Production of Humanized Fab Fragment against Human High Affinity IgE Receptor in<i>Pichia pastoris</i>

Takahashi, Kyôko; Yuuki, Toshifumi; Takai, Toshiro; Ra, Chisei; Okumura, Katsuhiko; Yokota, Toyokazu; Okumura, Yasushi

doi:10.1271/bbb.64.2138

Cited by 21 publications

(9 citation statements)

References 27 publications

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“…Wood et al (32) were also able to detect separate secretion of light ( ) chains and heavy ( ) chains in Saccharomyces cerevisiae. In contrast, Takahashi et al (25) were unable to detect secreted light or heavy (Fd fragment) chains when these were expressed individually in P. pastoris.…”

Section: Discussionmentioning

confidence: 91%

“…For example, quantities of 1 to 2 g of soluble, assembled FabЈ fragments/liter have been demonstrated in the periplasm of Escherichia coli (3). Secretion of ScFv fragments into the culture supernatant of Pichia pastoris at high titers, e.g., 250 mg/liter and 1.2 g/liter, has been reported by Eldin et al (6) and by Freyre et al (8), respectively, and up to 40 mg of secreted Fab/liter has been achieved with this yeast (16,25). In comparison to antibody fragments, there are very few reports of the production of full-length antibodies in microbial systems and, when reported, the titers have been very low (11,32).…”

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confidence: 77%

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Characterization of Humanized Antibodies Secreted byAspergillus niger

Ward¹,

Lin²,

Victoria³

et al. 2004

Appl Environ Microbiol

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Two different humanized immunoglobulin G1( ) antibodies and an Fab fragment were produced by Aspergillus niger. The antibodies were secreted into the culture supernatant. Both light and heavy chains were initially synthesized as fusion proteins with native glucoamylase. After antibody assembly, cleavage by A. niger KexB protease allowed the release of free antibody. Purification by hydrophobic charge induction chromatography proved effective at removing any antibody to which glucoamylase remained attached. Glycosylation at N297 in the Fc region of the heavy chain was observed, but this site was unoccupied on approximately 50% of the heavy chains. The glycan was of the high-mannose type, with some galactose present, and the size ranged from Hex 6 GlcNAc 2 to Hex 15 GlcNAc 2 . An aglycosyl mutant form of antibody was also produced. No significant difference between the glycosylated antibody produced by Aspergillus and that produced by mammalian cell cultures was observed in tests for affinity, avidity, pharmacokinetics, or antibody-dependent cellular cytotoxicity function.

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Section: Discussionmentioning

confidence: 91%

mentioning

confidence: 77%

Characterization of Humanized Antibodies Secreted byAspergillus niger

Ward¹,

Lin²,

Victoria³

et al. 2004

Appl Environ Microbiol

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“…Functional Fab fragments could be efficiently secreted into the culture supernatant up to 40 mg/L in fed batch fermentations (Lange et al, 2001); however, only about 30% were correctly assembled to the heterodimer. Also Takahashi et al (2000) succeeded in Fab secretion driven by the P. pastoris phosphatase leader. Most of the Fab fragments could be detected in the supernatant and only little in the insoluble fraction of the cells.…”

Section: Introductionmentioning

confidence: 98%

Engineering of Pichia pastoris for improved production of antibody fragments

Gasser

Maurer

Gach

et al. 2006

Biotech & Bioengineering

176

133

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The methylotrophic yeast Pichia pastoris has been used for the expression of many proteins, including antibody fragments. However, limitations became obvious especially when secreting heterodimeric Fab fragments. Up-to-date, antibody fragments have only been expressed under control of the strong inducible alcohol oxidase 1 (AOX1) promoter, which may stress the cells by excessive transcription. Here, we examined the secretion characteristics of single chain and Fab fragments of two different monoclonal anti-HIV1 antibodies (2F5 and 2G12) with both the AOX1 and the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. Also, the influences of different secretion leaders and strains were evaluated. Interestingly, secretion was only achieved when using the GAP promoter and the Saccharomyces cerevisiae mating factor alpha (MFalpha leader), whereas there was no difference between the two P. pastoris strains. During fed batch fermentation of a 2F5 Fab expressing strain, intracellular retention of Fab heavy chains was observed, while both intact Fab and single light chain molecules were only detected in the supernatants. This led to the conclusion that protein folding and heterodimer assembly in the ER are rate limiting steps in Fab secretion. To alleviate this limitation, S. cerevisiae protein disulfide isomerase (PDI) and the unfolded protein response (UPR) transcription factor HAC1 were constitutively overexpressed in P. pastoris. While the overexpression of HAC1 led to a moderate increase of Fab secretion of 1.3-fold, PDI enabled an increase of the Fab level by 1.9-fold. Hence, the formation of interchain disulfide bonds can be seen as a major rate limiting factor to Fab assembly and subsequent secretion.

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“…The quantity of the recombinant Fab fragment was up to 420~458 mg/L in 5 L bioreactor, which is much higher than that of other Fab fragments expressed in P. pastoris (Takahashi et al, 2000;Lange et al, 2001). The recombinant humanized anti-HBsAg Fab fragment was purified effectively by ion-exchange chromatography from the culture supernatant.…”

Section: Discussionmentioning

confidence: 98%

Production of Recombinant Humanized Anti-HBsAg Fab Fragment from Pichia pastoris by Fermentation

Deng¹,

Jin²,

Zhang³

et al. 2005

BMB Reports

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In this report, we describe the high-yield secretory expression of the recombinant human anti-HBsAg Fab fragment from Pichia pastoris that was achieved by cointegration of the genes encoding the heavy and light chains (both under the control of alcohol oxidase promoter) into the genome of the yeast cells. The fed-batch fermentations were carried out in a 5 L scale. Both chains of the Fab were successfully expressed upon methanol induction. The absorbance (OD 6 0 0 ) of the broth can reach 350~500 at the end of fed-batch phase. After the induction, the expression level of the recombinant Fab (soluble) reached 420~458 mg/L. The recombinant Fab fragment was purified from the crude culture supernatant by ion exchange chromatography and the purity of the recombinant Fab fragment was over 95%. The affinity activities of the crude fermentation supernatant and the purified Fab were analyzed by indirect ELISA, which showed that the purified recombinant Fab fragment had high affinity activity with hepatitis B surface antigen.

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Production of Humanized Fab Fragment against Human High Affinity IgE Receptor inPichia pastoris

Cited by 21 publications

References 27 publications

Characterization of Humanized Antibodies Secreted byAspergillus niger

Characterization of Humanized Antibodies Secreted byAspergillus niger

Engineering of Pichia pastoris for improved production of antibody fragments

Production of Recombinant Humanized Anti-HBsAg Fab Fragment from Pichia pastoris by Fermentation

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