comparison of different growth data form different researches published.

1. Cultivation strategies to enhance productivity in
Pichia pastoris
Presentation by :
Mohammed Suleman
Pradipta Hore
Nilima Mahapatra

2. Introduction
• Methylotrophic yeast
• Expression system for production of heterologous proteins of
pro and eukaroyotes
• High biomass concentration.
• Highly regulated promoter
• Protein produced intracellularly or secretory in nature.
Kingdom- Fungi
Phylum- Ascomycota
Class- Saccharomycetes
Order- Saccharomycetales
Family- Saccharomycetaceae
Genus- Komagataella
Species- pastoris

3. • pPICZα A, B, and C are 3.6 kb vectors used to express and
secrete recombinant proteins in Pichia pastoris.
• Highly regulated inducible AOX1(Alcohol oxidase 1)
promoter-methanol inducible expression of the gene.
• AOX1 and AOX2 genes-genes enable the cells to assimilate
methanol as their sole carbon and energy source.
• Alpha factor-Recombinant proteins are expressed as fusion
to an N-terminal peptide encoding the Saccharomyces
cerevisiae α-factor secretion signal.
• C-terminal peptide containing the c-myc epitope and a
polyhistidine (6xHis) tag for detection and purification of a
recombinant fusion protein.
• Amp, Kan, Zeo used as Marker gene.
Structure of Pichia expression vector

5. • AOX1 promoter regulates 85% of AOX production while the
AOX2 promoter is less active
• Strains of Pichia :
Mut+ - AOX1 and AOX2 gene both expressed
Mut s- only AOX1 gene expressed
Mut - -AOX1 and AOX2 absent
• Some vectors such as pGAPZα A,B,C use constitutive
GAP(glyceraldehyde-3-phosphate dehydrogenase).
• pGAP-based expression system is more suitable for large-
scale production because the hazard and cost associated with
the storage and delivery of large volume of methanol are
eliminated.

7. Disadvantages of using Saccharomyces cerevisiae
• Low expression and modest yield
• Proteins often hyperglycosylated so turns antigenic
• Proteins retained in periplasmic space so protein purification becomes
costly affair
• Produces ethanol at high cell densities (toxic)
Disadvantages of using Escherichia coli
• Eukaryotic proteins cannot be expressed. No glycosylation and disulfide
bridge formation.
Disadvantages of using Drosophila melanogaster
• Need of complex media
Advantages of using Pichia pastoris
• Soluble proteins
• High cell densities/high protein yield
• Controllable process
• Minimal downstream purification or processing
• Products similar to those produced in mammalian systems
• Grows in simple media

8. • Biomass-The mass of living biological organisms in a
given system. Changes in biomass is depicted in form of
specific growth rate.
• Specific growth rate(µ)- Grams of biomass built per gram
of biomass present per hour of process time.
• Specific productivity rate - Milligrams of product formed
built per gram of biomass present per hour of process
time.
• Process time -Total time for the process to occur.
• Types of cultures
Batch
Fed batch(generally used with AOX1 strains)
Continuous cultures(generally used with GAP stains)
Production kinetics

11. • Product titre – Grams of product per litre of
supernatant or per litre of broth.

12. Development and implementation of an optimum process strategy
The crucial thing is to screen under “industrially relevant
conditions”.
Major steps to follow :
• screening of clones to reduce the number.
• characterisation of ‘typical’ behaviour to obtain
the relation between biomass growth and
product formation.
• implementation of a production process in
fedbatch mode

13. Screening in small scale:
• Screening can be done in batch type experiments.
• Screening “Best producer” does not always give high-
throughput in larger scale.
• For AOX1 controlled promoter, pulses of methanol are
given after the complete depletion of glucose.

14. • But for a bioreactor, the specific growth rate maintained
lower than the maximum value.
• In case of GAP controlled promoter, specific growth rate is
near to μmax.
• Selection of clones based on their performances , and
further characterisation by fedbatch process.
• For AOX1-promoter, the use of the MutS phenotype can be
advantageous because of smaller growth range.

15. Drawbacks of current screening process:
• Clonal differences due to chromosomally integrated system.
• Edge effects on 96 well plates.
• Strong transient response is unique to each strain.
• Small scale bioreactor is used , reflects the condition of large scale
production.
• Such as release of glucose from a polymer by an enzyme .

16. Development of production process of a newly developed strain requires-
Determination of maximum specific growth rate:
It determines the maximum substrate consumption or the upper limit
of substate addition.
Using batch culture, with excess available substrate.
For AOX1-controlled product formation, initial methanol pulse of 0.5%
(v/v) and at least 4 consecutive pulses of 1.0% (v/v) methanol are given.
Substrate depletion monitored by off gas analyses.
These production characteristics do not reflect the highest possible
productivity.
Characterisation of biomass growth and product formation :

17. • Relationship between specific product formation rate and the corresponding specific
growth rate is empirically for each strain/clone.
• Several different pre-set μ-values to establish the desired qp(μ)-relationship.
• Specific growth rate is controlled at a defined value lower than or equal to its
maximum (μmax).
• To maintain a constant specific growth rate feed rate must be increased exponentially.
• For AOX1-controlled recombinant product formation MutS strains with a considerably
lower μmax are choosen.
• Favourable for the design of a production process with a high titre and productivity
• Dynamic process conditions , entire production range of μ to be covered in a single
experiment.
Establishing production kinetics:

18. Establishing fedbatch production processes
Induction and production of heterologous protein are interconnected
to substrate utilization and biomass growth.
Commercially available expression kits by INVITROGEN dominate a
standard protocol for typical P. pastoris cultivation with three stage
strategies
Batch phase for biomass growth with glycerol or glucose
 Fedbatch phase for biomass enhancement with glycerol or
glucose
Transition phase an optional methanol induction followed by
fedbatch

20. Conventional protocol for simple recombinant protein production
What standard protocol is suitable for a
particular stain depends on the strain-specific
maximum specific growth rate with methanol
and optimum μ operational range for
product formation
Values of different substrate considered to be
standard for typical growth are
0.061g g-1 glycerol
0.33g g-1 methanol
Maintenance rate for methanol of 0.061g g-1
h-1 from a recombinant strain with μmax of
0.07 h-1 was used.

22. Customized process strategies
 Considering the maximum specific growth rate μmax as the upper limit and
the production kinetics qp(μ) as an indicator of the optimum range of μ of
a customized fedbatch profile to maximize product titre is possible.

23. High productivity by fedbatch production processes

24. Limiting factors for high density cultivations (P. pastoris)
Oxygen and heat transfer are often limiting factors irrespective
of the substrate used.
However heat evolution and oxygen uptake strongly depend
on specific growth rate.

25. Recommendation for best practice in process development
Critical factors influencing the performance of production process are
 Specific productivity (qp)
 Maximum biomass in the reactor ( x.V )
 Productive time ( between induction and harvest )
A high initial concentration of biomass and low specific growth rate
during production are favourable.

26. Future development
In spite of increasing intensive research on P. pastoris system it
is still far from maturity of E. coli or S. cerevisiae.
Some innovative development in this field are as follows
Single cell level perspective (FACS, Microengraving)
Approaches to reduce experimental load (physiological
characterization based on software sensors)
Product quality (biological activity, half life, deamination,
glycosylation etc. )

27. Conclusion
both comparison and development of biotechnological production processes
with P. pastoris.
Optimum process strategy which can be applied for different strain and
bioreactors
relationship between biomass growth and protein production,