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Optimization of Enzymatic Antibody Fragmentation for Yield, Efficiency, and Binding Affinity.

ABSTRACT: Enzymatic antibody fragmentation has been well studied for various hosts and isotypes, but fragmentation patterns also vary unpredictably by clone, and optimizing Fab or F(ab')2 production by trial and error consumes large quantities of antibodies. Here, we report a systematic strategy for optimizing functional F(ab')2 production via pepsin digestion from small quantities of IgG. We tested three key parameters that affect fragmentation, pH, enzyme concentration (% pepsin w/w), and reaction time, and found that pH had the greatest impact on fragmentation yield and efficiency. We then developed a systematic approach to obtaining acceptable yields, digestion efficiency, and binding affinity. Three case studies are described to illustrate the approach. We anticipate that this work will provide a quick and cost-effective method for researchers to produce antibody fragments from whole IgG, avoiding haphazard trial and error.

SUBMITTER: Kinman AWL

PROVIDER: S-EPMC6718091 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Optimization of Enzymatic Antibody Fragmentation for Yield, Efficiency, and Binding Affinity.

Kinman Andrew W L AWL Pompano Rebecca R RR

Bioconjugate chemistry 20190128 3

Enzymatic antibody fragmentation has been well studied for various hosts and isotypes, but fragmentation patterns also vary unpredictably by clone, and optimizing Fab or F(ab')<sub>2</sub> production by trial and error consumes large quantities of antibodies. Here, we report a systematic strategy for optimizing functional F(ab')<sub>2</sub> production via pepsin digestion from small quantities of IgG. We tested three key parameters that affect fragmentation, pH, enzyme concentration (% pepsin w/ ...[more]

PMID: 30649877

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Project description:BackgroundNovel commercial kits for whole genome library preparation for next-generation sequencing on Illumina platforms promise shorter workflows, lower inputs and cost savings. Time savings are achieved by employing enzymatic DNA fragmentation and by combining end-repair and tailing reactions. Fewer cleanup steps also allow greater DNA input flexibility (1 ng-1 μg), PCR-free options from 100 ng DNA, and lower price as compared to the well-established sonication and tagmentation-based DNA library preparation kits.ResultsWe compared the performance of four enzymatic fragmentation-based DNA library preparation kits (from New England Biolabs, Roche, Swift Biosciences and Quantabio) to a tagmentation-based kit (Illumina) using low input DNA amounts (10 ng) and PCR-free reactions with 100 ng DNA. With four technical replicates of each input amount and kit, we compared the kits' fragmentation sequence-bias as well as performance parameters such as sequence coverage and the clinically relevant detection of single nucleotide and indel variants. While all kits produced high quality sequence data and demonstrated similar performance, several enzymatic fragmentation methods produced library insert sizes which deviated from those intended. Libraries with longer insert lengths performed better in terms of coverage, SNV and indel detection. Lower performance of shorter-insert libraries could be explained by loss of sequence coverage to overlapping paired-end reads, exacerbated by the preferential sequencing of shorter fragments on Illumina sequencers. We also observed that libraries prepared with minimal or no PCR performed best with regard to indel detection.ConclusionsThe enzymatic fragmentation-based DNA library preparation kits from NEB, Roche, Swift and Quantabio are good alternatives to the tagmentation based Nextera DNA flex kit from Illumina, offering reproducible results using flexible DNA inputs, quick workflows and lower prices. Libraries with insert DNA fragments longer than the cumulative sum of both read lengths avoid read overlap, thus produce more informative data that leads to strongly improved genome coverage and consequently also increased sensitivity and precision of SNP and indel detection. In order to best utilize such enzymatic fragmentation reagents, researchers should be prepared to invest time to optimize fragmentation conditions for their particular samples.

Project description:Since 2020, the receptor-binding domain (RBD) of the spike protein of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been constantly mutating, producing most of the notable missense mutations in the context of "variants of concern", probably in response to the vaccine-driven alteration of immune profiles of the human population. The Delta variant, in particular, has become the most prevalent variant of the epidemic, and it is spreading in countries with the highest vaccination rates, causing the world to face the risk of a new wave of the contagion. Understanding the physical mechanism responsible for the mutation-induced changes in the RBD's binding affinity, its transmissibility, and its capacity to escape vaccine-induced immunity is the "urgent challenge" in the development of preventive measures, vaccines, and therapeutic antibodies against the coronavirus disease 2019 (COVID-19) pandemic. In this study, entropy-enthalpy compensation and the Gibbs free energy change were used to analyze the impact of the RBD mutations on the binding affinity of SARS-CoV-2 variants with the receptor angiotensin converting enzyme 2 (ACE2) and existing antibodies. Through the analysis, we found that the existing mutations have already covered almost all possible detrimental mutations that could result in an increase of transmissibility, and that a possible mutation in amino-acid position 498 of the RBD can potentially enhance its binding affinity. A new calculation method for the binding energies of protein-protein complexes is proposed based on the entropy-enthalpy compensation rule. All known structures of RBD-antibody complexes and the RBD-ACE2 complex comply with the entropy-enthalpy compensation rule in providing the driving force behind the spontaneous protein-protein docking. The variant-induced risk of breakthrough infections in vaccinated people is attributed to the L452R mutation's reduction of the binding affinity of many antibodies. Mutations reversing the hydrophobic or hydrophilic performance of residues in the spike RBD potentially cause breakthrough infections of coronaviruses due to the changes in geometric complementarity in the entropy-enthalpy compensations between antibodies and the virus at the binding sites.

Dataset Information

Optimization of Enzymatic Antibody Fragmentation for Yield, Efficiency, and Binding Affinity.

Publications

Optimization of Enzymatic Antibody Fragmentation for Yield, Efficiency, and Binding Affinity.

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