Introduction, Materials and Methods, Results, References

1. A Simplified and Efficient
Process for Insulin Production
in Pichia pastoris
Presented By
Naveed Ur Rehman
BS 7TH, Microbiology

2. A Simplified and Efficient Process for Insulin
Production in Pichia pastoris
• Introduction
• Insulin is a relatively low-priced drug
• Chronic nature of Diabetes means the cost for insulin treatment is high, and
together with an increasing number of patients
• This financial burden challenges healthcare systems worldwide
• Its price reduction is needed in order to improve its availability especially in lower
to lower-middle income countries
Fig. 1 Insulin in the form of infusion

3. A Simplified and Efficient Process for Insulin
Production in Pichia pastoris
• Introduction
• Two major pathways for large-scale production of recombinant human
insulin are currently used
• First pathway
• Escherichia coli used as an expression host
• Where the overexpressed insulin precursor
(IP) forms inclusion bodies requiring
solubilisation and oxidative refolding
Fig. 1 Pathway for production recombinant human insulin

4. A Simplified and Efficient Process for Insulin
Production in Pichia pastoris
• Introduction
• Second pathway
• They used yeast as expression systems (mainly Saccharomyces cerevisiae)
• Where the IP is directly secreted in the culture supernatant in its correctly folded
conformation
Fig. 2 Structure Saccharomces cerevisae

5. A Simplified and Efficient Process for Insulin
Production in Pichia pastoris
• Introduction
• Half of the world insulin supply derives from S. cerevisiae
• However, the standard recovery and purification process of human insulin
produced in S. cerevisiae can include up to fifteen steps
• Methylotrophic yeast Pichia pastoris has a number of attractive characteristics for
heterologous protein production

6. A Simplified and Efficient Process for Insulin
Production in Pichia pastoris
• They Used simple two-phase cultivation process
• A glycerol batch and a constant methanol fed-batch phase for secretory IP
production
• In a result ~2 fold enhancement of IP production compared to the highest values
reported, significantly increasing the efficiency of insulin manufacture

7. A Simplified and Efficient Process for Insulin
Production in Pichia pastoris
Selection of strain
and vector
Culture growth conditions
and IP production
Recovery of IP from culture
broth with tangential flow
filtration
IP purification and
concentration
Transpeptidation reaction,
deprotection and
purification of human
insulin
Mass spectrometry
Materials and Methods

8. A Simplified and Efficient Process for Insulin
Production in Pichia pastoris
• Results
• Production of Insulin precursor in fed-batch fermentation
• High-density fermentation was performed in 30 L bioreactor using 15 L of culture
media
• Expression level of IP in the culture supernatant of 2.26 g L-1.
• This high-level secretory production of IP in the culture supernatant compares
favorably to the highest yields previously published of 0.3 to 1.5 g L-1

9. A Simplified and Efficient Process for Insulin
Production in Pichia pastoris
• Clarification of bioreactor’s cell culture in tangential flow
filtration system
• Separate cells from the culture broth a tangential flow stacked plate membrane
device with open feed channels in the tangential flow filtration (TFF) system was
utilized

10. A Simplified and Efficient Process for Insulin
Production in Pichia pastoris
• IP purification and concentration
• The material recovered by TFF was loaded directly onto two different cation
exchange resins for purification i.e.
• Sepharose SP Fast Flow and Toyopearl GigaCap S-650 M
• Toyopearl GigaCap S-650 M showed a higher loading capacity for IP (110 g L-1)
along with IP binding at relatively high conductivity (15 mS cm-1)
• In comparison to Sepharose SP Fast Flow matrix (36 g L-1 loading capacity at the
conductivity 8 mS cm-1)
• Analysis of the 3 peaks coming out from Toyopearl GigaCap S-650 M was
performed by analytical RP-HPLC and show that IP is present only in peak 3

11. A Simplified and Efficient Process for Insulin
Production in Pichia pastoris
• IP purification and concentration
• Storage of IP in the extraction buffer at +4˚C did not compromise
quality of IP as determined by RP-HPLC and mass spectrometry even
after 6 months
• Use of Toyopearl GigaCap S-650 M resin show that extraction
strategy as a novel procedure to capture IP at high load
• Also extraction at high concentration (~80 mg/ml) in a solvent mixture
• It is suitable for transpeptidation without intermediate step of
diafiltration/buffer exchange.

12. A Simplified and Efficient Process for Insulin
Production in Pichia pastoris
• Conversion of IP to human insulin
• The insulin precursor was converted enzymatically into insulin ester
• Through transpeptidation in the presence of trypsin and an excess of O-t-butyl-L-
threonine t-butyl ester (H-Thr (tBu)-OtBu) acetate salt
• Trypsin mediates cleavage of the N-terminal spacer peptide and the Ala-Ala-Lys
connecting linker peptide
• The transesterification reaction where threonine ester is added to B-29 residue
• We developed cation exchange chromatography extraction conditions such that the
concentrated IP extraction in the organic solvent could be directly used in the
transpeptidation

13. Reference