Monoclonal antibodies (mAbs) are widely used in research, diagnosis, and pharmaceutical purposes. Lately, the relatively lower quality of the research-purpose mAbs is a point of concern within the research community¹,². The problems include the lack of validation and low reproducibility. As most of the validation is done by immunoassay, the extent of protein contamination, including undesired antibody forms, remains unknown.

Mass spectrometry based protein sequencing technology enables the discovery and characterization of undesired antibody forms in mAb reagents. In this study, we examine the existence of a second light chain in addition to the expected primary light chain. To our surprise, a significant portion of the mAbs we studied include a second light chain that can be completely or partially de novo sequenced by mass spectrometry method.


Each antibody sample was digested with six different enzymes: Trypsin, Lys C, Chymotrypsin, Pepsin, Asp N, and Elastase. Tandem mass spectrometry data were collected with an Orbitrap Q Exactive for each digest. Novor³ software was used to de novo sequence each peptide. The protein sequence is assembled with the REmAb™ sequencing platform. After the sequencing of the first pair of heavy and light chains for the antibody, the software attempts to sequence a second light chain from the data. A second light chain is reported as found if it has significant sequence coverage based on LC-MS/MS data and is sufficiently different from the first one.

Fig. 1. General workflow of REmAb™ includes (1) multiple enzyme digests, (2) mass spec., (3) de novo peptide sequencing. and (4) sequence assembly.

Preliminary Data

Eighty distinct research-purpose monoclonal antibodies were sequenced. The majority of the antibodies are from mouse and rat, accounting for 58 and 19 of the antibodies, respectively. The other 3 antibodies are from three other mammals.

For all the 80 distinct antibodies, REmAb™ could confidently de novo sequence the complete sequences for the primary pair of heavy and light chains. In addition, a second different light chain sequence could be detected in 11 out of the 80 antibodies, or approximately 14% of the cases. All of the 11 secondary light chains could be supported by at least ten unique peptide spectrum matches with the mass spectrometry data. For 9 of the 11 cases, the second light chain was abundant enough to get a complete sequence coverage with the mass spectrometry data. For the other 2 cases, the MS data could confidently cover 96% and 88% of the amino acids in the variable regions, respectively.

Fig 2. Approximately 14% of the studied mAb samples have an abundant secondary light chain.


  • A significant portion (14%) of the studied research-purpose mAbs have more than one light chain present in the sample
  • The mass spectrometry based protein sequencing method, REmAb™, can sequence both the primary and secondary chains in the protein sample


  • Baker, M (2015). Blame it on the antibodies. Nature, 521(7552), 274-276.
  • Bradbury, A., & Plückthun, A. (2015). Reproducibility: Standardize antibodies used in research. Nature, 518(7537), 27-29.
  • Ma, B. (2015). Novor: Real-Time Peptide de Novo Sequencing Software. Journal of the American Society for Mass Spectrometry, 26(11).

Reproduced from McDonald, Z., Liu Q., Xie, M., Ma, B., Taylor, P., & Stajduhar, A. (2018). Studying the Prevalence of Secondary Light Chains in Research Purpose Monoclonal Antibodies with MS-Based De Novo Protein Sequencing. ASMS 2018 San Diego, MP 063, with permission.