Overview of Biotechnology at the End of the 20th Century

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.