Delta Biotechnology Limited

Novozymes Biopharma UK Limited

Novozymes Delta, Limited

Andrew Robert Goodey

Richard A Johnson

Darrell Sleep

John Rodney Woodrow

Alan Victor Quirk

Stephen Berezenko

Patricia Carol Wood

Hendrik Van Urk

Stephen James Burton

Darell Sleep

Mark J Wilson

Steven James Burton

David J Ballance

David St Coghlan

Graham P Belfield

Dina Vakeria

A process is provided for the preparation of albumin from a yeast culture medium which has extremely low levels of or is essentially free of colorants, metal ions, human proteins, host proteins, fragments of albumin, polymers or aggregates of albumin and viruses, and which is essentially non-glycated, relatively high in free thiol and with an intact C-terminus. The process comprises separating the yeast from the culture medium to yield an albumin solution; subjecting the albumin solution to positive mode cation exchange, affinity, and positive mode anion exchange chromatography; and concentrating and formulating the albumin with sodium octanoate and sodium chloride.

Process of high purity albumin production

A process is provided for the preparation of albumin which has extremely low levels of or is essentially free of colorants, metal ions, human proteins, host proteins, fragments of albumin, polymers or aggregates of albumin and viruses, and which is essentially non-glycated, relatively high in free thiol and with an intact C-terminus. The process comprises passing albumin (preferably expressed and secreted by transformed yeast) through positive mode cation exchange and then positive mode anion exchange chromatography. Other steps may also be employed, ultrafiltration, gel permeation chromatography, affinity chromatography binding the albumin by using blue dyes) and affinity chromatography binding contaminants by using an aminophenylboronic acid resin. Elution of albumin, with a compound having affinity for albumin, from a material having no specific affinity for albumin is also disclosed, as is removal of ammonium ions with a counter-ion.

The present invention relates to a process for purifying the protein human serum albumin extracted from serum or recombinant human albumin produced by transforming a microorganism with a nucleotide coding sequence encoding the amino acid sequence of human serum albumin. The present invention provides highly purified albumin.

A fusion polypeptide comprising, as at least part of the N-terminal portion thereof, an N-terminal portion of HSA or a variant thereof and, as at least part of the C-terminal portion thereof, another polypeptide except that, when the said N-terminal portion of HSA is the 1-n portion where n is 369 to 419 or a variant thereof then the said polypeptide is one of various specified entities. The HSA-like portion may have additional N-terminal residues, such as secretion leader sequences (signal sequences). The C-terminal portion is preferably the amino terminal fragment of human urokinase-type plasminogen activator. The N-terminal and C-terminal portions may be cleavable to yield the isolated C-terminal portion, with the N-terminal portion having served to facilitate secretion from the host. Such cleavage can be achieved in yeast using a sequence cleavable by the KEX2 protease of S. cerevisiae.

Polypeptides

A process for the preparation of albumin which has extremely low levels of or is essentially free of colorants, metal ions, human proteins, fragments of albumin, polymers or aggregates of albumin, and viruses, and which is relatively non-glycated, relatively high in free thiol and with an intact C-terminus. The process comprises passing albumin (preferably expressed and secreted by transformed yeast) through two chromatography purifications, ultrafiltering the product, passing through two further chromatography steps and again ultrafiltering the product.

High purity albumin and method of producing

A yeast promoter comprising the promoter of the (cytoplasmically located glycerol-3-phosphate dehydrogenase gene (GPD 1) possesses advantageous capacity to regulate the expression of heterologous polypeptides in yeasts transformed therewith. Expression of such polypeptides utilizing the GPD1 promoter can be regulated by the presence(repressed) or absence(derepressed) of high levels of sucrose or glucose in the fermentation medium. Alternatively, a non-repressing carbon source, such as glycerol or ethanol, can be added to the fermentation medium.

Yeast promoter for expressing heterologous polypeptides

Secretory leader sequences, for use in secreting heterologous polypeptides in yeast, are formed by fusing part of the human serum albumin pre-sequence or part of the Kluyveromyces lactis killer toxin pre-sequence to the Saccharomyces cerevisiae mating factor alpha-1 KEX2 cleavage recognition site. The resulting fusion leader sequences are:

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Mapperley Park, United Kingdom

Andrew Robert Goodey