Efficient production of recombinant parathyroid hormone (rPTH) fragment 1-34 in the methylotrophic yeast Hansenula polymorpha

Abstract

Background: Osteoporosis is a bone disease of the elderly that leads to increased risk of fracture. Currently, it aff ects more than 200 million adults worldwide, placing an enormous economic burden on healthcare providers. Present approved drug treatments of osteoporosis, for example, using bisphosphonates, only reduce bone mineral loss, are variable in prevention of future fractures and can induce serious complications long term. From 2002, availability of the recombinant active, N-terminal 1-34 fragment of human parathyroid hormone 1-34, which directly stimulates bone formation, has provided an alternative, potentially more eff ective therapeutic agent for treating osteoporosis, especially in its most severe form. We have adopted the methylotrophic yeast strain Hansenula polymorpha (H. polymorpha) for production of recombinant parathyroid hormone (rPTH) 1-34 since its expression system is highly inducible and target proteins are secreted intact into the fermentation broth without the need for enzymatic cleavage of a fusion protein. Methods: Culture conditions for the growth of selected clones of H. polymorpha super-transformants and their rPTH production capacity were sequentially optimized through the microtitre plate, shake flask, small tank bioreactor scales to the large bioreactor scale. Results: The production of rPTH 1-34 by transformed H. polymorpha was greatly enhanced by adding the calnexin chaperone gene to the expression plasmid. Implementation of final optimized culture medium constituents and operational fermentation conditions, of which maintenance of constant oxygen tension (pO(2)) was found to be a critical factor at the 80 litre scale, resulted in yields up to 150 mg active rPTH 1-34 per liter of culture supernatant. Conclusion: A robust fermentation process for the effi cient production of active rPTH 1-34 based on a calnexin super-transformed H. polymorpha clone was developed. Potentially, this has cost-saving benefi ts for the production and marketing of clinical-grade rPTH 1-34