In contrast to natural antibodies, monoclonal antibodies (mAbs) offer less variability11, but even so, their production still makes their protein sequence susceptible to problems. Take hybridomas for instance, a common method used to produce mAbs11. In hybridoma technology, plasma cells are fused with myeloma cells. Hybridoma alternatives also rely on immortalization with an oncogene, or oncovirus11.
Figure 3. Illustration showing the typical hybridoma development process. De novo protein sequencing and mass spectrometric analysis at Rapid Novor (REpAb®, REmAb®, MATCHmAbTM) is routinely used to complement various stages of the hybridoma development process shown above.
The proteome of hybridoma cells is naturally prone to non-canonical splicing, mutations, fusions, and PTMs12. As such, through time, mAbs produced through these means result in the accumulation of problems4,5,13,14 that require further analysis through mass spectrometry-based proteomics means14-19.
Furthermore, hybridomas are often lost or misplaced2,3, and/or the antibody sequence has not been obtained in time from nucleotide sequencing means. The latter two are common and frequently given reasons for using de novo protein sequencing technology.
Next Generation Sequencing
Antibody discovery through next generation sequencing (NGS) is known as B cell repertoire (BCR) and single B cell sequencing. Both are slightly different approaches to obtain the immunoglobulin (Ig) gene sequences20,21.
In single B-cell sequencing, isolated peripheral blood mononuclear cells (PBMCs) are sorted using flow cytometry (e.g., florescence-activated cell sorting or FACS) to sequence individual B lymphocytes’ DNA or RNA with next generation sequencing22. Additional quantification and characterization are often performed to further understand the humoral response20.
When B cells are individually selected, light chain and heavy chain information per Ig sequence is retained20. In contrast, NGS of the BCR does not offer antibody heavy and light chain pairing information because all cells are homogenized prior to sequencing20,22,23. In both cases, resulting sequences are recombinantly expressed in mammalian cell lines (e.g., CHO, HEK 293)24,25.
Figure 4. Schematic illustrating an overview of next generation sequencing.
Single B-cell sequencing or NGS relies on culturing individual clones prior to sequencing the antibody genes; as a result, there is a potential for missing key B-cell clones from the BCR20,26,27. Novel mAb discovery also often requires spleen collection for NGS and thus, the death of the producer animal28. Thus, NGS on its own does not completely reflect the circulating antibody repertoire. Despite its high throughout nature, NGS has significant limitations as an antibody discovery tool26.
Synthetic Library Generation
B-cells are isolated from plasma to extract the mRNA and perform RT-PCR and other Ig isotype specific PCR to generate a synthetic repertoire displayed by phage, or bacteria, or yeast for affinity maturation in vitro28,29. These libraries can produce synthetic antibodies that exceed the affinities and specificities of natural antibodies30. Moreover, these libraries can then be “recycled” to screen against other target protein28,29.
Figure 5. Diagram showing how phage display affinity maturation occurs in vitro.
Display technology is dependent on nucleotide sequencing (e.g., sequencing of the BCR, or single B cell NGS approaches) to generate synthetic repertoire libraries for subsequent affinity maturation. As such, it is subject to the same limitations of nucleotide sequencing approaches mentioned in the previous section. Pairing of the heavy and light chains during library construction may not reflect the natural antibody repertoire31. Moreover, key B cell clones’ light and heavy chains might be missing from the final library prior to affinity maturation31. Finally, because affinity maturation happens in vitro, antibodies generated do not have the relevant PTMs (glycans) required for stability, and thus initial testing is blind to the impact PTMs may have in vivo. As a result, display technology’s artificial nature may prolong target validation before antibody drugs can be adapted to the clinic.
Figure 6. Illustration comparing different antibody discovery technologies.