Hybridomas: All Steps Lead to Improvement

Hybridomas and related services are, with phage display, the main routes to discovering and developing monoclonal antibodies (mAbs) to specific antigens. Both methods have advantages and drawbacks, which is perhaps the topic of a future article.

As fusion products between myeloma cells and B cells, hybridomas retain the relevant characteristics of both—infinite expansion and mAb generation—while overcoming B cells' short lifespan. These characteristics have, in large part, supported the industrialization of mAb production and its billion-dollar markets.

Before hybridomas, researchers relied on polyclonal antibodies, which are collections of antibodies from different B cell populations, each recognizing a different epitope on the antigen. Polyclonal antibodies, which are part of a mammal's natural response to infection, generally provide greater coverage of an antigen's epitopes than monoclonal antibodies, roughly corresponding to natural immunity vs. induced (i.e., vaccinated) immunity.

Polyclonal antibodies are still relevant, however. Many diagnostic and research-use assays employ them, as well as drugs like Regeneron's REGN-COV2, a polyclonal treatment for COVID-19.

Polyclonals are easier to produce and expand than mAbs and generally more robust, advantages that translate to lower cost and shorter development times. Although they provide the most complete antigen/epitope coverage, polyclonal antibodies are heterogeneous and their component antibodies vary from batch to batch. Variability is normally the kiss of death for development-stage drugs, but that will probably change under the current expedited approval paradigm.

Regardless, says Dr. Weilin Liu, a Field Application Scientist at GenScript Biotech (Netherlands), hybridoma technology will remain the platform of choice for mAb work. "The stability of hybridoma methods, their easy maintenance, relatively low cost, and more importantly the reliable and never-ending supply of mAbs they make possible has fueled and accelerated the pace of antibody production and discovery."

Optimization opportunities

Multi-step hybridoma mAb protocols provide numerous opportunities for optimization, beginning with the earliest step of antigen optimization, which actually involves selection rather than improvement. Note that the "best" antigen for an immunoassay may not be the optimal antigen for a therapeutic. Improvements may similarly occur at any step, for example directing B cell maturation toward specific subtypes, during myeloma-B cell fusion, and of course during expansion through typical cell culture optimization.

"Optimization may occur at any step and requires multi-disciplinary expertise," says Dr. Liu.

One step recognized as critical from very early on is mAb humanization, which overcomes well-known immunogenic differences between humans and other species. "Our approach, which uses humanized transgenic mice and rats, together with B cells from immunized humans, largely resolves those issues," Dr. Liu says. The company uses a variety of immunogens, including DNA, peptides, recombinant proteins, whole cells, and virus-like particles.

Another area for improvement is self-tolerance, the process through which the immune system recognizes self-generated (but non-human) antigens as non-threatening. GenScript's ImmumoPlus^TM technology uses immunomodulators to circumvent this process and allow B cells to generate high-affinity mAbs. GenScript has tackled fusion and immortalization as well through Electrofusion, an alternative to more standard polyethylene glycol-based fusion methods. Electrofusion exposes B cells and cancer cells together to a pulsed electric field that brings the cells into close proximity and alignment, at which point a second pulse causes the cells to fuse.

GenScript also optimizes at the screening stage through a combination of proprietary and off-the-shelf selection methods. The company's combinatorial screening platforms, which include high-throughput flow cytometry, solve capacity and screening bottlenecks, which Dr. Liu regards as "the main hurdles in mAb development." Recently, GenScript incorporated a chip-based technology, Beacon^TM Single B cell, for isolating functional antibody-producing B cells in days instead of weeks.

Hybridomas as a service

Most hybridoma projects today are outsourced to specialty companies which, Dr. Liu says, provide greater consistency, higher throughput, more-rapid turnaround time, and relatively lower cost compared with an in-house development. "Outsourcing this has resulted in the establishment of well-characterized production cell lines and further innovations. Each stage, including cell fusion, screening, and subcloning, as well as characterization and production of the target mAb, presents unique challenges, and correlates closely with the success of subsequent steps."

"Many biopharmaceutical companies have internal hybridoma capabilities, though there has been a continued increase in the reliance on third-party services in recent years," says Barry Duplantis, Sr. Scientific Leader at Immunoprecise. "The choice to outsource hybridoma development is driven by lower costs, faster turnaround times, proprietary technologies, and access to highly qualified expertise." According to Duplantis, demand for hybridomas—the "workhorses for development of monoclonal antibodies," is driven by burgeoning development pipelines for therapeutic drugs to immunodiagnostic kits for cancer and infectious diseases, plus the pure research that supports these initiatives.

Immunoprecise uses a proprietary electrofusion platform which, Duplantis says, results in a greater number of hybridomas compared with traditional methods. "When combined with our proprietary semi-solid media workflow, we can ensure that every potential target-specific monoclonal antibody is identified within industry-leading timelines. Our rapid detection of target-specific clones allows for immediate validation, subcloning, cryopreservation, and DNA sequencing of valuable clones."

Hybridomas are not a perfect technology. Duplantis cites genetic drift and variability, and stability as potential drawbacks. "These issues are of concern, particularly when antibodies secreted from the hybridoma show altered characteristics over time, or the hybridoma stops producing the antibody of interest altogether. These concerns may be overcome through more-rapid identification of clones and the ability to sequence, cryopreserve, and isolate the genetic material required to convert a hybridoma into recombinant monoclonal antibody. Once in recombinant form, a monoclonal antibody is not subject to these challenges."