Overcoming Obstacles to Antibody Production with Hybridoma Sequencing

At Absolute Antibody, we’ve successfully sequenced more than 1,900 hybridomas in the last two years. We use high-throughput (NGS) sequencing to deliver all variable heavy (VH) and variable light (VL) domains of hybridoma cell lines, as well as a full report characterizing the hybridoma’s sequences and any heterogenicity or other antibody isotypes that might be present.

Our novel NGS approach offers several key advantages compared to traditional sequencing methods such as V-region PCR or 5’ RACE sequencing. It ensures you receive the correct variable domains even if the original hybridoma was not truly monoclonal, works on any species or isotype, and can provide sequencing results from low quality or quantity starting material.

Read the case studies below to see how our next-generation sequencing has helped some of our clients sequence their hybridomas and overcome obstacles to antibody production.

Unknown Isotype

A clone was provided to us by our client 4TEEN4 Pharmaceuticals and upon sequencing, two heavy chains and one light chain were identified. This client had previously worked with a CRO on this mouse IgG1. NGS had identified the primary full-length sequence to be a mouse IgG2b, with a secondary mouse IgG1 expressed at a very low level. Our method identifies variable heavy and light chains separately. It is also able to determine relative abundance of each identified gene reported as transcripts per million (TPM).

The CRO had performed peptide mapping and, although uncertain, incorrectly told the client that it was mouse IgG1. Our RNA-Seq method identifies all antibody related genes within the transcriptome, while the PCR/Sanger method would have failed to identify the mouse IgG2b based on its reliance on primer specificity.

Resurrecting a Dead Hybridoma

Dr. Jake Kurtis, Professor of Pathology and Laboratory Medicine at Brown University, studies malaria and had identified potential vaccine candidates. One monoclonal antibody was particularly effective at killing malaria in culture but then suddenly stopped producing antibodies.

We began to work with the Kurtis laboratory to obtain the sequence from the limited amount of remaining non-viable cells. The material was heavily degraded, but we were able to produce a sequence using RNA Seq. We were able to recombinantly produce the antibody in gram quantities and the research continued. PCR/Sanger sequencing would have relied on primer binding to conserved sites in CH/CL, which is not always possible when working with such a limited amount of degraded material. Read the full case study here.

In April 2020, the Kurtis laboratory published a paper in Nature on the anti-malaria target,  a protein called PfGARP. Absolute Antibody is cited in the methods section of the paper (PMID 32427965).

Contamination in a Cell Line

Unbeknownst to our client in the biopharmaceutical industry, contamination of their cell line had occurred during passage before the cells were frozen. When our laboratory sequenced the hybridoma, we identified the presence of duplicate heavy and light chains. Our client then asked us to sequence an older vial from their cell bank. This time there was only one isotype, the expected IgG1. The PCR/Sanger sequencing method is not capable of separating multiple genes of the same species and isotype. Using this transitional method would not have identified the two separate antibodies and the contamination would have remained undiscovered.

Our method involves RT-PCR, using a random hexamer prior to high-throughput NGS sequencing, followed by assembly of contigs with our proprietary algorithm. We then mine the data for all viable antibody sequences identifying variable heavy and light chains separately. This is how we discovered the two antibodies present in the hybridoma cell line.

A Species Mix-Up

One of our biotechnology clients provided us with a murine clone where the variable regions were identified following 5’RACE (Rapid amplification of cDNA ends). We identified both the heavy and light chains but achieved no expression during production. During a second sequencing run, this time using our NGS method, we identified the identical light chain but a different variable heavy chain. Homology in the primer binding site is likely what allowed us to identify the same light chain, but when full length constant regions were obtained for both heavy and light chains, they aligned to a hamster rather than a mouse.

This time, the expression of the hamster VH/VL was successful. Traditional PCR is dependent on primer specificity to species and isotype. Our platform is neither species nor isotype specific, thus eliminating dependence on conserved primer binding sites. In this case, our client did not have accurate information on their clone, which can be a common problem.

 

Learn more about our next-generation sequencing on our website or contact us if you have any questions. We are always happy to help!