CHBE 242

Biotechnology and Bioprocess Engineering

James Piret
Michael Smith Laboratories Chemical & Biological Engineering

Biotechnology
Using living organisms, or parts of organisms, to make or modify products, to improve microorganisms, plants or animals.
Ask for examples, includes basic research trying to understand and use mechanisms

includes

Biological Engineering
Broad-based engineering discipline that encompasses engineering applied to living systems. includes

- Biomedical Engineering
Application of engineering principles and techniques to medicine. &

- Bioprocess or Biochemical Engineering

Extension of chemical engineering principles to the design of processes that involve biological organisms or molecules.
Myself and Charles Haynes - production like chem. eng. but using enzymes or cells as catalyst also developing technologies for biological analysis - Proteomics e.g.

Biotechnology Industry Recombinant Proteins

Often using mammalian cells that provide needed post-translational modifications such as glycosylation should find images to show without and with

Monoclonal Antibodies therapy of cancer & arthritis

rheumatoid arthritis

Tissue Plasminogen Activator therapy of heart attacks & stroke

Diverse Amino Acid Structures

MSG traditional Asian cuisine supplement, isolated and patented in 1907 by Ajinomoto >1 million metric tons produced/yr

Bioprocessing Products
(Enzymes - proteins with catalytic activity) polymers of amino acids

Enzymes
Egyptian Cheese (4000 BC) Proteases
Jenner 96 cow pox

Bacteria
Amino Acids
MSG

Fungi
Sumerian Wine (6000 BC)

Animal Cells
Sumerian wine. low alcohol content, safe water, nutrients, painkiller <1900 in the West

High-fructosecorn-syrup (60s)

Vaccines (1796) Cholera (1870s) Penicillin (1928) Pasteur Antibiotics hInsulin (82) hGrowth Hormone (85) Hep. B vaccine (86) G-CSF (91)

Polio Vaccine (1949) Antibodies (75) t-PA (87) EPO (89) PhD Rituxin (97) Herceptin (98) Gene & Cell Therapy

Biofuels Unless from wastes will not influence global warming Tour de France scandals 98+ ~18 deaths linked to EPO abuse from '87 to '91

Mild Reactions Conditions

Often required for heat sensitive enzyme substrates and products (glucose, proteins)

Chemical Hydrolysis of Proteins

Protein 24 h, 6 N HCl, 100C Amino Acids
[usually 110C under vacuum]
(H3O+ is catalyst, highly corrosive, glass lined vessels)

vs. Enzymatic in minutes at 25C, pH 7

("ambient conditions)

many types of "proteases" e.g. Trypsin - narrow substrate specificity "after" arginine or lysine Papain - broad substrate specificity, cleave all peptide bonds
Still much more specific, e.g. chemicals since H3O+ would hydrolyze starch too And non-specific glutamine residue ->glutamate degradation proteins would not

Chemical NH3 process vs. N2 fixing by enzymes at 1 atm, 25C

Ask vs. Chemical NH3 process at?... 100 atm, 350C to produce fertilizer - DANGER, entire sessions at AIChE

Biodiversity of Archaea
Conversely actually biocatalysts provide many opportunities due to diversity of environments

Low temperatures Marinibacillus marinus (4C) Diversity of life in extreme environments Rich source of biocatalysts

High salt B. halophilus (2M NaCl)

Low pH B. tusciae pH 4 High temperatures (121C)

High pH B. alcalophilus pH 10

Bioethanol

Produced by fermentation of sugars derived from wheat, corn, sugar beets, sugar cane, molasses, i.e. sugar, starch or cellulose

Greenhouse gas incentives to obtain ethanol from cellulose First break down cellulose to sugars:

Then fermentation to ethanol by yeast S. cerevisiae:

C6H12O6

2 C2H5OH

+ 2 CO2

Mendels genetic research published (1866) DNA proven to store genetic information (1943)

R&D of Genetic Engineering

Transcription DNA gene mRNA Translation

Protein

DNA Double helix structure (Watson and Crick 1953) published - Replicate each strand yielding a new double helix

Genetic code translates triplets of DNA bases 1965 Nobel Prize to 20 specific amino acids in proteins (Khorana, 1952-60 BC Research) First recombinant DNA experiment (1973) First recombinant DNA product (1982, human Insulin)
1980 genetic engineering patentable, now >30 billion $/yr industry 1982 Mike Smith DNA site-directed mutagenesis 1993 Nobel Prize allows modifying proteins in desired ways

Batch Culture Growth Phases

Balanced Growth m constant

mnet = m - kd = 0 Secondary metabolites

Cell Death & Lysis

Decreasing Growth Rate

Stationary
Death
(or Decline)

[X] Viable Cell # or DCW Lag

Adaptation

Exponential
Accumulated metabolites and/or substrate nutrients limiting growth

Time
Underline contrast with most catalysts that decline over time, appear sequentially, stationary arrow last

Small Molecules (points) and Reactions (lines) of E. coli Metabolism

Point out parts will show - glycolysis, TCA, biosynthesis

Ask # genes in coli (at least 1 per line) ~1000 since lots of other functions besides metabolism

Engineers working on math modeling to improve understanding of systems biology, but bioprocess engineers generally use useful approximations, though empirical

Exponential Growth Phase

Growth rate independent of nutrient concentration, in excess
Balanced growth of cells, all components increase equally The first order exponential growth rate expression is:

dX m net X dt
Integrating,

Where (e.g. Cells/L) X=X0 @ t=0

X ln m net t X0

or

X X0 e

m net t

Substrate Limited Growth

Saturated dependence of specific growth rate (m) on substrate concentration (S), e.g. Glucose Kinetics most often described by the Monod equation

mm S
Ks S

mm - max specific growth rate

when S >> Ks Ks saturation constant

Chemostat
(Continuous Culture)
Technological advance for analysis of substrate limitation of growth A continuous stirred tank reactor (CSTR) Cellular growth typically limited by one essential nutrient; others in excess At steady state, nutrient, product and cell concentrations are constant, well mixed

Chemostat (CSTR)
A material balance on the limiting substrate:
IN OUT Cell Growth Product Formation

FS0 FS VR m g X

1 YXMS

1 dS VR q p X VR YP S dt

S0 and S - feed and effluent substrate concentrations (g/L)

qp - specific rate of extracellular product formation (g P/g cell-h) Y MX/S and YP/S - yield coefficients (g cells/g S and g P/g S)
Allows prediction of X and S dependence on the flow rate, for process modeling

Culture Production Processes

Medium Feed

Medium Feed

Batch

Fed-Batch

Perfusion

Batch for microbial processes, fed-batch for antibiotics and MAb Perfusion for cellular therapy and some protein production

Fermentation Flow Sheet

Media & Inoculum
Cell engineering Sterilization Process Models Aeration Process control Reactor design

CHBE 381 Bioprocess Engineering I

Enzymes too

Fermentation

Separations

Cell disruption Solid removal Purification Concentration

CHBE 481 Bioprocess Engineering II

CHBE 365 Biotechnology Laboratory Design, thesis, TEs

Product

Mammalian Cell Bioengineering

Since 1900 Tissue culture (>$109/year market) 1953+ Mammalian cell vaccines (>$5x109/yr)

~1980+ Recombinant proteins ($30 x109/yr) Tissue Engineering & Gene Therapy
~1990+ Stem Cell & Regenerative Medicine

Gene Therapy with Stem Cells & Retroviral Vectors

Ex Vivo Protocols
Harvest CD34+ cells

Retroviral Therapy

Long-term expression
Risk of oncogenesis Low vector titres and transduction rates - clinical retransduction - R&D complication

Transduce Cells

Return cells (~30 x 106 cells/ kg body weight)

Hematopoietic cells

Endothelial cells

Cardiomyocytes

1998 Human Embryonic Stem Cells 2007 induced Pluripotent Stem Cells
Neurons Osteoblasts Islet cells

A. Nagy

Transplantation Medicine

Tissue Engineering

Human Donors

Stem Cells

Solid Organ Transplants 30,000/year (US & Canada)

Bone Marrow & Stem Cell Transplants 15,000/year

Blood Transfusions 5,000,000/year

Cell Processing for Islet Transplantation

Top

Liver of a type 1 diabetic patient

Islet

Middle

Donor pancreas

Bottom

Duct - rich

Ball mill & enzymatic dissociation

Density fractionation

Acinar - rich

Reprogrammed or Embryonic Stem Cells

Progenitors

Ask engineering applicable to bioprocess systems, mass balances, BIOREACTOR mainly stirred tank PROCESS CONTROL AND OPTIMIZATION MASS TRANSPORT - oxygen DOWNSTREAM PROCESSING

Summary

Bioprocess manufacture of valuable products Design of bioprocesses requires biological understanding and the application of engineering technology Biotechnology has great potential to further influence health care and global warming CHBE 365/381/481 Bioprocess Engineering ?technical electives non-Biological Option students should consider
(seems only cant take 381 in 4th year and asked Joanne if could fix)

If have time Ask if can think of potential for abuse? Note abuse of EPO - >>20 deaths among cyclists in Europe positive impact on 100,000s, but abuse leads to trouble for few Could add more bone marrow culture or HFBR spiel