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Use of Cyclodextrin as a Novel Agent in the SEC-HPLC Mobile Phase to Mitigate the Interactions of Proteins or Peptide or their Impurities with the Residual Silanols of Commercial SEC-HPLC Columns with Improved Separation and Resolution

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Abstract

Purpose

Accurate quantification of the intact proteins, antibodies or peptides and their impurities without interaction to silanols of HPLC column.

Methods

Hydroxypropyl ß Cyclodextrin (HPCD) is added in the mobile phase at different concentrations. Different commercial SEC-HPLC columns and biologics with a molecular weight ranging from 5.8 kDa to 150kDa were assessed with and without cyclodextrin.

Results

Addition of non-ionic sugars such as Hydroxypropyl ß Cyclodextrin in the mobile phase, resulted improved peak performance such as theoretical plates, peak resolution, peak width, peak height, and improved quantification of aggregates in biologics such as antibodies Humira and Actemra, and peptides such as insulin. There is an increase in peak height, reduced retention time, increased plate and reduced peak width with increasing concentration of cyclodextrin studied.

Discussion

High ionic strength, basic amino acids such as arginine, organic solvents (with a concentration low enough not to precipitate protein), sodium perchlorate and ion pairing agents in the mobile phase used for separation of peptides, proteins and antibodies to prevent silanol interaction. These commonly used solutions are not always successful, as they not only interact with the biologic, but are sometimes, not compatible. The non-ionic cyclodextrin itself does not cause protein aggregation but prevents the nonspecific binding or interaction of protein itself and thereby allowing for improved resolution, and accurate quantification of aggregates in antibodies, and peptides. The data on the separation in presence of cyclodextrin in the mobile phase showed higher peak resolution, improved peak shape, accurate apparent molecular weight, improved efficiency, and less peak tailing for biological products.

Conclusion

Hydroxypropyl ß Cyclodextrin in the mobile phase, resulted improved SEC-HPLC resolution, and quantitation of aggregates in biologics by preventing the interaction of biologics to silanol of the commercial SEC-HPLC columns.

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Abbreviations

°C:

Degree Centigrade

AUC:

Area under the curve

BEH:

Ethylene-bridged hybrid

CHO:

Chinese Hamster Ovary

DAD:

Diode-Array Detector

EP:

European Pharmacopeia

FDA:

Food and Drug Administration

HCl:

Hydrochloric acid

HMW:

High Molecular Weight

HPCD:

Hydroxypropyl ß Cyclodextrin

IgG:

Immunoglobulin G

kDa:

Kilo Dalton

mL:

Milli liter

mM:

Milli molar

PBS:

Phosphate buffered saline

PI:

Isoelectric point

pH:

Potential Hydrogen

SEC-HPLC:

Size-Exclusion High-Performance Liquid Chromatography

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ACKNOWLEDGMENTS AND DISCLOSURES

All authors are employee of CuriRx. The authors declare that they have no conflict of interest

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Correspondence to Kaliappanadar Nellaiappan.

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Javeri, I., Nellaiappan, K., McNemar, C. et al. Use of Cyclodextrin as a Novel Agent in the SEC-HPLC Mobile Phase to Mitigate the Interactions of Proteins or Peptide or their Impurities with the Residual Silanols of Commercial SEC-HPLC Columns with Improved Separation and Resolution. Pharm Res 35, 168 (2018). https://doi.org/10.1007/s11095-018-2446-x

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  • DOI: https://doi.org/10.1007/s11095-018-2446-x

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