N‐terminal or signal peptide sequence engineering prevents truncation of human monoclonal antibody light chains

Abstract

Monoclonal antibodies (mAbs) contain short N-terminal signal peptides on each individual polypeptide that comprises the mature antibody, targeting them for export from the cell in which they are produced. The signal peptide is cleaved from each heavy chain (Hc) and light chain (Lc) polypeptide after translocation to the ER and prior to secretion. This process is generally highly efficient, producing a high proportion of correctly cleaved Hc and Lc polypeptides. However, mis-cleavage of the signal peptide can occur, resulting in truncation or elongation at the N-terminus of the Hc or Lc. This is undesirable for antibody manufacturing as it can impact efficacy and can result in product heterogeneity. Here, we describe a truncated variant of the Lc that was detected during a routine developability assessment of the recombinant human IgG1 MEDI8490 in Chinese hamster ovary cells. We found that the truncation of the Lc was caused due to the use of the murine Hc signal peptide together with a lambda Lc containing an SYE amino acid motif at the N-terminus. This truncation was not caused by mis-processing of the mRNA encoding the Lc and was not dependent on expression platform (transient or stable), the scale of the fed-batch culture or clonal lineage. We further show that using alternative signal peptides or engineering the Lc SYE N-terminal motif prevented the truncation and that this strategy will improve Lc homogeneity of other SYE lambda Lc-containing mAbs. Biotechnol. Bioeng. 2017;114: 1970-1977. © 2017 Wiley Periodicals, Inc.

Macro- and Micro-Heterogeneity of Natural and Recombinant IgG Antibodies

“…Although it is less common, N-terminal truncation has been reported. A combination of a murine signal peptide and antibody lambda light chain causes an alternative cleavage of the signal peptide resulting in a mAb with the loss of three amino acids from the light chain [17]. With the use of a specific tag to label N-terminal primary amine in combination with liquid chromatography mass spectrometry (LC-MS), an mAb variant with the loss of one amino acid from the light chain was observed [11].…”

Analytical comparability study of recombinant monoclonal antibody therapeutics

“…11,16 In rare cases, miscleavages result in truncation. 17,18 The presence of N-terminal pyroGlu or a leader sequence is not expected to affect the overall structure and function of recombinant mAbs The presence of N-terminal glutamine or pyroGlu has no impact on potency. 11 In human endogenous immunoglobulin (IgG), the N-terminal glutamine is almost completely converted into pyroGlu, 19 minimizing immunogenicity concerns for this modification.…”

Antibody Upstream Sequence Diversity and Its Biological Implications Revealed by Repertoire Sequencing

“…An AUS contains two consecutive functional elements, namely 5' UTR and leader, both of which were found to implicate in mRNA transcription and translation [14][15][16][17][18][19][20][21]. Particularly, leader sequences play a vital role in antibody expression and are often engineered to improve the efficiency of monoclonal antibody production [16,21,22]. In Rep-Seq studies, the AUSs are often the targets of the PCR primers for obtaining full-length antibody variable regions [23,24].…”