Cleaning-in-place for immunoaffinity resin monitored by in situ ATR-FTIR spectroscopy
File(s)
Published version
Author(s)
Byrne, B
Kazarian, SG
Boulet-Audet, M
Type
Journal Article
Abstract
In the next ten years, the pharmaceutical industry anticipates that revenue from biotherapeutics will overtake those generated from small drug molecules. Despite effectively treating a range of chronic and life-threatening diseases, the high cost of biotherapeutics limits their use. For biotherapeutic monoclonal antibodies (mAbs), an important production cost is the affinity resin used for protein capture. Cleaning-in-place (CIP) protocols aim to optimise the lifespan of the resin by slowing binding capacity decay. Binding assays can determine resin capacity from the mobile phase, but do not reveal the underlying causes of Protein A ligand degradation. The focus needs to be on the stationary phase to examine the effect of CIP on the resin. To directly determine both the local Protein A ligand concentration and conformation on two Protein A resins, we developed a method based on attenuated total reflection (ATR) Fourier Transform Infrared (FTIR) spectroscopy. ATR-FTIR spectroscopic imaging revealed that applying a carefully controlled load to agarose beads produces an even and reproducible contact with the internal reflection element. This allowed detection and quantification of the binding capacity of the stationary phase. ATR-FTIR also showed that Protein A proteolysis does not seem to occur under typical CIP conditions (below 1M NaOH). However, our data revealed that concentrations of NaOH above 0.1 M cause significant changes in Protein A conformation. The addition of >0.4 M trehalose during CIP significantly reduced NaOH-induced ligand unfolding observed for one of the two Protein A resins tested. Such insights could help to optimise CIP protocols in order to extend resin lifetime and reduce mAb production costs.
Date Issued
2015-07-10
Date Acceptance
2015-06-18
Citation
Analytical and Bioanalytical Chemistry, 2015, 407 (23), pp.7111-7122
ISSN
1618-2650
Publisher
Springer
Start Page
7111
End Page
7122
Journal / Book Title
Analytical and Bioanalytical Chemistry
Volume
407
Issue
23
Copyright Statement
© The Author(s) 2015. This article is published with open access at Springerlink.com. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
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Publication Status
Published