Antibody Sequencing and High Throughput Production for Hybridoma Libraries

In vitro diagnostic and reagent production companies have historically relied on hybridomas for the continuous production of their monoclonal antibodies. At face value, these seemingly sturdy cell lines can produce an infinite amount of antibody protein. However, the hidden risks of hybridomas are only realized when production begins to dwindle. Even “immortal” hybridoma cell lines can be affected by outside factors, rendering them no longer useful. And if that hybridoma supplies an in vitro diagnostic antibody or validated reagent, the results can be devastating.

Companies with hybridoma libraries will often utilize cell storage and cryopreservation as a back-up. However, neither of these options are risk free. The most secure option for protecting your antibody is hybridoma sequencing and recombinant expression.

Hybridoma Security and Recombinant Antibody Benefits

Hybridomas secreting highly validated antibodies take significant time and money to develop. In vitro diagnostic and reagent companies can have hybridoma libraries comprising hundreds, even thousands, of clones and these assets may not be as secure as needed to prevent catastrophic loss.

Threats to Hybridomas

The fragility of hybridoma production is often downplayed or not fully understood, and back-up storage options can provide a false sense of security. Hybridomas can be adversely affected by:

  • Cell line instability – Over time, antibody production from established hybridomas can diminish, requiring enrichment or re-cloning.
  • Genetic drift – Loss of binding, isotype switching, and complete loss of antibody expression can occur.
  • Cross contamination – Cells from a different species can contaminate and annex the antibody production. (Learn more: Using Antibody Sequencing to Save a Contaminated Hybridoma for Recombinant Expression)
  • Physical loss or destruction – Cell storage and cryopreservation are not 100% guaranteed to prevent the loss of hybridomas. Flooding, fires, power failures, and even human error can have dire consequences for long term storage.

Risk Mitigation

Hybridoma sequencing and animal-free recombinant production mitigates the risks to your antibody. Sequencing the antibody genes of your hybridoma offers the advantages of:

  • Security – The antibody can be indefinitely produced recombinantly even if the hybridoma line is lost.
  • Flexibility – Convert the antibody to any species, isotype, or format.
  • Intellectual property – Patent the sequences of unique variable domains.

Once the sequence is obtained, the antibody can be produced recombinantly. Antibody production in our animal-free transient expression system provides benefits compared to monoclonals from hybridomas.

  • Biological definition – Recombinant antibodies are defined at the sequence level and production guarantees high purity monoclonal antibodies. Research has shown that 30% of hybridomas are not monoclonal and can contain one or more additional productive heavy or light chains (Bradbury et al. 2018).
  • Lot-to-lot consistency – Genetically defined antibody sequences enable minimal variation between production lots.
  • Control of supply chain – Recombinant technology mitigates manufacturing risk by “digitizing” antibody assets and guaranteeing long-term reproducible production.

To simplify the sequencing of large numbers of antibodies, Absolute Antibody has developed a service tailor made for clients with extensive hybridoma portfolios.

Antibody Sequencing and In vitro Production

Moving a hybridoma collection to recombinant production does not need to be a difficult process. With 5000+ hybridomas sequenced and more than 23,000 different recombinant antibodies produced, Absolute Antibody has the expertise suited to working with reagent and IVD companies.

Antibody Sequencing

Absolute Antibody’s high-throughput Next Generation Sequencing (NGS) allows us to deliver all variable heavy (VH) and variable light (VL) domains of the hybridoma cell lines, regardless of species or isotype. We provide a full report characterizing the hybridoma’s sequences and heterogenicity or other antibody isotypes that may be present. This gives you the ability to select the correct variable domains before moving forward with an antibody candidate.

Antibody Expression

The production of recombinant antibodies involves cloning the antibody sequence into vectors, generating plasmid DNA for transfection, transiently transfecting cells, and finally expressing and purifying the antibody. Absolute Antibody’s recombinant antibody production service utilizes a serum-free mammalian expression system that generates high purity antibodies in an absolutely defined protein concentration, with all production occurring in our ISO 9001:2015-certified manufacturing facility in the UK. This animal-free method allows for the consistent and continual production of antibodies without the risk of genetic drift, contamination, or cell line death.

Hybridoma Conversion Service

Absolute Antibody enables companies with a monoclonal antibody portfolio to start converting their hybridoma library to a recombinant protein library, using our proven recombinant technology. Our high-throughput sequencing method can sequence up to 50 hybridomas concurrently, with bulk sequencing packages available. Our FleXpress™ high-throughput antibody production service offers customers the flexibility to rapidly express numerous recombinant antibodies, providing a cost-effective way to test recombinant production and take advantage of its many benefits.

The pilot project would start with ~100 hybridoma clones. Once functionality of the recombinant antibodies (equivalent to the original hybridoma-derived parent versions) is confirmed, additional bulk hybridoma batches can be converted to recombinant protein.

Additionally, our antibody engineering service can alter the species, isotype, and subtype of the antibody or reformat it into fragments, bispecifics, and multispecifics. Our team of antibody engineering experts can advise the best options for your antibodies.

Contact us today to discuss your antibody needs and to get started on your next project.

 

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