An efficient process of generating bispecific antibodies via controlled Fab-arm exchange using culture supernatants

Paul, Suparna; Connor, Judy; Nesspor, Tom; Haytko, Peter; Boakye, Ken; Chiu, Mark L.; Jiang, Haiyan

doi:10.1016/j.pep.2016.01.014

Cited by 15 publications

(13 citation statements)

References 24 publications

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“…A limited number of methodologies have been developed for such purpose and they mainly rely on LC‐based techniques such as RPLC‐MS following reduction and enzymatic digestion of polyclonal antibody product and CEX analysis of intact antibodies . Similarly, CEX and HIC analysis have been performed to separate the two parental mAbs from a bi‐specific antibody in order to determine the heterodimerization efficiency . CE‐based techniques may gain more popularity for the separation of mAb mixtures, especially when antibodies are strongly adsorbed on the LC column stationary phase as for belimumab and ixekizumab in CEX .…”

Section: Resultsmentioning

confidence: 99%

High‐resolution separation of monoclonal antibodies mixtures and their charge variants by an alternative and generic CZE method

et al. 2018

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The determination of mAb critical quality attributes (CQA) is crucial for their successful application in health diseases. A generic CZE method was developed for the high-resolution separation of various mAb charge variants, which are often recognized as important CQA. A dynamic coating of the capillary was obtained with polyethylene oxide (PEO), whereas Bis-Tris allowed the analysis of mAbs under native conditions at pH 7.0. The effect of PEO and Bis-Tris concentrations, as well as the nature of the acidic counter ion on the method performance was systematically studied. The %RSD on migration times was below 5% on three different CE instruments using the optimized method. Additional charge variants (in particular acidic variants) were resolved for 10 out of 17 mAbs compared to a reference CZE approach involving the use of ε-amino-caproic acid (EACA), triethylenetetramine (TETA), and hydroxypropylmethyl cellulose (HPMC). The amount of basic and acidic charge variants of 17 Food and Drug Administration (FDA) approved mAbs covering a wide range of physico-chemical properties, e.g., pI between 8.0 and 9.4 and different hydrophobicity, were mainly comprised between 5-15% and 15-30%, respectively. It is noteworthy that applications for the quality control in hospitals as well as for the combination of the immune checkpoint inhibitors nivolumab and ipilimumab were presented.

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Section: Resultsmentioning

confidence: 99%

High‐resolution separation of monoclonal antibodies mixtures and their charge variants by an alternative and generic CZE method

et al. 2018

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“…Since synthesis of BsAbs using chemical procedures requires highly purified preparations (usually using affinity chromatography) of the two antibodies and twin affinity purification steps to isolate the pure BsAbs, final yields are generally quite poor. To address the issue, Paul et al 39. prepared BsAbs by subjecting the culture supernatants of Human IgG 1 and Kappa antibodies (with point mutations) to Fab arm exchange in the unfractionated culture supernatant followed by affinity purification of the resulting bifunctional.…”

Section: Discussionmentioning

confidence: 99%

A detergent-based procedure for the preparation of IgG-like bispecific antibodies in high yield

Gupta

Hoque

Zaman

et al. 2016

Sci Rep

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Bispecific antibodies (BsAbs), with the ability to recognize two different epitopes simultaneously, offer remarkable advantages in bioassays, cancer therapy, biosensors, and enzyme electrodes. Preparation and purification of BsAbs in adequate quantities remains a major hurdle in their use in various applications. Poor yield is also the principal limitation in the preparation of BsAbs by the redox procedure. IgG with reduced inter-heavy chain disulfides do not dissociate into half molecules at neutral pH. In this study, we report that the dissociation occurs in presence of sodium dodecyl sulphate (SDS) and inclusion of the detergent during the redox procedure results in remarkable increase in the formation of the BsAbs. Exposure of antibodies to 0.1% (w/v) SDS causes only minor loss in secondary/tertiary structure and the ability to bind the antigen. The BsAbs prepared using the modified redox procedure that recognize the antigens HRP and α-LA were prepared and successfully employed for detecting α-LA in milk/dairy products by ELISA and dot blot techniques. BsAbs were also prepared from partially purified immunoglobulin gamma (IgG). This work shows for the first time that SDS, by dissociating IgG with reduced inter-heavy chain disulfides into half molecules, markedly enhances the formation of BsAbs by the redox procedure.

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“…As discussed in Section 2, nonspecific interactions between the proteins and the SEC stationary phase are the most critical measures of the applicability of SEC independently on the antibody type. To the best of our knowledge -and surprisingly -, very few SEC studies have been published for other therapeutic proteins than mAbs and ADCs, except Waxman et al who performed stability testing for Fc-fusion proteins and a cytokine [48] as well as Paul et al who evaluated the efficacy of a BsAb formation from two parental mAbs [49]. Interestingly, the two parent mAbs had different SEC elution/retention times, while the BsAb had an elution/retention time in between the two mAbs.…”

Section: Application Of Modern Uhp-sec To Real Case Studymentioning

confidence: 99%

“…Interestingly, the two parent mAbs had different SEC elution/retention times, while the BsAb had an elution/retention time in between the two mAbs. As discussed in Section 5, the significant difference of hydrophobicity between the two parent mAbs (HIC retention times of around 15 min vs. 45 min observed by running a generic linear inverse salt gradient) was likely to explain the unexpected SEC behavior [49]. Last but not least, biosimilarity evaluation also often includes SEC comparability studies for mAb [50,51], ADC [52], granulocyte colony stimulating factor (G-CSF) [53] and erythropoietin (EPO) [54] products.…”

Section: Application Of Modern Uhp-sec To Real Case Studymentioning

confidence: 99%

Unraveling the mysteries of modern size exclusion chromatography - the way to achieve confident characterization of therapeutic proteins

Goyon

Fekete

Beck

et al. 2018

Journal of Chromatography B

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Modern size exclusion chromatography (SEC) can be defined by the use of relatively small columns (e.g., 150 × 4.6 mm) packed with sub-3 μm particles, allowing a 3- to 5-fold increase in method throughput compared to that of conventional SEC. The quick success of the first sub-2 μm SEC column introduced in 2010 led to the development of numerous ultra-high performance (UHP)-SEC columns for the analysis of therapeutic monoclonal antibody (mAb)-based products. Aggregates also known as high-molecular-weight species (HMWS) are indeed one of the most important critical quality attributes (CQAs) of mAbs, as HMWS may decrease the product efficacy or cause immunogenicity effects. Therefore, the confident characterization of mAbs requires strong knowledge of not only modern SEC performance (i.e., selectivity and efficiency) but also the inherent limitations caused by non-specific interactions more likely to occur with complex antibody drug conjugates (ADCs) and some commercial mAb products. This review discusses the importance of liquid chromatographic (LC) instrumentation in order to exploit the full potential of modern SEC columns and current trends to hyphenate SEC to mass spectrometry (MS). Recent applications for antibody-based products (i.e., mAbs, ADCs, Fc-Fusion proteins and bispecific antibodies) are presented. Finally, tips and tricks are provided to further optimize SEC separations and maintaining their performance over time with better understanding of unexpected SEC results.

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An efficient process of generating bispecific antibodies via controlled Fab-arm exchange using culture supernatants

Cited by 15 publications

References 24 publications

High‐resolution separation of monoclonal antibodies mixtures and their charge variants by an alternative and generic CZE method

High‐resolution separation of monoclonal antibodies mixtures and their charge variants by an alternative and generic CZE method

A detergent-based procedure for the preparation of IgG-like bispecific antibodies in high yield

Unraveling the mysteries of modern size exclusion chromatography - the way to achieve confident characterization of therapeutic proteins

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