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Australasian Biotechnology, Vol. 11 No. 2, 2001, pp. 35-36 BIOPROCESSING PERSPECTIVES ON BIOPROCESSING II Michael Zachariou Code Number: au01029 Like Martin, my perspective on bioprocessing is broad and has largely been shaped by my career experiences within the biopharmaceutical industries of Australia and the US, as well as my academic experiences. It is also an area that we are focusing on within the newly formed Fermentation and Bioprocessing Group (FBG) at CSIRO Molecular Science in Clayton. When downstream processing (DSP) scientists have found it difficult to isolate a target molecule, such as a particular chiral compound, or when organic chemists need a multitude of steps to synthesize a compound, they have sometimes turned to a form of bioprocessing termed biotransformation. In many ways, biotransformation has been touched upon in Martin’s perspective but I would like to expand on that to include an area that we are investigating in the FBG. One area of biotransformation we have embarked upon is the use of microbes to synthesize novel building blocks for use in synthetic organic chemistry to use. In this instance, a microbe can be viewed as a first rate synthetic organic chemist which can, for example, convert an aniline to a cis-1,2-dihydrocatechol, using an dioxygenase enzyme as catalyst. Since it can produce such a product in >99.8% enantiomeric excess and at levels of 35g/L over a 48 hour period, relatively insignificant levels of DSP and no synthetic organic chemist are required. The dihydrocatechol can be used to make a variety of compounds that otherwise would have been difficult to make synthetically. With the world’s supply of petrochemicals anticipated to become severely depleted within the next 30 to 40 years, the chemical industry is looking for new feedstocks for making its chemicals. It’s also looking for more efficient and environmentally friendly approaches to its synthetic capabilities. An example of this new direction is the announcement recently by the Cargill-Dow Chemical Bioprocessing joint venture, to build a $300-million plant in the US for the production of polylactide polymers from a fermentation that uses corn sugar feedstocks. Furthermore, with an ethanol producing plant of capacities at 260,000L per day from Escherichia coli being built in the US, using sugar cane waste and rice hulls for feedstocks, ethanol is back on the table as a viable alternative fuel. All these social issues can be investigated from the perspective of bioprocessing. The FBG at CSIRO Molecular Science will be spending a significant amount of its research time addressing these aspects of bioprocessing. I could also mention the area of chiral synthons as precursors for important drug manufacturing by the pharmaceutical industry but an overview of how biotransformation (bioprocessing) can be used to assist in this area is given in the second article on biotransformation. I did my PhD within the Center for Bioprocessing Technology at Monash University where a main focus of study was understanding and developing novel separation tools. Within that environment the definition of bioprocessing was clearly the processing of biopolymers and in particular proteins. Together with Professor Milton Hearn we developed a new mode of protein purification using immobilized metal ion affinity chromatography (IMAC), that was not based on the traditional histidine interaction but on aspartate and glutamate participation. The processing of biological materials is as varied as the types of biological materials, which would include not only biopolymers such as proteins, polysaccharides and polynucleotides but also chemicals derived from biological systems such as sugars, fatty acids, oils etc. The isolation or purification of biological materials is necessary to further understand the nature of the molecule structurally and functionally. For industries such as the pharmaceutical industry it is also a regulatory requirement so as to meet safety standards. For the diagnostic kits industry it is often a market demand to ensure optimal performance for reactions such as PCR. For the chemical industry it is the perusal of the biological arena for enzymes that will make one form of a racemic mixture in preference over another, so as to avoid lengthy and costly separation processes such as simulated moving beds. These industries are all willing to invest heavily in developing economical, optimized, robust and reproducible processes for isolating biological materials. I will use the pharmaceutical industry as my example of what are the various stages normally involved in developing a bioprocess (also termed downstream process (DSP)). There is quite often dispute between the fermentation scientist and the DSP scientist as to where the fermentation ends and where the DSP begins, even though from a bioprocessing perspective it can be viewed as a continuum. As a DSP scientist, I will begin the downstream process from cell separation. Generally, the target molecule is made in the cell and either secreted or maintained within the cell. In either case the cells need to be separated from the media in which they were grown. Processing using continuous flow centrifugation in combination with filtration is commonly used for this purpose within the pharmaceutical industry. A significant advance has been the introduction of fluidized/expanded beds that allow the simultaneous separation of cells and capture of target molecules. The main stage after cell or cell-debris removal involves a processing step sometimes referred to as de-watering so as to effectively begin to manage the large volumes of harvest. The type of processing involved here can be by membrane filtration, precipitation or chromatography. A very effective way of achieving de-watering whilst also obtaining some degree of purification is chromatography. With the advent of the giga-porous stationary phases such as the Poros™ and Hyper D™ supports, or the ceramic based hydroxylapatite support from Bio-Rad, that maintain good dynamic capacities at linear velocities as high as several thousand cm/hr, the processing of large volumes for de-watering purposes became passé Other stationary supports from Amersham-Pharmacia such as the Streamline series used in fluidized/expanded beds and the Lichrosphere supports from EM Merck have all contributed to rapid de-watering steps. The next stages of processing all involve a variety of steps, generally chromatographic, that aim to purify the target molecule to levels that demonstrate host protein background, DNA and viral clearance, whilst maintaining target-molecule integrity and activity. These goals have to be achieved within the constraints of an economical process. Along with the appropriate validation required, the development of such a process becomes time-consuming and contributes significantly to the 5-10 year development time required for a human therapeutic to get to market. Development of new tools that will accelerate the DSP are always in demand by the pharmaceutical industry as well as other industries such as the food, dairy, chemical, diagnostic and many other industries. It is an area that forms part of the backbone of many product developments but unfortunately is only an afterthought at the initial stages of product development. The FBG at CSIRO Molecular Science will focus some of its research toward the development of new tools that will assist in this type of bioprocessing and in particular in the areas of affinity chromatography. We believe that this area is not only a useful producer of tools for DSP and consequently product development, but can also be useful in the upcoming area of proteomics. With the human genome mapped out, last year the key question on everyone’s mind is, so what do all those genes do? The proteins that these genes code for will need to be isolated and their function determined. The Chairman of Incyte Genomics, Dr. Randal Scott, a few weeks ago said “Proteins are the core source of valuable data for disease understanding and drug development.’’ Isolation techniques will therefore play a pivotal role in the area of proteomics and drug development. Instruments such as the Protein Chip from Ciphergen whose function is to identify disease markers utilizing separations technologies at its core, are already on the market in the US and are just being introduced into Australia. The processing of biological information such as genomic data bases and protein data bases, an area known as bioinformatics, can also be viewed as a type of bioprocessing. In conclusion, bioprocessing has many definitions of which I hope to have given a fair perspective.
Copyright 2001 - AusBiotech |
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