thermodynamics_biotech & bioprocess engineering

7/30/2019 Thermodynamics_Biotech & Bioprocess Engineering

1/23

CHBE 242

Biotechnology and

Bioprocess Engineering

James PiretMichael Smith LaboratoriesChemical & Biological Engineering


2/23

BiotechnologyUsing 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 EngineeringBroad-based engineering discipline that encompassesengineering applied to living systems.includes

- Biomedical EngineeringApplication of engineering principles and techniques to medicine.

&

- Bioprocess or Biochemical EngineeringExtension 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 catalystalso developing technologies for biological analysis - Proteomics e.g.


3/23

Biotechnology Industry

Recombinant Proteins

Monoclonal Antibodies

therapy of cancer & arthritis

rheumatoid arthritis

Tissue Plasminogen Activator

therapy of heart attacks & stroke

Often using mammalian cells that provide needed post-translational modifications

such as glycosylationshould find images to show without and with


4/23

Diverse Amino Acid Structures


5/23

Biofuels

hInsulin (82)

hGrowth Hormone

(85)

Hep. B vaccine (86)

G-CSF (91)

t-PA (87)

EPO (89)PhD

Rituxin (97)

Herceptin (98)

Gene & Cell Therapy

Bioprocessing Products

Enzymes Bacteria Fungi Animal Cells

Egyptian Cheese

(4000 BC)

Sumerian Wine

Amino Acids (6000 BC)

MSG

Sumerian wine. low alcohol

content, safe water, nutrients,

painkiller 1 million metric tons produced/yr

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


6/23

Ask vs. Chemical NH3process at?... 100 atm, 350C to produce fertilizer

- DANGER, entire sessions at AIChE

Mild Reactions ConditionsOften required for heat sensitive enzyme substrates and products (glucose, proteins)

Chemical NH3process vs. N2fixing by enzymes at 1 atm, 25C

Chemical Hydrolysis of ProteinsProtein Amino Acids

24 h, 6 N HCl, 100C [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 bondsStill much more specific, e.g. chemicals sinceH3O

+ would hydrolyze starch too

Andnon-specificglutamine residue ->glutamate degradation proteins would not


7/23High temperatures (121C)

Diversity of life

in extreme

environments

Rich source ofbiocatalysts

High pH

B. alcalophilus

pH 10

Low pH

B. tusciae

pH 4

Biodiversity of Archaea

Low temperatures

Marinibacillus marinus(4C)

High salt

B. halophilus(2M NaCl)

Conversely actually biocatalysts provide many opportunities due to diversity of environments


8/23

Greenhouse gas incentives toobtain ethanol from cellulose

First break down cellulose to sugars:

Then fermentation to ethanol

by yeast S. cerevisiae:

C6H12O6 2 C2H5OH + 2 CO2

BioethanolProduced by fermentation of sugars derivedfrom wheat, corn, sugar beets, sugar cane,molasses, i.e. sugar, starch or cellulose


9/23

R&D of Genetic Engineering

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 Prizeto 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 mutagenesis1993 Nobel Prize

allows modifying proteins in desired ways

Mendels genetic research published (1866)

DNA proven to store genetic information (1943)

DNA gene mRNA Protein

Transcription Translation


10/23

DecreasingGrowth

Rate

Batch Culture Growth Phases

Adaptation

Balanced

Growth

m constant

Accumulated

metabolites and/or

substrate nutrients

limiting growth

mnet = m - kd = 0Secondary

metabolitesCell Death

& Lysis

Lag

Exponential

Stationary

Death(or Decline)

Time

[X]

Viable

Cell #

or DCW

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


11/23

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 mathmodeling to improve

understanding of systems

biology, but bioprocess

engineers generally use useful

approximations, though

empirical

Small Molecules (points) and Reactions (lines) ofE. coli Metabolism


12/23

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:

Xdt

dX

netm Where (e.g. Cells/L) X=X

0@ t=0

Integrating,t

netneteXXort

X

X mm

0

0

ln


13/23

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

SK

S

s

m

m

m

mm - max specific growth rate

when S >> Ks

Ks

saturation constant


14/23

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(Continuous Culture)

Technological advance

for analysis of substrate

limitation of growth


15/23

Chemostat (CSTR)

A material balance on the

limiting substrate:

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

qp - specific rate of extracellular product formation (g P/g cell-h)

YMX/Sand YP/S- yield coefficients (g cells/g Sand gP/g S)

Allows prediction of X and S dependence on the flow rate, for process modeling

0

1 1R g R p RM

X S P S

dSFS FS V X V q X V

Y Y dt m

IN OUT Cell Growth Product Formation


16/23

Culture Production Processes

Batch Fed-Batch Perfusion

Medium Feed Medium Feed

Batch for microbial processes, fed-batch for antibiotics and MAb

Perfusion for cellular therapy and some protein production


17/23

Fermentation Flow Sheet

Fermentation

Media & Inoculum Cell engineering Sterilization

Process Models

Aeration Process control

Reactor design

Separations

Cell disruption

Solid removal

Purification

Concentration

CHBE 381

Bioprocess

Engineering I

Enzymes too

CHBE 481

BioprocessEngineering II

CHBE 365

Biotechnology

Laboratory

Design, thesis, TEs

Product


18/23

Since 1900Tissue 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

Mammalian Cell Bioengineering


19/23

Gene Therapy with

Stem Cells & Retroviral Vectors

Harvest CD34+ cells

Transduce

Cells

Return cells

(~30 x 106 cells/ kg body weight)

Ex Vivo Protocols Retroviral Therapy

Long-term expression

Risk of oncogenesis

Low vector titres and

transduction rates

- clinical retransduction- R&D complication


20/23

Hematopoietic cells

Endothelial cells

Osteoblasts

Cardiomyocytes

Neurons Islet cells

1998 Human Embryonic Stem Cells 2007 induced Pluripotent Stem Cells

A. Nagy


21/23

Transplantation Medicine

Human

DonorsTissue

Engineering

Blood

Transfusions

5,000,000/year

Solid Organ

Transplants

30,000/year

(US & Canada)

Bone Marrow

& Stem Cell

Transplants

15,000/year

Stem

Cells


22/23

Islet

Duct - rich

Acinar - rich

?Progenitors

Ball mill &enzymatic

dissociation

Densityfractionation

Donor pancreas

Liver of a type 1diabetic patient

Top

Mid

dle

Bottom

Reprogrammed or

Embryonic Stem Cells

Cell Processing for Islet Transplantation


23/23

Summary

Bioprocess manufacture of valuable products

Design of bioprocesses requires biological understandingand the application of engineering technology

Biotechnology has great potential to further influencehealth care and global warming

CHBE 365/381/481 Bioprocess Engineering

?technical electives non-Biological Option students should consider(seems only cant take 381 in 4thyear and asked Joanne if could fix)

Ask engineering applicable to bioprocess systems, mass balances,

BIOREACTOR mainly stirred tank

PROCESS CONTROL AND OPTIMIZATION

MASS TRANSPORT - oxygen

DOWNSTREAM PROCESSING

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

thermodynamics_biotech & bioprocess engineering

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