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Investigation of an Uncommon Artifact during Reducing Capillary Electrophoresis-Sodium Dodecyl Sulfate Analysis of a Monoclonal Antibody with Dynamic Light Scattering and Reversed Phase High-Performance Liquid Chromatography

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Abstract

Purposes

In reducing capillary electrophoresis sodium dodecyl sulfate (CE-SDS) analysis of a monoclonal antibody (mAb-1), the peak area ratio of heavy chain (HC) to light chain (LC) was out of balance, while multiple artifact peaks were observed following the migration of HC. The main purposes of this study were to describe the techniques utilized to eliminate this artifact and clarify the root cause for this interesting phenomenon.

Methods

We optimized the CE-SDS analysis of mAb-1 by a vairety of techniques including changing the concentration of protein or replacing SDS with a more hydrophobic surfactant (i.e., sodium hexadecyl sulfate (SHS) or sodium tetradecyl sulfate (STS) instead of SDS) in sample and/or the sieving gel buffer. Dynamic light scattering (DLS) and reversed phase high-performance liquid chromatography (RP-HPLC) were used to study the protein-surfactant complex.

Results

The artifact could be partially mitigated by reducing the protein concentration and replacing SDS with SHS or STS in the sample and/or the sieving gel buffer solutions. Due to replacing a more hydrophobic surfactant, the HC-surfactant complex formed was more resistant to dissociation, preventing additional hydrophobic HC-HC interaction and aggregation, thus eliminating the artifact problem.

Conclusions

DLS and RP-HPLC are powerful supplementary techniques in characterizing the protein-surfactant complex, and hydrophobic surfactants such as SHS and STS could afford more normal electropherograms during the analysis of mAbs.

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Abbreviations

AHC :

Heavy chain peak area

ALC :

Light chain peak area

BME:

β-Mercaptoethanol

CE:

Capillary electrophoresis

ChP:

Chinese Pharmacopoeia

DLS:

Dynamic light scattering

HC:

Heavy chain

HMW:

High molecular weight

LC:

Light chain

mAb:

Monoclonal antibody

MALDI-TOF-MS:

Matrix-assisted laser desorption/ionization time of flight mass spectrometry

NGHC:

Non-glycosylated heavy chain

PAGE:

Polyacrylamide gel electrophoresis

RP-HPLC:

Reversed phase high-performance liquid chromatography

SDS (SC12S):

Sodium dodecyl sulfate

SHS (SC16S):

Sodium hexadecyl sulfate

STS (SC14S):

Sodium tetradecyl sulfate

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Acknowledgements

Fundings were generously provided by the Ministry of Science and Technology of China (Grant No. 2018ZX09J18107-002) and the Scientific Research Fund of Zhejiang Provincial Education Department (Grant No. Y202148326). We also would like to thank Youping Xu at the Analysis Center of Agrobiology and Environmental Sciences at Zhejiang University and Ms. Haihong Hu for MALDI-TOF-MS analysis and lab management, respectively. The authors have no conflicts of interest to declare.

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Authors and Affiliations

  1. Institute of Drug Metabolism and Pharmaceutical Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China

    Han Gao, Si-Tao Wang, Bin-Bin Shen, Min-Fei Sun & Wei-Jie Fang

  2. Hangzhou Institute of Innovative Medicine, Zhejiang University, Hangzhou, 310016, China

    Han Gao, Bin-Bin Shen, Min-Fei Sun & Wei-Jie Fang

  3. Zhejiang Bioray Biopharmaceutical Co., Taizhou, 317000, China

    Feng Hu, Haibin Wang & Lei Li

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  1. Han Gao
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Gao, H., Wang, ST., Hu, F. et al. Investigation of an Uncommon Artifact during Reducing Capillary Electrophoresis-Sodium Dodecyl Sulfate Analysis of a Monoclonal Antibody with Dynamic Light Scattering and Reversed Phase High-Performance Liquid Chromatography. Pharm Res 39, 1959–1968 (2022). https://doi.org/10.1007/s11095-022-03303-0

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