Cooverexpression of chaperones for enhanced secretion of a single-chain antibody fragment in Pichia pastoris

Damasceno, Leonardo M.; Anderson, Kyle A.; Ritter, Gerd; Cregg, James M.; Old, Lloyd J.; Batt, Carl A.

doi:10.1007/s00253-006-0652-7

Cited by 126 publications

(72 citation statements)

References 42 publications

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“…Then Potgieter et al (2009) presented the production of more than 1 g L À1 of a functional, full length antibody by a glycoengineered yeast strain in a robust, scaleable cultivation process with uniform N-linked glycans. However, for antibody fragments, expression levels in excess of 8 g L À1 have been achieved in P. pastoris (Damasceno et al, 2007) using a combination of strain improvements and process optimization techniques. Strain improvements included (i) co-expression of folding chaperones (Damasceno et al, 2007) such as immunoglobulin binding protein (BiP) and protein disulfide isomerase (PDI), (ii) protease deletions (Brankamp et al, 1995), (iii) optimization of secretion leaders , and (iv) the use of fusion proteins (Reitinger et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

Antibody expression kinetics in glycoengineered Pichia pastoris

Potgieter

Kersey²,

Mallem³

et al. 2010

Biotech & Bioengineering

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Growth of the antibody market has fueled the development of alternative expression systems such as glycoengineered yeast. Although intact antibody expression levels in excess of 1 g L(-1) have been demonstrated in glycoengineered yeast, this is still significantly below the titers reported for antibody fragments in fungal expression systems. This study presents a simplified approach to estimate antibody secretion kinetics and oxygen uptake rate requirements as a function of growth-rate controlled by a limiting methanol feed rate in glycoengineered Pichia pastoris. The yield of biomass from methanol and the specific oxygen requirements predicted in this study compare well with values reported in the literature for wild-type P. pastoris, indicating the intrinsic nature of these yields independent of glycoengineering or the heterologous protein expressed. Specific productivity was found to be a non-linear function of specific growth rate. Based on comparison with relationships between specific growth rate and specific productivity reported in the literature this correlation seems empirical in nature and cannot be established a priori. These correlations were then used in a simple mass balance based model to predict the cultivation performance of carbon limited cultivations under oxygen transfer limited conditions to indicate the usefulness of this approach to predict large scale performance and aid in process development.

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Section: Introductionmentioning

confidence: 99%

Antibody expression kinetics in glycoengineered Pichia pastoris

Potgieter

Kersey²,

Mallem³

et al. 2010

Biotech & Bioengineering

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“…To attain this goal, the introduction of chaperones may improve the assembly of the antibody in the ER, resulting in efficient secretion. However, introduction of the chaperones was not sufficient to yield high amounts of full-length antibody in yeast, while the overexpression of chaperones such as disulfide isomerase have been reported to contribute partially to the enhancement of the secretability of the antibody fragment (8,14,36). In contrast, an increase in secretion was confirmed with several kinds of different antibodies by the addition of R3AD to O. minuta cultures (data not shown).…”

Section: Discussionmentioning

confidence: 65%

Efficient Antibody Production upon Suppression of O Mannosylation in the Yeast Ogataea minuta

Kuroda

Kobayashi

Kitagawa

et al. 2008

Appl Environ Microbiol

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When antibodies were expressed in the methylotrophic yeast Ogataea minuta, we found that abnormal O mannosylation occurred in the secreted antibody. Yeast-specific O mannosylation is initiated by the addition of mannose at serine (Ser) or threonine (Thr) residues in the endoplasmic reticulum via protein O mannosyltransferase (Pmt) activity. To suppress the addition of O-linked sugar chains on antibodies, we examined the possibility of inhibiting Pmt activity by the addition of a Pmt inhibitor during cultivation. The Pmt inhibitor was found to partially suppress the O mannosylation on the antibodies. Surprisingly, the suppression of O mannosylation was associated with an increased amount of assembled antibody (H2L2) and enhanced the antigen-binding activity of the secreted antibody. In this study, we demonstrated the expression of human antibody in O. minuta and elucidated the relationship between O mannosylation and antibody production in yeast.Antibodies for pharmaceutical applications have recently received a great deal of attention due to their specific antigenbinding activities, antibody-dependent cellular cytotoxicity, and other useful characteristics. Indeed, sales of antibodies for pharmaceuticals have grown rapidly over the past decade and are expected to exceed 30 billion U.S. dollars by 2010 (1). A large volume of antibodies is required for pharmaceuticals, as antibodies are primarily marketed for chronic conditions. Antibodies possess relatively low potency, which results in the need for accumulating doses over time (10). Although therapeutic antibodies have great potential value, they have also been viewed with some skepticism. Antibodies are expensive to produce, in part because they are commonly manufactured by using batch/fed-batch cultures of mammalian cells. The increasing demand to reduce the cost of antibody production has promoted research into the development of an antibody expression system that is more practical than expression with mammalian cells.Development of transgenic plants and animals as alternative hosts is therefore a promising field of study. Monoclonal antibodies have, thus far, been successfully produced from a number of sources, including plants, the milk of transgenic goats, the eggs of transgenic chickens, etc. (4, 12, 32). Moreover, antibodies derived from certain newly developed transgenic systems share physical characteristics that are similar to those of antibodies from mammalian cells such as Chinese hamster ovary (CHO) cells while exhibiting higher antibody-dependent cellular cytotoxicity activity due to the absence of fucose residues in N-linked sugar chains (6, 45). These alternative transgenic expression systems could reduce the cost of large-scale antibody production. However, the prolonged construction of transgenics remains a major disadvantage in terms of market demands.The production of antibodies and antibody fragments has been studied by using various microorganism expression systems, including Escherichia coli, fungus, and yeast (7,13,37,41). Since microorg...

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“…In eukaryotic cells the focus has been more on the ability of chaperones to reduce the cell toxicity of polyQ and asynuclein aggregates linked with the occurrence of neurodegenerative diseases such as Huntington and Parkinson (Auluck et al, 2002;Cummings et al, 2001;Muchowski and Wacker, 2005;Willingham et al, 2003). Fewer studies (Damasceno et al, 2007;Gasser et al, 2007;Smales et al, 2004) have examined the consequences of manipulation of the chaperone network in the context of recombinant protein production in cultured cell lines.…”

Section: Introductionmentioning

confidence: 97%

Transient expression of human TorsinA enhances secretion of two functionally distinct proteins in cultured Chinese hamster ovary (CHO) cells

Jossé

Smales

Tuite

2009

Biotech & Bioengineering

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Cultured mammalian cells, particularly Chinese hamster ovary (CHO) cells, are widely exploited as hosts for the production of recombinant proteins, but often yields are limiting. Such limitations may be due in part to the misfolding and subsequent degradation of the heterologous proteins. Consequently we have determined whether transiently co-expressing yeast and/or mammalian chaperones that act to disaggregate proteins, in CHO cell lines, improve the levels of either a cytoplasmic (Fluc) or secreted (Gluc) form of luciferase or an immunoglobulin IgG4 molecule. Over-expression of the yeast 'protein disaggregase' Hsp104 in a CHO cell line increased the levels of Fluc more significantly than for Gluc although levels were not further elevated by over-expression of the yeast or mammalian Hsp70/40 chaperones. Over-expression of TorsinA, a mammalian protein related in sequence to yeast Hsp104, but located in the ER, significantly increased the level of secreted Gluc from CHO cells by 2.5-fold and to a lesser extent the secreted levels of a recombinant IgG4 molecule. These observations indicate that the over-expression of yeast Hsp104 in mammalian cells can improve recombinant protein yield and that over-expression of TorsinA in the ER can promote secretion of heterologous proteins from mammalian cells.

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Cooverexpression of chaperones for enhanced secretion of a single-chain antibody fragment in Pichia pastoris

Cited by 126 publications

References 42 publications

Antibody expression kinetics in glycoengineered Pichia pastoris

Antibody expression kinetics in glycoengineered Pichia pastoris

Efficient Antibody Production upon Suppression of O Mannosylation in the Yeast Ogataea minuta

Transient expression of human TorsinA enhances secretion of two functionally distinct proteins in cultured Chinese hamster ovary (CHO) cells

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