Engineering of Biological Processes Lecture 4: Production kinetics

1
Engineering of Biological ProcessesLecture 4
Production kinetics

• Mark Riley, Associate Professor
• Department of Ag and Biosystems Engineering
• The University of Arizona, Tucson, AZ
• 2007

2
Objectives Lecture 4

• Investigate production kinetics and limiting
  factors.
• Characterize product formation based on yields

3
Production kinetics

• Classified based on the relationship between
  product synthesis and energy generation in the
  cell
• Growth associated
• Non-growth associated
• Mixed-growth associated

4
Products

• Growth-associated
• produced at the same time as cell growth
• constitutive enzymes (ones that are normally
  present)
• glucose isomerase
• metabolic intermediates
• pyruvate, citrate, acetate
• Non-growth-associated
• takes place during the stationary phase (m0)
• secondary metabolites
• antibiotics
• Mixed - growth associated
• takes place during growth and stationary phases
• metabolic byproducts
• lactate, ethanol
• secondary metabolites

5
Product generation
Growth-associated
Non-growth associated
Mixed-growth associated
6
Protein production

• Antibody (MAb) production by mammalian cells
  (hybridomas)?
• Growth associated
• Non-growth associated
• Other?
• In 1990, Suzuki and Ollis (NCSU) developed a
  structured model that suggested "negatively
  growth associated" MAb production kinetics.

Biotechnol Prog. 1990 May-Jun6(3)231-6. Suzuki
E, Ollis DF.
7

• Hybridoma cultures where growth was slowed by
  either a DNA synthesis inhibitor or by a
  selective inhibitor of initiation of nonantibody
  protein exhibited 50-130 MAb production rate
  enhancement for growth slowed up to 50.
• Experiments inconsistent with this approach
  showed other behavior general inhibition of
  protein chain elongation (by cycloheximide) or
  inhibition of ribosomal RNA (rRNA) synthesis (by
  actinomycin D) each slowed both growth and the
  specific MAb production rate, leading to net
  "positive" growth associated MAb production
  rates.

8
How can we account for this behavior?
Generalized production equation
9
Direct coupling to energy metabolism

• For products formed in pathways which generate
  ATP, rate of production is related to cellular
  energy demand.
• Growth is usually the major energy-requiring
  function of cells therefore, if production is
  coupled to energy metabolism, product will be
  formed whenever there is growth.

10
Direct coupling to energy metabolism
The above is a gross over-simplification of
production rates.
11
Maintenance

• ATP is also required for other activities called
  maintenance.
• cell motility
• turnover of cellular components
• adjustment of membrane potentials and internal pH

12
Kinetic expressions require growth-associated and
maintenance-associated production
YP/X is the theoretical yield of product from
biomass, mp is the specific rate of product
formation due to maintenance, and x is biomass
concentration.
13
Observed
Theoretical
Result Yp/x is higher than anticipated based
on growth alone
14
Effect of incorporating maintenance terms

• Gives observed yields rather than theoretical
  yields
• Accounts for unusual behavior
• negative association with growth

15
Cell growth stages in a batch culture
Limited by the depletion of a resource (nutrient,
space, oxygen).
16
Product formation indirectly or not coupled to
energy metabolism

• Product Formation Indirectly Coupled With Energy
  Metabolism
• Relationship between product formation and growth
  can be complicated. Beyond the scope here.
• Product Formation Not Coupled With Energy
  Metabolism
• Production not involving energy metabolism is
  difficult to relate to growth because growth and
  product synthesis are dissociated.
• Rate of formation of non-growth-associated
  product can be directly proportional to biomass
  concentration,
• constant qp
• qp complex function of growth rate
• empirical equations derived from experiment.

17
Substrate uptake

• Used for
• making biomass (x)
• making product (p)
• maintenance (ms)

18
Substrate uptake
Result rs is higher than anticipated based on
only growth and product formation
19
Lot's of parameters to estimate

• Need values for

20
Yield of cells from substrate
21
If there is no product generated (qp0)
Plot (1/Yx/s) vs. 1/m slope ms
22
With production
rp decreases Yx/s
23
Cancel out X s
24
(No Transcript)
25
Note

• Yx/s Yp/x Yp/s

26
To determine the metabolic parameters

• Need data on
• substrate uptake with time
• with and without product formation
• product generation with time
• with and without cell growth
• cell growth with time

27
So, what do these yields yield?

• Basic estimation of nutritional requirements
• Targets for manipulation
• Growth rates
• Maintenance terms
• ms, mp
• Fudge factors to explain why Yp/s gt Yp/s

28
Maximize production
29
How do we alter these parameters?

• Control nutrient and oxygen supply
• Cultivation methods fed batch
• Strain selection high producers
• Strain optimization
• Recombinant DNA techniques
• Metabolic engineering

30

• "Growth, metabolic, and antibody production
  kinetics of hybridoma cell culture Effects of
  serum concentration, dissolved oxygen, and pH in
  a batch reactor."
• The effects of serum, dissolved oxygen (DO)
  concentration, and medium pH on hybridoma cell
  growth, viability, cell density, carbohydrate and
  amino acid metabolism, respiration and energy
  production rates, and antibody production rates
  were studied.
• Cell growth was enhanced and cell death was
  decreased by increasing the serum level. The
  growth rates followed a Monod-type model with
  serum being the limiting component.
• Specific glucose, glutamine, and oxygen uptake
  rates and specific lactate and ammonia production
  rates did not change with serum concentrations.
  Amino acid metabolism was slightly influenced by
  the serum level.
• Oxidative phosphorylation accounted for about 60
  of total energy production. This contribution,
  however, increased at low pH values to 76.
• The specific antibody production rate was not
  growth associated and was independent of serum
  and DO concentrations. A 2-fold increase in
  specific antibody production rates was observed
  at pH values below 7.2.
• Higher concentrations of antibody were obtained
  at high serum levels, between 20 and 40 DO, and
  at pH 7.20 due to higher viable cell numbers
  obtained.

• Biotechnol Prog. 1991 Nov-Dec7(6)481-94.
  Ozturk SS, Palsson BO.

31
Example

• Yield example, modifying m, S

32
Impact of S on P
33
Effects of parameters

• ? S, ? P
• ? mmax, ? P
• ? mmax, ? P
• ?Y'p/s, ? P
• ?Y'x/s, ? P

34
Impact of Yx/s on product formation