the close linkage among these bioprocess developing stages.

Overall Bioprocessing:
Integrated Systems
Although aspects of each of the three essential cell factory bioprocessing stages have been discussed separately, it is evident that eventually any changes in a given element impact the other two (Figure 1). Also, a particular stream could become relatively unimportant, e.g., upstream as in biomedical or agricultural applications. Successful development of bioprocessing for biomanufacturing relies on an integrative approach on all the stages. Therefore, it is important to include the bioprocessing tools (often computer-aided) in a tool kit for the integration and optimization of a total cell-factory system for economic sensitivity analyses, which indicate commercial feasibility or identify areas for further research.2, 15

Conclusion:
The recent surge in the need for bioprocessing expertise in industrial biotechnology is reflected in the want ads of journals and magazines.16 Unfortunately, most universities do not have the critical mass of multidisciplinary resources to adequately meet the current challenges. Pooling facilities and expertise between universities, as exemplified by the Canadian Cell-Factory Bioprocessing Research Network,4 is a fairly uncommon scenario, unique to Canada.

Compared to the complementary areas of the life sciences, the bioprocessing arena is not expected to produce spectacular new discoveries. Rather, the development of improved tools that meet the challenges of industrial biotechnology is expected to be the norm for this increasingly important industrial sector in terms of corporate wealth creation and job employment generation in OECD countries.3 Our research group at UW is aimed in this direction.

References:
(1)     Moo-Young, M. Comprehensive Biotechnology: The Principles, Applications and Regulations of Biotechnology in Industry, Agriculture and Medicine. Pergamon/Elsevier, 1985.
(2)     BIO.COM newsletter, May 17, 2006.
(3)     Aldridge, S. "Biomanufacturing faces new set of challenges." GEN 24, 1 (2004).
(4)     The Canadian Cell-Factory Bioprocessing Research Network (Cellnet; www.cellnet.ca).
(5)     Chou C. P., C.C. Yu, J.H. Tseng, M. I. Lin, H.K. Lin. "Genetic manipulation to identify limiting steps and develop strategies for high-level expression of penicillin acylase in Escherichia coli." Biotechnol Bioeng 63 (1999): 263-72.
(6)     Lin W.J., S.W. Huang, C.P. Chou. "DegP coexpression minimizes inclusion body formation upon overproduction of recombinant penicillin acylase in Escherichia coli." Biotechnol Bioeng 73 (2001): 484-92.
(7)     Pan K.L., H. C. Hsiao, C. L. Weng, M.S. Wu, C. P. Chou. "Roles of DegP in prevention of protein misfolding in the periplasm upon overexpression of penicillin acylase in Escherichia coli." J Bacteriol 185 (2003): 3020-30.
(8)     Xu Y., C. L. Weng, N. Narayanan, M. Y. Hsieh, W. A. Anderson, Scharer J. M, et al. "Chaperone-mediated folding and maturation of penicillin acylase precursor in the cytoplasm of Escherichia coli." Appl Environ Microbiol 71 (2005): 6247-53.
(9)     Higgins D. R., J. M. Cregg, ed. Pichia Protocols. New Jersey: Humana Press, 1998.
(10)     Benchapattarapong N., W. A. Anderson, F. Bai, M. Moo-Young. "Rheology and hydrodynamic properties of Tolypocladium inflatum fermentation broth and its simulation." Bioprocess Biosys Eng 27 (2005): 239-247.
(11)     Wang L. P., D. Ridgway, T. Y. Gu, M. Moo-Young. "Bioprocessing strategies to improve heterologous protein production in filamentous fungal fermentations." Biotechnol Adv 23 (2005): 115-29.
(12)     Huang H. J., D. Ridgway, T. Y. Gu, M. Moo-Young. "Enhanced amylase production by Bacillus subtilis using a dual exponential feeding strategy." Bioprocess Biosys Eng 27 (2004): 63-9.
(13)     Skolpap W., J. M. Scharer, P. L. Douglas, M. Moo-Young. "Fed-batch optimization of á-amylase and protease-producing Bacillus subtilis using Markov chain methods." Biotechnol Bioeng 86 (2004): 706-17.
(14)     Gheshlaghi R., J. M. Scharer, M. Moo-Young, P. L. Douglas. "Medium optimization for hen egg white lysozyme production by recombinant Aspergillus niger using statistical methods." Biotechnol Bioeng 90 (2005): 754-60.
(15)     Superpro Designer: Intelligen Inc. (www.intelligen.com).
(16)     GEN 2005, 25, Jan. 1,

Murray Moo Young, PhD, is a professor emeritus at the UW, director of the Canadian Cell-Factory Bioprocessing Research Network, and president of the International Society for Environmental Biotechnology.

C. Perry Chou, PhD is an associate professor at UW, and Canada Research Chair in Novel Strategies for High-Level Recombinant Protein Production.