Optimizing antibody expression: The nuts and bolts

Ayyar, B. Vijayalakshmi; Arora, Sushrut; Ravi, Shiva Shankar

doi:10.1016/j.ymeth.2017.01.009

Cited by 30 publications

(28 citation statements)

References 136 publications

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“…The large size and crystallizable fragment (Fc) domain of mAbs results in slow tissue penetration, long half‐lives, and expensive and sometimes difficult production methods, which are often not suitable for therapeutic and imaging applications . rAb fragments can be engineered to overcome these issues by controlling rAb size and configuration, binding affinity and specificity, and effector properties, and by fusing rAbs to other proteins or tags that facilitate detection and purification …”

Section: Methodsmentioning

confidence: 99%

“…Genetic‐engineering approaches have been used to construct rAb fragments that can be expressed in bacteria, yeast, or mammalian cells . Extensive studies on antibody structure have identified domains with discrete functions that can be expressed independently.…”

Section: Methodsmentioning

confidence: 99%

“…[1] rAb fragments can be engineered to overcome these issues by controlling rAb size and configuration, binding affinity and specificity,a nd effector properties, and by fusing rAbs to other proteins or tags that facilitated etectiona nd purification. [2] Genetic-engineering approaches have been used to construct rAb fragments that can be expressed in bacteria, yeast, or mammalian cells. [2] Extensive studies on antibody structure have identified domains with discrete functions that can be expressed independently.Anumber of these rAb domainsh ave been expressed, including variable heavy (VH), variable light (VL), antigen-binding fragment (Fab), and Fc domains.…”

mentioning

confidence: 99%

“…[2] Genetic-engineering approaches have been used to construct rAb fragments that can be expressed in bacteria, yeast, or mammalian cells. [2] Extensive studies on antibody structure have identified domains with discrete functions that can be expressed independently.Anumber of these rAb domainsh ave been expressed, including variable heavy (VH), variable light (VL), antigen-binding fragment (Fab), and Fc domains. [3] Relative to mAbs, smaller rAb fragments penetrate tissues faster and are rapidlyc leared through the kidney.…”

mentioning

confidence: 99%

“…[4] rAbs, rAb fragments, and unnatural rAb fusion proteins are often difficult to express and have poor solubility,a ggregate, and are toxic. [2] The productiono fr Ab andr Ab fragments is influenced by an umber of factors including size, sequence, Figure 1. Analysis of rAb SpyTag and SpyCatcher partsand devices.…”

mentioning

confidence: 99%

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Synthetic Modular Antibody Construction by Using the SpyTag/SpyCatcher Protein‐Ligase System

et al. 2017

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Efforts to engineer recombinant antibodies for specific diagnostic and therapy applications are time consuming and expensive, as each new recombinant antibody needs to be optimized for expression, stability, bio-distribution, and pharmacokinetics. We have developed a new way to construct recombinant antibody-like "devices" by using a bottom-up approach to build them from well-behaved discrete recombinant antibody domains or "parts". Studies on antibody structure and function have identified antibody constant and variable domains with specific functions that can be expressed in isolation. We used the SpyTag/SpyCatcher protein ligase to join these parts together, thereby creating devices with desired properties based on summed properties of parts and in configurations that cannot be obtained by using genetic engineering. This strategy will create optimized recombinant antibody devices at reduced costs and with shortened development times.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Synthetic Modular Antibody Construction by Using the SpyTag/SpyCatcher Protein‐Ligase System

et al. 2017

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Tailoring translational strength using Kozak sequence variants improves bispecific antibody assembly and reduces product‐related impurities in CHO cells

Blánco¹,

Williams²,

Tang³

et al. 2020

Biotech & Bioengineering

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Optimal production of bispecific antibodies (bsAb) requires efficient and tailored co‐expression and assembly of two distinct heavy and two distinct light chains. Here, we describe a novel technology to modulate the translational strength of antibody chains via Kozak sequence variants to produce bsAb in a single cell line. In this study, we designed and screened a large Kozak sequence library to identify 10 independent variants that can modulate protein expression levels from approximately 0.2 to 1.3‐fold compared with the wild‐type sequence in transient transfection. We used a combination of several of these variants, covering a wide range of translational strength, to develop stable single cell Chinese hamster ovary bispecific cell lines and compared the results with those obtained from the wild‐type sequence. A significant increase in bispecific antibody assembly with a concomitant reduction in the level of product‐related impurities was observed. Our findings suggest that for production of bsAb it can be advantageous to modify translational strength for selected protein chains to improve overall yield and product quality. By extension, tuning of translational strength can also be applied to improving the production of a wide variety of heterologous proteins.

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Effects of variable domain orientation on anti‐HER2 single‐chain variable fragment antibody expressed in the Escherichia coli cytoplasm

Koçer

Cox

DeLisa

et al. 2020

Biotechnology Progress

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Single-chain variable fragment (scFv) antibodies have great potential for a range of applications including as diagnostic and therapeutic agents. However, production of scFvs is challenging because proper folding and activity depend on the formation of two intrachain disulfide bonds that do not readily form in the cytoplasm of living cells. Functional expression in bacteria therefore involves targeting to the more oxidizing periplasm, but yields in this compartment can be limiting due to secretion bottlenecks and the relatively small volume compared to the cytoplasm. In the present study, we evaluated an anti-HER2 scFv, which is specific for human epidermal growth receptor 2 (HER2) overexpressed in breast cancer, for functional expression in the cytoplasm of Escherichia coli strains BL21(DE3) and SHuffle T7 Express, the latter of which is genetically engineered for cytoplasmic disulfide bond formation. Specifically, we observed much greater solubility and binding activity with SHuffle T7 Express cells, which likely resulted from the more oxidative cytoplasm in this strain background. We also found that SHuffle T7 Express cells were capable of supporting high-level soluble production of anti-HER2 scFvs with intact disulfide bonds independent of variable domain orientation, providing further evidence that SHuffle T7 Express is a promising host for laboratory and preparative expression of functional scFv antibodies.

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Optimizing antibody expression: The nuts and bolts

Cited by 30 publications

References 136 publications

Synthetic Modular Antibody Construction by Using the SpyTag/SpyCatcher Protein‐Ligase System

Synthetic Modular Antibody Construction by Using the SpyTag/SpyCatcher Protein‐Ligase System

Tailoring translational strength using Kozak sequence variants improves bispecific antibody assembly and reduces product‐related impurities in CHO cells

Effects of variable domain orientation on anti‐HER2 single‐chain variable fragment antibody expressed in the Escherichia coli cytoplasm

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