Bioprocessing, Microorganisms and Enzymes: From Discovery To Industrial Translation

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By Denis Groleau

Introduction

When I graduated from the Université Laval in 1973 with a BSc (Microbiology/Biochemistry), the hottest scientific news in the field was the famous enzyme reverse transcriptase. Later, I learnt that the first genetic engineering paper was published during the 1972-73 period. The word ‘biotechnology’ was still unknown. Then came the wave of recombinant proteins and of the first commercial therapeutic proteins/peptides produced via genetic engineering. Other waves followed: protein engineering, genomics, proteomics, other –omics, metabolic engineering and systems biology. Something else also materialized during the same period: bioprocessing as an ingredient of biotechnology.

Today, it is generally recognized that the discovery of antibiotics gave birth to biochemical engineering. Along the same line, one may say that genetic engineering or modern biotechnology gave gradual birth to a discipline today called bioprocessing.

Bioprocessing is somewhat different from biochemical engineering in the sense that it places more emphasis on the ‘bio’ component of the process i.e. the biocatalyst, be it an enzyme, a microorganism or a more advanced cellular system. Having a ‘high performance’ biocatalyst is the most important factor for success in bioprocessing. With the ‘right biocatalyst’, significantly less time and efforts will need to be spent on bioprocess optimization.

Bioprocessing may be defined as the mixture of science, technology and of some ‘art’ that is needed to convert lab discoveries into promising bioprocesses and bioproducts.

Depending on the field of application, various challenges need to be met: regulatory, environmental, scalability, robustness, competitiveness, etc. Many of the failures of modern biotechnology can be traced back to lack of attention paid to bioprocessing aspects. In a few words, bioprocessing ‘makes things happen’.

NRC mandate and involvement in bioprocessing

In 1983, I was in the room at the National Research Council when the first Biotechnology Strategy for Canada and the creation of the NRC Biotechnology Research Institute (NRC-BRI) were announced.

The ‘thinkers’ behind the Biotechnology Strategy had already concluded that bioprocessing, then called biochemical engineering, is an important element of biotechnology. NRC-BRI’s original plan included, therefore, a Biochemical Engineering R&D program and a major Fermentation Pilot Plant facility. The focus, then, was on natural resource-linked processes and products. The Biochemical Engineering R&D program has become today a Bioprocess R&D program supervised by the Bioprocess Center, comprised of roughly 80 people active in various branches of bioprocessing, from molecular biology to enzymology, microbiology, insect and animal cell technology, viral vector development and production, bioprocess scale-up and intensification, process control, product recovery and purification. As it can be assumed, pilot scale work is at the core of our activities and these activities make us rather unique in Canada and in North America, especially in the public domain.

NRC-BRI’s bioprocessing expertise and facilities cover both the ‘R’ and ‘D’ sides of R&D. Flexibility and versatility are two important values.

In the last 20 years, NRC-BRI’s bioprocessing facilities have served numerous Canadian companies through contractual and collaborative agreements; they are regularly used as magnet to attract major foreign investment to Canada; they have been used as a model to design and build similar bioprocessing facilities and finally they have served as training ground for an important number of industrial guest workers, graduate students and Canadian/non-Canadian trainees of all kinds. Although the NRC-BRI offers mostly hands-on training, two types of formal training in bioprocessing have been designed and offered in the past.

NRC-BRI’s bioprocessing experts are also regularly invited to act as industrial consultants or reviewers of major biotech initiatives.

Bioprocessing: From discovery to industrial translation

Converting promising discoveries into commercial products and processes (industrial translation) is very risky, difficult, costly and time-consuming. In recent years, this has been recognized as one of Canada’s main weaknesses in the high tech area, especially in biotechnology.

Moving from discovery to industrial translation represents a continuum of activities. Nevertheless, these activities may be separated into three major blocks: Discovery; Development; and Commercial production. Bioprocessing can contribute to each one of these three blocks of activities but its importance naturally increases along the commercialization chain. At NRC-BRI, the Microbial and Enzymatic Technology Group has contributed to the three areas.

In the Discovery area, the Group carries out original and strategic research related to bioprocessing and supports both NRC-BRI research and industrial research through production and purification of various and precious research reagents (examples: peptides, recombinant proteins, therapeutic or process enzymes, pigments, polymers, antibodies, etc.). In terms of original research output, for instance, the Group has performed research on microbial strain development and optimization for recombinant protein production, on polymer production by fermentation, including design and production of functionalized polymers, on the design and development of enzymatic bioprocesses run under synthetic conditions, on the monitoring of fermentation processes and, finally, on a better understanding of the basis for hydrophobic interaction chromatography.

In the Development area, most of the Group’s activities touch the development, optimization and scale-up of bioprocesses for the production and purification of biotech products, using either microbial cells or enzymes as biocatalysts. The Group also possesses the expertise and facilities for producing and purifying GLP-grade (bio)pharmaceuticals in order to support pre-clinical research. Our Microbial Fermentation Pilot Plant has fermentors of various volumes, up to the 1500L-scale, and the Group also maintains a modern pilot-scale laboratory for the extensive purification of proteins/peptides and of similar products. This pilot-scale laboratory is rather unique in Canada. The availability of such pilot-scale infrastructures is of great strategic importance for the Canadian biotech industry as it permits to have access to significant quantities of materials for supporting commercialization efforts while reducing time-to-market and production costs. Readily access to pilot-scale bioprocessing infrastructures and expertise saves at least 18-24 months of developmental time, in my opinion, by comparison to designing, building and operating its own bioprocessing facility, not including the cost of recruiting and training the necessary personnel.

Finally, in the Commercial Production area, the Group cannot contribute directly since NRC’s mandate totally excludes commercial manufacturing. However, the Group contributes indirectly via development and testing of production bioprocesses of a ‘semi-industrial’ nature and via personnel training, consulting activities and provision of engineering data. Examples of such activities are: development of a bioprocess for antibody fragment prodution by E. coli, from sub-cloning to pilot-scale testing, within an eight to nine month period, before successful translation to commercial production; development and scale-up of a fermentation process for the production of a new microbial insecticide, approved later by the US EPA; process development and production of several kilos of dried microbial biomass containing pigments for experimental testing on fish; contributions to the development and scale-up of a fermentation process for the production of a biodegradable plastic together with incubation of the emerging company on NRC-BRI’s premises; repeated production of plasmid DNA at large-scale to support gene therapy research. Whatever the project, the Group tries to develop bioprocesses that will fit with the particularities of the expected manufacturing location.

A few scientific/technological accomplishments

The Group can claim several scientific/technical accomplishments over the years, such as: the development of a proprietary prokaryotic expression system, based on the methylotrophic bacterium Methylobacterium extorquens, for applications in the biopharma and bioproduct fields; the development of an alternative regulated expression system, based on the cumate switch, for universal application in E. coli and in M. extorquens; the comprehensive understanding of the behavior of various hydrolases under different sets of operating conditions, from aqueous conditions to non-aqueous conditions, mono- versus multi-phase systems; expertise and infrastructure for the safe handling of methanol-utilizing microbial expression systems (P. pastoris, M. extorquens) up to pilot-scale; the development of an online sensor for monitoring methanol and other volatile compounds in fermentors; the development of a process for polishing commercial lactoferrin to yield ultra-pure lactoferrin using a little-used mode of flowthrough chromatography. These accomplishments combined with the experience and knowledge gained while working on industrial projects and on other projects of various kinds make the Group an ideal partner for both scientific interactions and industrial translation of promising discoveries.

Our top priority objectives

The Group will continue to develop bioprocessing tools and technologies of a generic nature that may be applied to various fields of biotechnology. However, a shift towards industrial biotechnology (bioproducts from biomass), also called ‘white biotechnology’, has already begun. Bioprocessing R&D is of utmost importance in the bioproducts arena due to an absolute need for very efficient biocatalysts, cost-effective production, low environmental footprint, robust and resilient bioprocesses able to sustain process disturbances, low energy consumption and overall sustainability. To meet these challenges, the Group will: develop a core expertise in metabolic engineering in order to develop efficient and robust microbial cell factories starting with microorganisms oxidizing methanol and/or methane, two important non-food substrates; increase the Group’s expertise in process engineering; set-up a downstream processing (DSP) laboratory dedicated to bioproducts and develop more expertise in the area; upgrade our pilot-scale infrastructure to make it a 21sth century facility; develop bioprocesses and bioproducts of potentially very high impact.

Conclusion

The economic and societal benefits of research of the discovery type come mostly from the commercialization or application of the discoveries. In the case of biotechnology, bioprocessing R&D and bioprocess development must become essential elements of Canada’s strategy aiming at increasing the commercialization of Canadian discoveries. Canadian biotech companies must become convinced that investing in bioprocess R&D and bioprocess development is money very well spent. This will be even more so in the emerging bioproducts sector.

Whether biotechnology is red (health), green (environment) or white (industrial bioproducts), bioprocessing should be part of the action plan.

Denis Groleau, PhD (Microbiology) is a Senior Research Officer and the Group Leader of the, Microbial and Enzymatic Technology Group of the Bioprocess Center of the National Research Council Biotechnology Research Institute (NRC-BRI), in Montréal, QC