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Case Study


Gigagen leverages the power of Ranomics' combinatorial variant libraries

In a groundbreaking collaboration, Gigagen harnessed the capabilities of the Ranomics VariantFind platform to elevate their antibody affinity maturation process.

 

The collaboration's success is documented in the publication titled:

"Affinity Maturation of Antibodies by Combinatorial Codon Mutagenesis versus Error-Prone PCR"

This case study highlights the pivotal role played by Ranomics VariantFind in creating variant libraries for accelerating antibody engineering. The publication showcases the unparalleled efficiency and precision of our platform in comparison to traditional methods such as error prone PCR.

Revolutionizing Antibody Affinity Maturation Of Therapeutic Monoclonal Antibodies 

affinity maturation workflow_gigagen publication.jpg

Affinity maturation workflow

The VariantFind method aimed to mutate two amino acids within the six CDRs of the light and heavy chains, whereas the epPCR method aimed to mutate three amino acids anywhere along the entire scFv sequences 

Key points:

Objective:

The primary objective of this study is to rigorously assess the utility and effectiveness of two distinct methodologies for antibody affinity maturation. Specifically, the study aims to compare the traditional approach of Error-Prone PCR (epPCR) with Ranomics' combinatorial NNK codon mutagenesis targeted at the Complementarity Determining Regions (CDRs).

Methodologies:

  • The study utilized yeast surface display to create eight yeast single-chain variable fragment (scFv) libraries for affinity maturation of antibodies targeting immuno-oncology markers PD-1, PD-L1, CTLA-4, and OX40.

  • This involved employing both error-prone PCR (epPCR) and degenerate NNK combinatorial codon mutagenesis. The combinatorial codon mutagenesis was facilitated by the VariantFind platform from Ranomics Inc. VariantFind libraries contained 3–5 × 10e5 possible scFv variants, and the epPCR libraries theoretically as many as 10e10 potential variants.

  • The yeast libraries were screened for improved affinity, and long-read high throughput sequencing (Pacific Biosciences) before and after sorting was used to accurately quantify the frequency of each amino acid mutation across the full heavy and light chain V(D)J sequences.

  • The iterative optimization process, guided by sequencing data, aimed at refining the libraries and identifying promising antibody variants.

 

This methodology, integrating advanced mutagenesis, yeast display, and sequencing technologies, contributed to the in-depth exploration of antibody affinity maturation.

Results and Comparative Analysis

  1. VariantFind NNK-directed Mutagenesis:

  • No restrictions on spatial separation between mutated codons.

  • Absence of strong positional biases.

  • Occasional low presence of specific amino acids in given positions.

  • Substantial representation of amino acids observed across all templates.

   2. Comparative Analysis with epPCR

  • Similar patterns across all four targets.

  • Slightly higher tendency for epPCR to incorporate negatively charged and polar side chains.

  • More substantial differences for individual amino acids.

   3. Amino Acid Class Distribution:

  • VariantFind reflects the expected NNK codon distribution.

  • Not detectably influenced by the original amino acid.

   4. epPCR amino acid base

  • Biases observed for particular amino acids.

  • Likelihood of a derived amino acid codon influenced by the parental sequence's codon.

   5. Key Distinction:

  • VariantFind lacks parental sequence codon influence on derived amino acid codons.

  • Flexibility and unbiased representation distinguish VariantFind from epPCR mutagenesis.

Significance and Implications

Gigagen's "Affinity Maturation of Antibodies by Combinatorial Codon Mutagenesis versus Error-Prone PCR" is a seminal work that not only highlights the nuanced intricacies of two prominent techniques but also contributes significantly to the evolving field of antibody engineering.

This publication, enriched by the combinatorial mutagenesis libraries provided by Ranomics Inc, serves as a valuable resource for researchers seeking to optimize their methods in antibody affinity maturation and advance the frontiers of biotechnological innovation.

We recently performed specific mutagenesis of all six CDRs in a single scFv antibody sequence with Ranomics. We were very pleased with the mutation diversity that was obtained and were impressed with how specific (and fast) the mutations could be introduced to make a new library.

Adam Adler, CSO at Gigagen

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