High-throughput screening of antibody variants for chemical stability: identification of deamidation-resistant mutants

DiCara, Danielle; Andersen, Nisana; Chan, Ronald; Ernst, James A.; Ayalon, Gai; Lazar, Greg A.; Agard, Nicholas J.; Hilderbrand, Amy E.; Hötzel, Isidro

doi:10.1080/19420862.2018.1504726

Cited by 14 publications

(15 citation statements)

References 39 publications

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“…58,59 In this work, we have shown that point mutations of select aromatic residues may dramatically reduce viscosity. Advancing these engineered antibodies into clinical development would require assessment and mitigation of immunogenicity risk 60,61 and any manufacturing liabilities, [62][63][64] which are beyond the scope of this study. Indeed, assessment of whether these viscosity-lowering mutants also have improved behavior with respect to other properties affected by protein-protein interactions (e.g., aggregation, opalescence, turbidity) could further streamline the removal of antibody liabilities and provide insight into the interactions involved in these phenomena.…”

Section: Discussionmentioning

confidence: 99%

Dissecting the molecular basis of high viscosity of monospecific and bispecific IgG antibodies

Tilegenova¹,

Izadi²,

Yin³

et al. 2019

mAbs

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Some antibodies exhibit elevated viscosity at high concentrations, making them poorly suited for therapeutic applications requiring administration by injection such as subcutaneous or ocular delivery. Here we studied an anti-IL-13/IL-17 bispecific IgG4 antibody, which has anomalously high viscosity compared to its parent monospecific antibodies. The viscosity of the bispecific IgG4 in solution was decreased by only ~30% in the presence of NaCl, suggesting electrostatic interactions are insufficient to fully explain the drivers of viscosity. Intriguingly, addition of arginine-HCl reduced the viscosity of the bispecific IgG4 by ~50% to its parent IgG level. These data suggest that beyond electrostatics, additional types of interactions such as cation-π and/or π-π may contribute to high viscosity more significantly than previously understood. Molecular dynamics simulations of antibody fragments in the mixed solution of free arginine and explicit water were conducted to identify hotspots involved in self-interactions. Exposed surface aromatic amino acids displayed an increased number of contacts with arginine. Mutagenesis of the majority of aromatic residues pinpointed by molecular dynamics simulations effectively decreased the solution’s viscosity when tested experimentally. This mutational method to reduce the viscosity of a bispecific antibody was extended to a monospecific anti-GCGR IgG1 antibody with elevated viscosity. In all cases, point mutants were readily identified that both reduced viscosity and retained antigen-binding affinity. These studies demonstrate a new approach to mitigate high viscosity of some antibodies by mutagenesis of surface-exposed aromatic residues on complementarity-determining regions that may facilitate some clinical applications.

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Section: Discussionmentioning

confidence: 99%

Dissecting the molecular basis of high viscosity of monospecific and bispecific IgG antibodies

Tilegenova¹,

Izadi²,

Yin³

et al. 2019

mAbs

View full text Add to dashboard Cite

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“…Our work provides a structural demonstration of how deamidation could affect antibody function. Furthermore, the saturation mutagenesis study results, together with other reports on surprising findings with mutants for deamidation stabilization, 8 indicates that extensive screening of stabilization mutants may be necessary to understand the structure-function relationship of the CDR deamidation sites, and ultimately obtain variants with increased resistance to deamidation for improved developability.…”

Section: Discussionmentioning

confidence: 79%

“…These liabilities include chemical degradations such as oxidation, deamidation, isomerization, and fragmentation. [6][7][8][9][10] Deamidation of asparagine (Asn) residues is a major posttranslational modification that can significantly impact protein structure and function. 11,12 The non-enzymatic modification proceeds via formation of a five-member ring succinimide intermediate, which is subsequently hydrolyzed into a mixture of isoaspartate (isoAsp) and aspartate (Asp).…”

Section: Introductionmentioning

confidence: 99%

Engineering an anti-CD52 antibody for enhanced deamidation stability

Qiu¹,

Wei²,

Bird³

et al. 2019

mAbs

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Deamidation evaluation and mitigation is an important aspect of therapeutic antibody developability assessment. We investigated the structure and function of the Asn-Gly deamidation in a human anti-CD52 IgG1 antibody light chain complementarity-determining region 1, and risk mitigation through protein engineering. Antigen binding affinity was found to decrease about 400-fold when Asn33 was replaced with an Asp residue to mimic the deamidation product, suggesting significant impacts on antibody function. Other variants made at Asn33 (N33H, N33Q, N33H, N33R) were also found to result in significant loss of antigen binding affinity. The co-crystal structure of the antigen-binding fragment bound to a CD52 peptide mimetic was solved at 2.2Å (PDB code 6OBD), which revealed that Asn33 directly interacts with the CD52 phosphate group via a hydrogen bond. Gly34, but sits away from the binding interface, rendering it more amendable to mutagenesis without affecting affinity. Saturation mutants at Gly34 were prepared and subjected to forced deamidation by incubation at elevated pH and temperature. Three mutants (G34R, G34K and G34Q) showed increased resistance to deamidation by LC-MS peptide mapping, while maintaining high binding affinity to CD52 antigen measured by Biacore. A complement -dependent cytotoxicity assay indicated that these mutants function by triggering antibody effector function. This study illustrates the importance of structure-based design and extensive mutagenesis to mitigate antibody developability issues.

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“…The development of a protein-based vaccine, therapeutic, or diagnostic reagent for a novel disease requires the screening of numerous expression cassettes, for example, to identify suitable regulatory elements (e.g., promoters) that achieve high levels of product accumulation, a sub-cellular compartment 1 https://coronavirus.jhu.edu/map.html that ensures product integrity, as well as different product candidates to identify the most active and most amenable to manufacturing in plants (Buyel et al, 2013a;Kohli et al, 2015;DiCara et al, 2018;Spiegel et al, 2019;Kerwin et al, 2020).…”

Section: Screening Of Product Candidatesmentioning

confidence: 99%

The Emergency Response Capacity of Plant-Based Biopharmaceutical Manufacturing-What It Is and What It Could Be

et al. 2020

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Several epidemic and pandemic diseases have emerged over the last 20 years with increasing reach and severity. The current COVID-19 pandemic has affected most of the world's population, causing millions of infections, hundreds of thousands of deaths, and economic disruption on a vast scale. The increasing number of casualties underlines an urgent need for the rapid delivery of therapeutics, prophylactics such as vaccines, and diagnostic reagents. Here, we review the potential of molecular farming in plants from a manufacturing perspective, focusing on the speed, capacity, safety, and potential costs of transient expression systems. We highlight current limitations in terms of the regulatory framework, as well as future opportunities to establish plant molecular farming as a global, de-centralized emergency response platform for the rapid production of biopharmaceuticals. The implications of public health emergencies on process design and costs, regulatory approval, and production speed and scale compared to conventional manufacturing platforms based on mammalian cell culture are discussed as a forward-looking strategy for future pandemic responses.

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High-throughput screening of antibody variants for chemical stability: identification of deamidation-resistant mutants

Cited by 14 publications

References 39 publications

Dissecting the molecular basis of high viscosity of monospecific and bispecific IgG antibodies

Dissecting the molecular basis of high viscosity of monospecific and bispecific IgG antibodies

Engineering an anti-CD52 antibody for enhanced deamidation stability

The Emergency Response Capacity of Plant-Based Biopharmaceutical Manufacturing-What It Is and What It Could Be

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