TY - JOUR
T1 - Engineering a High-Affinity Anti-Methoxy Poly(ethylene glycol) (mPEG) Antibody for Sensitive Immunosensing of mPEGylated Therapeutics and mPEG Molecules
AU - Hsiao, Chiao Yu
AU - Meng, Jun Lun
AU - Hong, Jung Zhe
AU - Ly, Xuan Huong
AU - Lin, Meng Hsuan
AU - Chang, Chin Yuan
AU - Nguyen, Minh Tram T.
AU - Cheng, Tian Lu
AU - Lin, Wen Wei
AU - Burnouf, Pierre Alain
AU - Al-Qaisi, Talal Salem
AU - Liu, En Shuo
AU - Su, Yu Cheng
N1 - Funding Information:
The authors gratefully acknowledge the financial support from the Young Scholar Fellowship (Columbus) Program under grant numbers MOST108-2636-B-009-003, MOST109-2636-B-009-003, MOST110-2636-B-009-010, and MOST111-2636-B-A49-005. This work is also supported in part by the Kaohsiung Medical University NYCU-KMU Joint Research Project (NYCUKMU-111-I005) and the “Center for Intelligent Drug Systems and Smart Bio-devices (IDSB)” from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan. The mouse anti-PEG IgM monoclonal antibodies (clone: AGP4) were kindly offered by Dr. Steve R. Roffler (Academia Sinica, Taipei, Taiwan). The authors also thank the technical services provided by the Synchrotron Radiation Protein Crystallography Facility of the National Core Facility Program for Biotechnology, MOST, and the National Synchrotron Radiation Research Center (NSRRC), a national user facility supported by MOST, Taiwan. 2
Publisher Copyright:
© 2022 American Chemical Society. All rights reserved.
PY - 2022/11/16
Y1 - 2022/11/16
N2 - Sensitive quantification of methoxy poly(ethylene glycol) (mPEG)-conjugated therapeutics for pharmacokinetic determination is critical for mPEGylated drug development. However, sensitive measurement of low-molecular-weight (lmw) mPEG compounds remains challenging due to epitope competition between backbone-specific anti-PEG antibodies. Here, we engineered a high-affinity methoxy-specific anti-mPEG antibody for sensitive quantification of free mPEG molecules and mPEGylated therapeutics. The affinity-enhanced h15-2Y antibody variant shows a 10.3-fold increase in mPEG-binding activity compared to parental h15-2b. h15-2Y-based sandwich ELISA can effectively quantify lmw mPEG5Kand high-molecular-weight (hmw) mPEG20Kat concentrations as low as 3.4 and 5.1 ng mL-1, respectively. Moreover, lmw mPEG compounds (560, 750, 1000, and 2000 Da) can be efficiently quantified via h15-2Y-based competitive ELISA with detection limits at nanomolar levels. This study provides a promising approach for application in the quantitative analysis of the various sizes of mPEG molecules to accelerate the timeline of mPEG-conjugated drug development.
AB - Sensitive quantification of methoxy poly(ethylene glycol) (mPEG)-conjugated therapeutics for pharmacokinetic determination is critical for mPEGylated drug development. However, sensitive measurement of low-molecular-weight (lmw) mPEG compounds remains challenging due to epitope competition between backbone-specific anti-PEG antibodies. Here, we engineered a high-affinity methoxy-specific anti-mPEG antibody for sensitive quantification of free mPEG molecules and mPEGylated therapeutics. The affinity-enhanced h15-2Y antibody variant shows a 10.3-fold increase in mPEG-binding activity compared to parental h15-2b. h15-2Y-based sandwich ELISA can effectively quantify lmw mPEG5Kand high-molecular-weight (hmw) mPEG20Kat concentrations as low as 3.4 and 5.1 ng mL-1, respectively. Moreover, lmw mPEG compounds (560, 750, 1000, and 2000 Da) can be efficiently quantified via h15-2Y-based competitive ELISA with detection limits at nanomolar levels. This study provides a promising approach for application in the quantitative analysis of the various sizes of mPEG molecules to accelerate the timeline of mPEG-conjugated drug development.
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U2 - 10.1021/acs.bioconjchem.2c00416
DO - 10.1021/acs.bioconjchem.2c00416
M3 - Article
C2 - 36320124
AN - SCOPUS:85141640265
SN - 1043-1802
VL - 33
SP - 2180
EP - 2188
JO - Bioconjugate Chemistry
JF - Bioconjugate Chemistry
IS - 11
ER -