Scalable Fermentation Process

a fermentation process and scalable technology, applied in the field of protein expression and fermentation technology, can solve the problems of reducing the yield of capsid protein of 50 litres and more, hampering further downstream processes, and recombinant capsid protein may have a negative impact on the yield of correctly assembled vlps, so as to improve the repression of the promoter, and improve the yield of soluble capsid protein

Inactive Publication Date: 2012-12-20
CYTOS BIOTECHNOLOGY AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This approach results in improved yield and purity of soluble capsid protein, enabling the scalable production of VLPs with consistent quality and self-assembly capability, suitable for commercial production scales.

Problems solved by technology

An scale up of these systems comprising volumes of 50 litre and more is expected to diminish in a great extent the respective capsid protein yield due to increased promoter leakage and / or lowered plasmid retention.
A further problem associated with commercially desired high-level expression and rapid accumulation of recombinant capsid proteins of bacteriophages is the formation of incorrectly folded protein species and the formation of so called inclusion bodies, i.e. protein aggregates, which are insoluble and which may hamper further downstream processes.
High expression rates of the recombinant capsid protein may therefore have a negative impact on the yield of correctly assembled VLPs.

Method used

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  • Scalable Fermentation Process

Examples

Experimental program
Comparison scheme
Effect test

example 1

Cloning Strategy for the Expression Plasmid pTac-nSD-Qb-mut (SEQ ID NO:1)

[0142]The coat protein-encoding gene (C) of E. coli RNA bacteriophage Qβ is amplified from plasmid pSDQb-mut (SEQ ID NO:33). The plasmid contains the sequence of gene C coding for the 133-aa Qβ coat protein (CP) and the 329-aa read through protein (A1). To prevent read-through, nucleotides 445-450 according to NCBI GenBank Acc. No. M99030 TGAACA (SEQ ID NO:31) are replaced by the sequence TAATGA (SEQ ID NO:32).

[0143]The coat protein-encoding gene C from plasmid pSDQb-mut is amplified by PCR. Oligonucleotide Qb-FOR3 / 2 (SEQ ID NO:34) with an internal EcoRI site and a synthetic Shine-Dalgarno (SD, SEQ ID No:4) sequence anneals to the 5′ end of the Qβ CP gene. Oligonucleotide Qblang-REV2 / 2 (SEQ ID NO:35) contains an internal HindIII site and primes to the 3′ end of the noncoding region of gene C. The 1054 by amplified PCR fragment includes nucleotides 46-1062 of NCBI GenBank Acc. No. M99039 (except the nucleotide c...

example 2

Cloning Strategy for the Expression Plasmid pTac-nSD-AP205 (SEQ ID NO:30)

[0145]The coat protein-encoding gene of Acinetobacter bacteriophage AP205 is amplified from plasmid pAP205-58. This plasmid contains the sequence of the coat protein gene (corresponding to nucleotides 1908-2303 of NCBI GenBank Acc. No. AF334111) coding for the 131-amino acid capsid protein of bacteriophage AP205.

[0146]The coat protein-encoding gene is amplified by PCR. Oligonucleotide nSDAP238-EcoRI for (SEQ ID NO:38) with an internal EcoRI site and a synthetic Shine-Dalgarno (nSD) sequence anneals to the 5′ end of the coat protein gene. Oligonucleotide AP238HindIIIrev (SEQ ID NO:39) contains an internal HindIII site and primes to the 3′ end of the coat protein gene. This oligonucleotide introduces a second stop codon behind the naturally occurring stop codon of the coat protein. The 438 bp amplified PCR fragment includes nucleotides 1908-2303 of NCBI GenBank Acc. No. AF334111 and the synthetic nSD sequence. Th...

example 3

Expression of Qβ CP Under Control of the tac Promoter and nSD

[0147]The E. coli strain RB791 was transformed with plasmids pTac-nSD-Qb-mut (SEQ ID NO:1). The clone was grown in shake flasks. Each flask contained 100 ml of R40 medium (main culture medium, Hypep 7455, glycerol, see Example 5) with kanamycin (25 μg / ml) and was inoculated with over night cultures at a start OD600 of 0.3. The shake flasks were incubated for 4 h (OD600 between 4 and 5) at 30° C. and an agitation of 220 rpm. The induction was carried out with 0.5% of lactose for 4 h. Protein production was determined by SDS-PAGE. The gel showed a strong protein band which was identified as Qβ CP.

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Abstract

This invention provides a robust fermentation process for the expression of a capsid protein of a bacteriophage which is forming a VLP by self-assembly, wherein the process is scalable to a commercial production scale and wherein the expression rate of the capsid protein is controlled to obtain improved yield of soluble capsid protein. This is achieved by combining the advantages of fed-batch culture and of lactose induced expression systems with specific process parameters providing improved repression of the promoter during the growth phase and high plasmid retention throughout the process.

Description

FIELD OF THE INVENTION[0001]This invention is related to the field of protein expression and fermentation technology. A process for the efficient expression of recombinant bacteriophage capsid protein in a bacterial host is described. The process leads to high yield of recombinant capsid protein which is capable of forming a virus-like particle (VLP) by self-assembly. Furthermore, the process is scalable from laboratory scale to fermenter volumes larger than 50 litres.BACKGROUND OF THE INVENTION[0002]Recent vaccination strategies make use of viruses or virus-like-particles (VLPs) to enhance the immune response towards antigens. For example, WO02 / 056905 demonstrates the utility of VLPs as a carrier to present antigens linked thereto in a highly ordered repetitive array. Such antigen arrays can cause a strong immune response, in particular antibody responses, against the linked antigen and are even capable of breaking the immune system's inherent tolerance towards self antigens. Such ...

Claims

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Application Information

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Patent Type & AuthorityApplications(United States)
IPC IPC(8): C12P21/02
CPCA61K2039/5256A61K2039/5258C12N7/00C12N2795/00023C12N2795/10051C07K14/005C12N2795/18022C12N2795/18052C12N2795/10061C12N2795/00051
InventorEMMERLING, MARCELHENNECKE, FRANKPFRUNDER, HOLGERRHIEL, MARTINSTEINER, PHILIPP
OwnerCYTOS BIOTECHNOLOGY AG