In recent years, as our understanding of antibodies deepens, antibody engineering technologies continue to advance. Techniques such as antibody mass spectrometry sequencing, single-cell sequencing and hybridoma cell sequencing have found widespread applications, providing more tools and avenues for antibody research and applications.

（Snapkov I, et al. Trends Biotechnol. 2022） 

Antibody sequencing addresses many challenges in antibody development. For example, it can be used when certain hybridomas have low expression levels or high-quality antibodies are lost, or when antibodies need to be further modified to enhance their performance. These are all areas where antibody sequencing technologies come into play. Let's take a look at the processes of hybridoma cell sequencing and antibody mass spectrometry sequencing:

Hybridoma Cell Sequencing:

Various methods are currently available for sequencing the variable regions of antibodies from hybridoma cells or lymphocytes. One such method is high-throughput RNA sequencing, which has proven to be highly accurate and enables in-depth analysis of antibody libraries. However, many laboratories may not be familiar with high-throughput sequencing technologies, as they require specialized knowledge in RNA-seq library preparation and computational analysis. Additionally, the cost of high-throughput library preparation and sequencing can be high, and the sequencing turnaround time may range from weeks to months. The mainstream approach to hybridoma cell sequencing involves designing universal primers for amplifying the variable regions of antibody clones from single-cell cultures. This PCR-based method allows for the next-generation sequencing of antibody variable regions, providing information on the antibody gene sequences.

（Hybridoma cell sequencing process） 

Antibody MS Sequencing

De novo antibody sequencing is a novel method based on high-quality LC-MS/MS spectra for direct sequencing. LC-MS/MS has become the standard technology for new protein sequencing, and capillary electrophoresis provides complementary information for liquid chromatography. It is rapidly evolving as a bottom-up proteomics technique and is widely used in peptide mapping of monoclonal antibodies, comprehensive antibody studies, and analysis of antibody-drug conjugates.

（Antibody MS Sequencing process）