Recombinant Antibody Technology

Karu, Alexander E.; Bell, Christopher W.; Chin, Tina E.

doi:10.1093/ilar.37.3.132

Cited by 13 publications

(11 citation statements)

References 60 publications

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“…Fab molecules are initially produced as pro-proteins and their ability to be transported to the periplasm depends on the 15-25 amino acid leader sequence (ompA). After the translocation, this leader sequence is cleaved by specific peptidases to form a mature Fab-pIII fusion protein of approximately 50,000-60,000 Da (Karu et al, 1995). In the pComb3X system, the presence of an amber codon upstream of the pIII gene and the use of non-amber suppressor E. coli can be exploited to encourage the production of soluble antibody (Barbas et al, 2001).…”

Section: Expression Purification and Characterization Of Fab C37mentioning

confidence: 99%

“…Based on an initial characterization of selected Fabs, Fab clone C37 found to be was able to neutralize proteolytic activity by up to 80%, and therefore, was selected for further expression and characterization in this study. The vast number of vectors available for expression, the manipulation of cloned genes, and complete information on the Escherichia coli genome, have encouraged the use of E. coli as a protein expression host (Karu et al, 1995). Both polyclonal and monoclonal antibodies allow for the analysis of the structure-function relationship of the protease in the pathogenesis of melioidosis.…”

Section: Introductionmentioning

confidence: 99%

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Neutralizing Chimeric Mouse-human Antibodies against Burkholderia pseudomallei Protease: Expression, Purification and Characterization

Chan¹,

Ong²,

Nathan³

2004

BMB Reports

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A recombinant Fab monoclonal antibody (Fab) C37, previously obtained by phage display and biopanning of a random antibody fragment library against Burkholderia pseudomallei protease, was expressed in different strains of Escherichia coli. E. coli strain HB2151 was deemed a more suitable host for Fab expression than other E. coli strains when grown in media supplemented with 0.2% glycerol. The expressed Fab fragment was purified by affinity chromatography on a Protein G-Sepharose column, and the specificity of the recombinant Fab C37 towards B. pseudomallei protease was proven by Western blotting, enzyme-linked immunosorbent assay (ELISA) and by proteolytic activity neutralization. In addition, polyclonal antibodies against B. pseudomallei protease were produced in rabbits immunized with the protease. These were isolated from high titer serum by affinity chromatography on recombinant-Protein A-Sepharose. Purified polyclonal antibody specificity towards B. pseudomallei protease was proven by Western blotting and ELISA.

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Section: Expression Purification and Characterization Of Fab C37mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Neutralizing Chimeric Mouse-human Antibodies against Burkholderia pseudomallei Protease: Expression, Purification and Characterization

Chan¹,

Ong²,

Nathan³

2004

BMB Reports

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“…The scFv that was expressed in 0.2% (v/v) glycerol showed the highest affinity towards exotoxin in contrast to the scFv that was expressed in 0.05% (v/v) glycerol, as previously described. This could be due to the overproduction of the scFv protein in the periplasm that leads to the formation of inclusion bodies when the expression is conducted in 0.05% (v/v) glycerol (Carrier et al, 1993;Karu et al, 1995). The inclusion bodies resulted in the accumulation of misfolded and incorrect conformation of the scFv, hence decreasing the affinity of the antibodies towards its antigen (Carrier et al, 1993).…”

Section: Anti-b Pseudomallei Exotoxin Scfv Affinity By Indirectmentioning

confidence: 99%

“…The scFv gene was subsequently subcloned from the original expression vector pComb3H to pComb3X (Lim et al, manuscript in preparation), because the latter vector allows better detection and purification of the expressed products (Rader and Barbas, 1997). The vast number of vectors available for the expression and manipulation of cloned genes, and complete information of the Escherichia coli genome have encouraged the use of E. coli as a protein expression host (Karu et al, 1995). In this study, we describe the optimization of the scFv expression towards B. pseudomallei exotoxin by utilizing various E. coli host cell strains and different concentrations of carbon sources.…”

Section: Introductionmentioning

confidence: 99%

Bacterial Expression of the scFv Fragment of a Recombinant Antibody Specific for Burkholderia pseudomallei Exotoxin

Su¹,

Lim²,

Nathan³

2003

BMB Reports

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The scFv antibody towards the Burkholderia pseudomallei exotoxin was previously constructed by phage display and exhibited good specificity towards the exotoxin. We report here the optimization of the scFv expression in an E. coli expression system. Four different E. coli strains (ER2537, TG1, HB2151, and XL1-Blue) were examined for optimal expression of the scFv protein. Two types of carbon source (i.e. 0.2% glucose and 0.2% glycerol) were also tested for their ability to induce the scFv expression. Cells that carried the scFv construct were grown at 30 degrees C and induced with 0.05 mM IPTG. The expression was then monitored by SDS-PAGE, Western blotting, and indirect ELISA. The Western blot profile showed different levels of the scFv expression among the host strains; XL1-Blue exhibited the highest level of the scFv protein expression. Glycerol at a concentration of 0.2% (v/v) significantly increased the scFv protein expression level when compared to 0.2% (w/v) glucose. Further optimization demonstrated that the scFv protein expression in XL1-Blue was the most optimal with a glycerol concentration as low as 0.05%. However, by indirect ELISA, only the scFv protein that was expressed in 0.2% (v/v) glycerol exhibited high specificity towards the Burkholderia pseudomallei exotoxin.

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“…Bioprocess. (2019) 6:34 and bacterial systems, but suffer when over expressed resulting in aggregation leading to lower product quality (Karu et al 1995;Nelson and Reichert 2009). In addition to aggregation, both molecules are subject to oxidation, another major concern for maintaining the quality of the product (Karu et al 1995;Nelson and Reichert 2009;Yan et al 2009;Moritz and Stracke 2017).…”

Section: Introductionmentioning

confidence: 99%

Cell-culture growth conditions resulting in the oxidation of a recombinant antigen-binding fragment

Siddiquee

Zhao

Stemler

et al. 2019

Bioresour. Bioprocess.

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Use of Quality-by-Design (QbD) tools is becoming an important part of the bioprocessing industry when developing a process for manufacturing operations to ensure the robustness and reproducibility of the biologic product. In the present study, a QbD tool, Design of Experiments (DOE), was utilized to optimize a bioprocess for the production of a CHO recombinant antigen-binding fragment (rFab) in small-scale bioreactors. DOE studies evaluated percent dissolved oxygen, temperature, and feeding strategy specific to this Chinese Hamster Ovary (CHO) clone. It was determined that these factors influenced cell viability, yield of the recombinant protein, and metabolic byproduct formation. To ensure the quality of the target molecule in the cell-culture process, small-scale purifications and analytical evaluation of the target molecule were completed prior to cell-culture scale-up to ensure that oxidation of the rFab, presence of free light chain, and truncation of thiol group were not observed. Analysis of the purified rFab by mass spectrometry indicated that rFab oxidation occurred under poor cell-culture conditions. PCR profile array results also revealed increased transcription of the oxidative genes Superoxide Dismutase 3, Myeloperoxidase, Dual Oxidase Like 2, Nuclear Receptor Coactivator 7, NADPH Oxidase Organizer 1, Mitochondria Uncouple Protein 3, Eosinophil Peroxidase, Lactoperoxidase Like, Serum Albumin Like, and Glutathione S-Transferase Pi 1 in this CHO strain. The present study suggests a mechanism and pathway for the oxidation of an rFab molecule during cell-culture bioprocess optimization. The present study also demonstrated the importance of utilizing the QbD tool of DOE to optimize the cell-culture bioprocess prior to scaling up into the large-scale production bioreactor. which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

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Recombinant Antibody Technology

Cited by 13 publications

References 60 publications

Neutralizing Chimeric Mouse-human Antibodies against Burkholderia pseudomallei Protease: Expression, Purification and Characterization

Neutralizing Chimeric Mouse-human Antibodies against Burkholderia pseudomallei Protease: Expression, Purification and Characterization

Bacterial Expression of the scFv Fragment of a Recombinant Antibody Specific for Burkholderia pseudomallei Exotoxin

Cell-culture growth conditions resulting in the oxidation of a recombinant antigen-binding fragment

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