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Expression of recombinant staphylokinase in the methylotrophic yeast Hansenula polymorpha.

Moussa M, Ibrahim M, El Ghazaly M, Rohde J, Gnoth S, Anton A, Kensy F, Mueller F - BMC Biotechnol. (2012)

Bottom Line: Therefore, development of an alternative fibrinolytic agent having superior efficacy to SK, approaching that of rt-PA, together with a similar or enhanced safety profile and advantageous cost-benefit ratio, would be of substantial importance.Potential glycosylation of rSAK-2 was successfully suppressed through amino acid substitution within its only N-acetyl glycosylation motif.The optimized production process described for rSAK-2 in Hansenula polymorpha provides an excellent, economically superior, manufacturing platform for a promising therapeutic fibrinolytic agent.

View Article: PubMed Central - HTML - PubMed

Affiliation: Minapharm Pharmaceuticals, Cairo, Egypt. mueller@minapharm.com

ABSTRACT

Background: Currently, the two most commonly used fibrinolytic agents in thrombolytic therapy are recombinant tissue plasminogen activator (rt-PA) and streptokinase (SK). Whereas SK has the advantage of substantially lower costs when compared to other agents, it is less effective than either rt-PA or related variants, has significant allergenic potential, lacks fibrin selectivity and causes transient hypotensive effects in high dosing schedules. Therefore, development of an alternative fibrinolytic agent having superior efficacy to SK, approaching that of rt-PA, together with a similar or enhanced safety profile and advantageous cost-benefit ratio, would be of substantial importance. Pre-clinical data suggest that the novel fibrinolytic recombinant staphylokinase (rSAK), or related rSAK variants, could be candidates for such development. However, since an efficient expression system for rSAK is still lacking, it has not yet been fully developed or evaluated for clinical purposes. This study's goal was development of an efficient fermentation process for the production of a modified, non-glycosylated, biologically active rSAK, namely rSAK-2, using the well-established single cell yeast Hansenula polymorpha expression system.

Results: The development of an efficient large scale (80 L) Hansenula polymorpha fermentation process of short duration for rSAK-2 production is described. It evolved from an initial 1mL HTP methodology by successive scale-up over almost 5 orders of magnitude and improvement steps, including the optimization of critical process parameters (e.g. temperature, pH, feeding strategy, medium composition, etc.). Potential glycosylation of rSAK-2 was successfully suppressed through amino acid substitution within its only N-acetyl glycosylation motif. Expression at high yields (≥ 1g rSAK-2/L cell culture broth) of biologically active rSAK-2 of expected molecular weight was achieved.

Conclusion: The optimized production process described for rSAK-2 in Hansenula polymorpha provides an excellent, economically superior, manufacturing platform for a promising therapeutic fibrinolytic agent.

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Typical RP-HPLC profile of a final rSAK-2 fermentation supernatant sample at 73.5 hours of cultivation.
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Figure 6: Typical RP-HPLC profile of a final rSAK-2 fermentation supernatant sample at 73.5 hours of cultivation.

Mentions: As sampling at different fermentation times has shown increasing rSAK-2 expression, fermentation to a total of 70–73 hours with constant feeding over 50 hours was evaluated. Due to the requirement for increased volume of feed solution, the initial volume of medium had to be reduced from 1.4 to 1.2 L, the feed rate re-adjusted to 20 mL/hour and the FS composition re-adjusted to 20% peptone (w/v), 22% glycerol (w/v), 20% yeast extract (w/v) and 15% methanol (w/v). An rSAK-2 concentration of 1212 mg/L was attained under these final optimized conditions. This production level was reproducible and consistent in several fermentations (data not shown). Figure 6 shows a typical RP-HPLC profile for an rSAK-2 containing supernatant at the end of fermentation.


Expression of recombinant staphylokinase in the methylotrophic yeast Hansenula polymorpha.

Moussa M, Ibrahim M, El Ghazaly M, Rohde J, Gnoth S, Anton A, Kensy F, Mueller F - BMC Biotechnol. (2012)

Typical RP-HPLC profile of a final rSAK-2 fermentation supernatant sample at 73.5 hours of cultivation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539880&req=5

Figure 6: Typical RP-HPLC profile of a final rSAK-2 fermentation supernatant sample at 73.5 hours of cultivation.
Mentions: As sampling at different fermentation times has shown increasing rSAK-2 expression, fermentation to a total of 70–73 hours with constant feeding over 50 hours was evaluated. Due to the requirement for increased volume of feed solution, the initial volume of medium had to be reduced from 1.4 to 1.2 L, the feed rate re-adjusted to 20 mL/hour and the FS composition re-adjusted to 20% peptone (w/v), 22% glycerol (w/v), 20% yeast extract (w/v) and 15% methanol (w/v). An rSAK-2 concentration of 1212 mg/L was attained under these final optimized conditions. This production level was reproducible and consistent in several fermentations (data not shown). Figure 6 shows a typical RP-HPLC profile for an rSAK-2 containing supernatant at the end of fermentation.

Bottom Line: Therefore, development of an alternative fibrinolytic agent having superior efficacy to SK, approaching that of rt-PA, together with a similar or enhanced safety profile and advantageous cost-benefit ratio, would be of substantial importance.Potential glycosylation of rSAK-2 was successfully suppressed through amino acid substitution within its only N-acetyl glycosylation motif.The optimized production process described for rSAK-2 in Hansenula polymorpha provides an excellent, economically superior, manufacturing platform for a promising therapeutic fibrinolytic agent.

View Article: PubMed Central - HTML - PubMed

Affiliation: Minapharm Pharmaceuticals, Cairo, Egypt. mueller@minapharm.com

ABSTRACT

Background: Currently, the two most commonly used fibrinolytic agents in thrombolytic therapy are recombinant tissue plasminogen activator (rt-PA) and streptokinase (SK). Whereas SK has the advantage of substantially lower costs when compared to other agents, it is less effective than either rt-PA or related variants, has significant allergenic potential, lacks fibrin selectivity and causes transient hypotensive effects in high dosing schedules. Therefore, development of an alternative fibrinolytic agent having superior efficacy to SK, approaching that of rt-PA, together with a similar or enhanced safety profile and advantageous cost-benefit ratio, would be of substantial importance. Pre-clinical data suggest that the novel fibrinolytic recombinant staphylokinase (rSAK), or related rSAK variants, could be candidates for such development. However, since an efficient expression system for rSAK is still lacking, it has not yet been fully developed or evaluated for clinical purposes. This study's goal was development of an efficient fermentation process for the production of a modified, non-glycosylated, biologically active rSAK, namely rSAK-2, using the well-established single cell yeast Hansenula polymorpha expression system.

Results: The development of an efficient large scale (80 L) Hansenula polymorpha fermentation process of short duration for rSAK-2 production is described. It evolved from an initial 1mL HTP methodology by successive scale-up over almost 5 orders of magnitude and improvement steps, including the optimization of critical process parameters (e.g. temperature, pH, feeding strategy, medium composition, etc.). Potential glycosylation of rSAK-2 was successfully suppressed through amino acid substitution within its only N-acetyl glycosylation motif. Expression at high yields (≥ 1g rSAK-2/L cell culture broth) of biologically active rSAK-2 of expected molecular weight was achieved.

Conclusion: The optimized production process described for rSAK-2 in Hansenula polymorpha provides an excellent, economically superior, manufacturing platform for a promising therapeutic fibrinolytic agent.

Show MeSH
Related in: MedlinePlus