Sonia Wallman PhD

New Hampshire Community Technical College

On-line resources for Bioprocessing

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 this course, 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.

In this course we will focus 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). We will culture three types of cells 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. We will also be using liquid chromatography and tangential flow/diafiltration 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 (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 student should realize that the education and training obtained in this course could be used in a number of fields including bioprocessing without the use of recombinant DNA and chemical processing. This is known as cross-training.

The following is an overview of modern bioprocessing:

Sonia Wallman, NHCTC. 1997

On-line Bioprocessing/fermentation resources

Compiled by Bio-Link Staff

EDUCATIONAL RESOURCES

New Hampshire Biotechnology Center

The New Hampshire Biotechnology Center provides two-year college programs to support the biomedical/biotechnology industry.  The Center's web-site contains four on-line Industry oriented courses.  Explorations is an introduction to the industry; Experience I reviews the state-of-the-art tools of biotechnology; Experience II focuses on the skills and know-how of a biotechnology technician in an atmosphere that simulates the biotechnology workplace; Bioethics discusses the major issues (ethical, legal and societal) that plague biotechnology today.

Industrial Microbiology
Skyline Community College has a two-year associate degree in Biotechnology.  The Industrial Microbiology course covers processes and techniques of industrial fermentation.  Topics include large-scale bioreactors and the importance of regulation and quality control.

Biotech Drugs via Fermentation
This is a short discussion and lots of good links compiled by the North Valley and Mountain Biotech Center.

Of Apples and Animals; An Introduction to Biotechnology
These are some lesson plans from the Ohio State University Extension Research.  They are designed as simple science activities for fourth to fifth grade.  It discusses Biotechnology and fermentation processes with an emphasis on food, agricultural and the environment.

ON-LINE COURSES

These are University level engineering courses in Bioprocessing and are well worth investigating for presentation of material and content.

Nam Sun Wang's ENCH482 & ENCH648B Biochemical Engineering
The Biochemical Engineering Department of the University of Maryland posts these courses for its students.  Theses are amazing sites; the entire course is on-line and includes the syllabus, notes, lab exercises and quizzes.  They are very detailed and contain lots of links.  The course covers the biological basis of fermentation, but the emphasis is on engineering and mathematical principles. 

Biochemical Engineering Course at Rensselar Polytechnical Institute
Rensselaer Polytechnic Institute has posted on-line its course outline and notes for Biochemical Engineering.  These courses cover engineering aspects of microbial processes.  Topics include cell culture, sterilization, aseptic techniques, bioprocess and engineering controls, and downstream processing.  Because this is a course for engineers who are learning industrial microbiology, it provides a different way of looking at biological systems.

Introduction to Biosystems Engineering
The University of Kentucky has an Industrial Bio engineering course on-line. This course is very agriculture based and is concerned with food and fiber production.  Included on the web site are the syllabus, lecture notes and some problems sets.

REGULATIONS, REPORTS and RESOURCES

Biotechnology for the 21st Century: New Horizons
This is a Report from the Biotechnology Research Subcommittee, Committee on Fundamental Science and the National Science and Technology Council July 1995.  This report by the federal government discusses the importance of industrial biotechnology development.  It outlines some of the government-funded projects and is a good review of the future of industrial biotechnology. 

Large Scale Biosafety Guidelines
This is a draft safety document compiled by ASM (American Society for Microbiology).   Although there are guidelines for working with large-scale recombinant microorganisms, there are at this time no specific biosafety guidelines that have been established for large-scale work with organisms that do not contain recombinant molecules or organism.  This document has lots of information that should be considered before and during work with large-scale fermentations. 

Sartorius Biotechnology
On this page is an interactive process chain diagram.  Although the links on the diagram are related to products sold by the company, it is a good example of a flow chart for a bio-system. 

Biotechnology
This web site is an "Information Resource" from the National Agricultural Library of the US Department of Agriculture.  It is no longer updated, but most of the links included are still active.  It is a list of selected sources, services and publications covering many aspects of agricultural biotechnology.  The page has no stand-alone information, but is a good resource. 

PHRMA
The Pharmaceutical Research and Manufacturers of America (PhRMA) represents the country's leading research-based pharmaceutical and biotechnology companies.  Phrma is decidedly pro-industry in their outlook.  This site has information on the drug industry and federal, state policies.  They address such public policy issues as the cost of pharmaceuticals.  The site also has information on genomics, bioinformatics and other related topics about modern biotechnology. 

The BioEnergy Home Page
This site is a resource for bioenergy, bioconversion, and bioprocess technology.  Throughout the pages compiled here there is detailed information on the conversion of organic matter to industrially useful products.  A section covers the conversion of biomass to ethanol production.  This site also contains many links to industry sites for biomass energy and renewable energy sources.

PROFESSIONAL SOCIETIES/ORGANIZATIONS

Society for In Vitro Biology (previously the Tissue Culture Association)
The Society for In Vitro Biology is a professional organization with dues paying members.  The society publishes a number of professional journals that deal with plant or animal tissue culture.  Although only paid members may take advantage of many of the benefits of this site, there is some information about workshops for educators that are sponsored by the society.  It has a history of fostering educational opportunities and is enthusiastic about emergent technologies. 

The Society for Industrial Microbiology
SIM is a professional association interested in the advancement of microbiological sciences, specifically as applied to industrial materials, processes, products and their associated problems.  Its members are composed of scientists employed in industry, government and university laboratories.  This is a membership organization and many of the benefits of the web site are limited to paid members.  They do have an open page on careers in industrial microbiology.  There are also some links to education resources although these links are of variable quality. 

Microbiology and Biotechnology Division of the American Society for Microbiology
The American society for Microbiology site is an organization for those microbiologists specializing in fermentation and large scale processes.  This site includes a newsletter for members and a page of useful links.

SUPPLIERS

Millipore Corporation
Millipore is a manufacturer of filtration devices and filters for research as well as industry.  The Biotech and Pharmaceutical section of this web site focuses on industrial applications that include downstream processing and water purification.  They include some flow charts for examples of industrial processes such as beer brewing.  Many of their products consider the need for quality control and assurance.  They also describe much of the terminology that accompanies filters and filter selection. 

NEW BRUNSWICK SCIENTIFIC CO., INC.
New Brunswick is a manufacturer of cell culture bioreactors, shakers, fermentors and other fermentation and bioprocessing equipment.  They also have an on-line technical library that is a must for anyone interested in this area.  Their technical literature includes theoretical basics and specifics about setting up cultures. 

Bellco Glass, Inc.
Bellco Glass is another supplier of fermentation and cell culture systems.

Stopford Fermentation Library Fermentation Manufacturer and Supplier
This is a list of links to Fermentation suppliers.

BIOPROCESS ENGINEERING - UNIVERSITY PROGRAMS/INSTITUTES

The mission of the Biological Process Technology Institute is to educate and train advanced students and to conduct innovative research at the interface of biology and engineering. 

Penn State Cooperative Extension runs a series of short courses in Biotechnology.  Titles include Fermentation and Bioseparation.

University of Kentucky Biosystems and Agricultural Engineering Department

The University of Arizona Agricultural and Biosystems Engineering

BIOREMEDIATION

Digital Learning Center for Microbial Ecology Microbe Zoo
The learning Center is a NSF funded Science Education Project developed by Michigan State University.  It is geared to the general public and is richly colored with numerous user-friendly illustrations.  Dirtland includes basic information on the use of microbes for Bioremediation. 

Bioremediation Resources
This is a list of resources compiled by the Biotechnology Information Center.  BIC is maintained by the National Agricultural Library of the US Department of Agriculture and provides access to selected sources, services and publications covering many aspects of agricultural biotechnology.  The site is composed of links that take you to resources both public and private.

Applications and Uses of Science: Biology
This site has links for teacher to sites that demonstrate bio-remediation.  If you need examples and want the references check here. 

Biological Process Technology Institute at the University of Minnesota, St. Paul Campus

Take a look at a Bioremediation experiment at the University of Minnesota with pictures and results.