Biotechnology at the End of the 20th Century

Modern biotechnology began when recombinant human insulin was first marketed in the United States in 1982. The effort leading up to this landmark event began in the early 1970's when research scientists developed protocols to construct vectors, by cutting out and pasting pieces of DNA together to create a new piece of DNA (recombinant DNA), that could be inserted into the bacterium, Escherichia coli (transformation). If one of the pieces of the new DNA included a gene which produced a protein enzyme that broke down a particular antibiotic, the bacterium would be resistant to that antibiotic and could grow in a medium containing it. To the piece of DNA that conferred resistance of Escherichia coli to a particular antibiotic was added the human gene for the making of insulin. If this recombinant DNA containing the human insulin gene was used to transform Escherichia coli,and the bacteria were plated on an agar plate containing the antibiotic, the bacteria that grew contained not only the antibiotic resistant gene but also the insulin gene. Additional new pieces of DNA were then added to promote the expression of the human insulin gene so that this new recombinant DNA (expression vector) could be used to transform Escherichia coli. Thus, large quantities of human insulin messenger RNA were formed, which in turn were translated into large quantities of the human insulin protein. This story of the beginning of modern biotechnology represents the research piece of modern biotechnology. Biotechnology Research is the topic of BT210 (Biotechnology Experience I: Research) which together with BT220 (Biotechnology Experience II: Manufacturing), comprises the Certificate in Biotechnology at NHCTC.

The next step in the development of modern biotechnology methods of protein production is what is now called process development. During process development the best growth conditions are identified that produce the most protein, as efficiently as possible. This best process is scaled-up to produce the quantities of human protein that are needed for pre-clinical and clinical trials and for manufacture. Process development also includes the development of media, buffers, reagents, solutions, and assays and the choice of tools, such as bioreactors and liquid chromatography equipment, for the growth of recombinant cells (upstream processing), for the isolation and purification of the recombinant protein (downstream processing), and for tests to insure that both the upstream and downstream processes are proceeding in a predictable manner (quality control). Toward the end of process development a master cell bank is laid down. The master cell bank is sized to last as long as the manufacture of the product will take place. Ordinarily the master cell bank is a large quantity of vials each containing 1ml of media within which there are about 1,000,000 recombinant cells (1,000,000 cells/ml) which are stored frozen in liquid nitrogen.

From process development, one proceeds to manufacture which starts with defrosting a vial from the master cell bank and adding it to a small amount of medium prepared in the media and buffer preparation division of the manufacturing facility. The cells are then grown under the conditions determined during process development and tested at the manufacturing facility during process validation. When the cells reach certain predetermined conditions, (for instance when the cells are in a particular place on the log phase of their growth curve, determined through OD readings using a spectrophotometer, and live cell counts using a microscope), they are transferred into a larger volume of growth medium. This process repeats itself (scale-up) until the final reactor volume is reached. In New England at this time the final reactor volume for human therapeutic proteins is typically 2500 to 5000 liters. This is upstream processing.

Following upstream processing, the cells are separated from the media in which they are growing and the protein is isolated from the cells or the media by a combination of techniques that include filtration, chromatography, and concentration. This process is termed downstream processing. The protein characteristics and purity must conform to certain conditions determined during process development and tested at the manufacturing facility during process validation. Upstream and downstream processing is monitored by the quality control division of the manufacturing facility. Quality control also handles environmental monitoring during production of the protein. The quality assurance division handles all the paper work generated by the various divisions of the manufacturing facility, which operates in compliance with current Good Manufacturing Practices determined by the Food and Drug Administration (FDA).

Of course once the protein is manufactured it must be formulated. Here excipients must be added to the purified protein to modify its activity or its storage qualities, for instance. If the protein is a therapeutic protein or a vaccine, after excipients are added the formulated preparation is filled into glass ampules, lyophilized, sealed, and labeled. This process known as formulate and fill is also regulated by the FDA under its cGMP regulations.

BT220 focuses on modern biotechnology manufacturing: on media, buffer, reagent, and solution preparation; and on the choice of tools for cell culture (upstream processing), for isolation and purification of proteins (downstream processing), and for testing to insure that a quality product is being manufactured and produced (quality control). In BT220 three types of cells are cultured, representing three of the Kingdoms of living things: the bacteria, Escherichia coli (Kingdom Monera); the yeast, Pichia pastoris (Kingdom Fungi); and mammalian Chinese hamster ovary (CHO) cells (Kingdom Animalia) using appropriate media and bioreactor tools that include test tubes, shake flasks, 50 and 500ml Bellco Spinner Flasks, and the 5000ml New Brunswick Bioflo 3000. Liquid chromatography and tangential flow/diafiltration are used to isolate, purify, and concentrate the protein produced from these cells. All of the cells contain expression vectors that carry a human gene for either tissue plasminogen activator (tPA) or human serum albumin (HSA). During cell culture (upstream processing), and protein isolation, purification, and concentration (downstream processing), tests will be made to insure that upstream and downstream processing are proceeding according to predetermined conditions (quality control).

Since the manufacture of human insulin using recombinant Escherichia coli began in 1982, many other proteins (for human and veterinary therapeutics, vaccines and diagnostics) are being manufactured. Today, 24 human therapeutic or vaccine proteins made by modern biotechnology methods have been approved by the FDA for marketing. This is the list and date of approval by the FDA: Actimmune (1990), Activase (this is human, recombinant tPA) (1990), Alferon N (1989), Betaseron (1993), Cerezyme (1994), Engerix-B (1989), EPOGEN (1993), PROCRIT (1993), Humatrope (1987), Humulin (1987), Intron A (1986-1992), KoGENate (1993), Leukine (1991), NEUPOGEN (1994), Nutropin (1994), OncoScint (1992), ORTHOCLONE (1993), Proleukin (1992), Protropin (1985), Pulmozyme (1993), RECOMBINATE (1992) RECOMBIVAX HB (1986), ReoPro (1994), Roferon-A (1986-1988) There are more than 200 other human therapeutic and vaccine proteins in clinical trials. Products are being tested to target the following diseases: cancer, AIDS, heart disease, multiple sclerosis, rheumatoid arthritis and viral diseases. Products are also being developed to reduce bleeding from surgical procedures, aid in wound healing and prevent organ transplant rejection. Vaccines are also being developed to prevent Lyme disease and herpes and against AIDS, rheumatoid arthritis, and cancer.

It is difficult to predict the future of this exciting new field of modern biotechnology using recombinant DNA to produce proteins. One direction the field seems to be moving, at least at the research level, is toward the synthesis of smaller molecules that are closer to organic chemicals than biochemicals, but contain the same or better activity compared to the native protein.

The following is an overview of modern bioprocessing:

Sonia Wallman, NHCTC. 1997