Application of simple fed-batch technique to high-level secretory production of insulin precursor using Pichia pastoris with subsequent purification and conversion to human insulin

Abstract

Background: The prevalence of diabetes is predicted to rise significantly in the coming decades. A recent analysis projects that by the year 2030 there will be ~366 million diabetics around the world, leading to an increased demand for inexpensive insulin to make this life-saving drug also affordable for resource poor countries.

Results: A synthetic insulin precursor (IP)-encoding gene, codon-optimized for expression in P. pastoris, was cloned in frame with the Saccharomyces cerevisiae alpha-factor secretory signal and integrated into the genome of P. pastoris strain X-33. The strain was grown to high-cell density in a batch procedure using a defined medium with low salt and high glycerol concentrations. Following batch growth, production of IP was carried out at methanol concentrations of 2 g L-1, which were kept constant throughout the remaining production phase. This robust feeding strategy led to the secretion of approximately 3 gram IP per liter of culture broth (corresponding to almost 4 gram IP per liter of cell-free culture supernatant). Using immobilized metal ion affinity chromatography (IMAC) as a novel approach for IP purification, 95% of the secreted product was recovered with a purity of 96% from the clarified culture supernatant. Finally, the purified IP was trypsin digested, transpeptidated, deprotected and further purified leading to approximately 1.5 g of 99% pure recombinant human insulin per liter of culture broth.

Conclusions: A simple two-phase cultivation process composed of a glycerol batch and a constant methanol fed-batch phase recently developed for the intracellular production of the Hepatitis B surface antigen was adapted to secretory IP production. Compared to the highest previously reported value, this approach resulted in an ~2 fold enhancement of IP production using Pichia based expression systems, thus significantly increasing the efficiency of insulin manufacture.

Figure 1

Construction of the recombinant P. pastoris X-33 strain. (A) The IP gene and polypeptide sequence. The bent arrows indicate the ends of the IP gene and polypeptide. The tripeptide linking the B and A chains is underlined. The nucleotides comprising the 5' and 3' restriction sites are shown in italics. The A and B chain aa residue numbers carry the cognate 'A' and 'B' prefixes, respectively. The black triangles denote the sites of trypsin cleavage. (B) Map of the yeast integrative vector encoding IP. The IP is cloned between the XhoI site in the α-factor signal encoding sequence and the NotI site in the polylinker.

Figure 2

Two-phase fed-batch cultivation of P. pastoris X-33 carrying the AOX1 promoter-driven IP gene. Cells were first grown in a batch phase with glycerol as carbon source followed by a methanol feeding phase to induce the production of IP. (A) Concentrations of glycerol (filled squares) and biomass (optical density: filled circles; CDM: filled triangles). (B) Concentration of methanol (solid line) and amount of methanol added to the bioreactor (dashed line). (C) Medium pH (solid line), amount of ammonium hydroxide (dashed line), and amount of phosphoric acid added to the bioreactor (dotted line). (D) Dissolved oxygen concentration (solid line), aeration rate (dotted line), and stirrer speed (dashed line). (E) Oxygen transfer (dashed line) and carbon dioxide evolution (solid line) rates and respiratory quotient (dotted line). Arrows indicate removal of culture broth. (F) Cell growth (filled circles) and accumulation of IP (filled squares). The dashed vertical line indicates the end of the glycerol batch and the start of the methanol feeding phase.

Figure 3

Time course analysis of secretory IP production. Cell-free culture supernatants were analyzed by 10% Tricine SDS-PAGE from samples taken directly before the addition of methanol (time 0) and 24, 48, 72, 96, 120 and 144 hours after the onset of methanol feeding. The arrow denotes the position of IP and M the lane of the molecular weight marker.

Figure 4

RP-HPLC profiles of insulin species recovered after IMAC, transpeptidation and deprotection reactions, and final purification. (A) RP-HPLC profile of the purified IP from IMAC. IP was eluted at a retention time of 19.4 minutes. (B) RP-HPLC profile of insulin species recovered after the transpeptidation reaction with i) pre-insulin H-Thr(tBu)-OtBu; ii) insulin species cleaved at B29 without the threonine ester; and iii) Insulin species cleaved at B22. (C) RP-HPLC profile of deprotection reaction in which the pre-insulin reaction mixture was incubated at room temperature for 5 minutes, with i) pre-insulin H-Thr(tBu)-OtBu; iv) insulin Thr-OtBu; and v) human insulin. (D) RP-HPLC profile of deprotection reaction in which the pre-insulin reaction mixture was incubated at room temperature for 60 minutes (E) RP-HPLC profile of purified human insulin. (F) RP-HPLC profile of mixture of human insulin and European Pharmacopoeia human insulin standard. The Phenomenex Jupiter C4 column was employed for quantification of insulin species. The identity of insulin species was determined by mass spectrometry as specified in the Materials and Methods section.