Procollagen-lysine 2-oxoglutarate 5-dioxygenases are responsible for 5R-hydroxylysine modification of therapeutic T-cell bispecific monoclonal antibodies produced by Chinese hamster ovary cells

被引：0

作者：

Bauer, Niels ^{[1
,2
,3
]}

Boettger, Marco ^{[1
]}

Papadaki, Styliani ^{[1
]}

Leitner, Tanja ^{[1
]}

Klostermann, Stefan ^{[4
]}

Kettenberger, Hubert ^{[1
]}

Georges, Guy ^{[1
]}

Larraillet, Vincent ^{[1
]}

von Kruechten, Dino Gluhacevic ^{[5
]}

Hillringhaus, Lars ^{[5
]}

Vogt, Annette ^{[1
]}

Auslaender, Simon ^{[1
]}

Popp, Oliver ^{[1
]}

机构：

[1] Roche Innovat Ctr Munich, Large Mol Res, Roche Pharm Res & Early Dev pRED, Penzberg, Germany

[2] Ludwig Maximilians Univ Munchen, Gene Ctr, Munich, Germany

[3] Ludwig Maximilians Univ Munchen, Dept Biochem, Munich, Germany

[4] Roche Innovat Ctr Munich, Data & Analyt, Roche Pharm Res & Early Dev pRED, Penzberg, Germany

[5] Roche Innovat Ctr Munich, Special Chem, Roche Diagnost, Penzberg, Germany

来源：

FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY | 2024年 / 12卷

关键词：

T-cell bispecific monoclonal antibodies; Chinese hamster ovary cells; hydroxylysine; CRISPR/Cas9; post-translational modification; mass spectrometry; metal cofactor; LYSYL HYDROXYLASE; PROTEIN HYDROXYLATION; CROSS-LINKING; CULTURE-MEDIA; EXPRESSION; PLOD2; MECHANISMS; FKBP65;

D O I：

10.3389/fbioe.2024.1414408

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

We present a detailed mass spectrometric analysis of three 2 + 1 T-cell bispecific monoclonal antibodies (TCB mAbs), where an unexpected +15.9950 Da mass shift in tryptic peptides was observed. This modification was attributed to the occurrence of 5R-hydroxylysine (Hyl) using a hybrid LC-MS/MS molecular characterization and CRISPR/Cas9 gene deletion approach. The modification was found at various sites within TCB mAbs, with a conspicuous hot spot motif mirroring a prior observation where Hyl was mapped to the CH1-VH Fab domain interface of IgGs. In contrast to the preceding report, our structural modeling analysis on TCB mAbs unveiled substantial differences in the orientation and flexibility of motifs in immediate proximity and across the artificial CH1-VL cross Fab interface and upstream elbow segment. Utilizing a hybrid database search, RNAseq, and a CRISPR/Cas9 knockout methodology in Chinese hamster ovary (CHO) production cell lines, procollagen-lysine, 2-oxoglutarate 5-dioxygenases (PLODs) were conclusively identified as the catalyzing enzymes accountable for the 5R-Hyl modification in TCB mAbs. To quantitatively inhibit Hyl formation in TCB mAbs, the activity of all three Chinese hamster PLOD isoenzymes needs to be depleted via CRISPR/Cas9 gene knockout. Moreover, our investigation identified cell culture iron availability, process duration, and clonal variability in CHO cells as elements influencing the levels of Hyl formation in TCB mAbs. This research offers a solution for circumventing Hyl formation in therapeutic complex mAb formats, such as TCB mAbs, produced in CHO cell culture processes, thereby addressing potential technical and biological challenges associated with unintended Hyl modification.

引用

页数：25