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Cleaning-in-place of immunoaffinity resins monitored by in situ ATR-FTIR spectroscopy.

ABSTRACT: In the next 10 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 spectroscopy also showed that Protein A proteolysis does not seem to occur under typical CIP conditions (below 1 M 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.

SUBMITTER: Boulet-Audet M

PROVIDER: S-EPMC4551555 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Cleaning-in-place of immunoaffinity resins monitored by in situ ATR-FTIR spectroscopy.

Boulet-Audet Maxime M Byrne Bernadette B Kazarian Sergei G SG

Analytical and bioanalytical chemistry 20150710 23

In the next 10 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 bind ...[more]

PMID: 26159572

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Project description:Extreme weather and globalisation leave our climate vulnerable to invasion by alien species, which have negative impacts on the economy, biodiversity, and ecosystem services. Rapid and accurate identification is key to the control of invasive alien species. However, visually similar species hinder conservation efforts, for example hybrids within the Japanese Knotweed complex.We applied the novel method of ATR-FTIR spectroscopy combined with chemometrics (mathematics applied to chemical data) to historic herbarium samples, taking 1580 spectra in total. Samples included five species from within the interbreeding Japanese Knotweed complex (including three varieties of Japanese Knotweed), six hybrids and five species from the wider Polygonaceae family. Spectral data from herbarium specimens were analysed with several chemometric techniques: support vector machines (SVM) for differentiation between plant types, supported by ploidy levels; principal component analysis loadings and spectral biomarkers to explore differences between the highly invasive Reynoutria japonica var. japonica and its non-invasive counterpart Reynoutria japonica var. compacta; hierarchical cluster analysis (HCA) to investigate the relationship between plants within the Polygonaceae family, of the Fallopia, Reynoutria, Rumex and Fagopyrum genera.ATR-FTIR spectroscopy coupled with SVM successfully differentiated between plant type, leaf surface and geographical location, even in herbarium samples of varying age. Differences between Reynoutria japonica var. japonica and Reynoutria japonica var. compacta included the presence of two polysaccharides, glucomannan and xyloglucan, at higher concentrations in Reynoutria japonica var. japonica than Reynoutria japonica var. compacta. HCA analysis indicated that potential genetic linkages are sometimes masked by environmental factors; an effect that can either be reduced or encouraged by altering the input parameters. Entering the absorbance values for key wavenumbers, previously highlighted by principal component analysis loadings, favours linkages in the resultant HCA dendrogram corresponding to expected genetic relationships, whilst environmental associations are encouraged using the spectral fingerprint region.The ability to distinguish between closely related interbreeding species and hybrids, based on their spectral signature, raises the possibility of using this approach for determining the origin of Japanese knotweed infestations in legal cases where the clonal nature of plants currently makes this difficult and for the targeted control of species and hybrids. These techniques also provide a new method for supporting biogeographical studies.

Dataset Information

Cleaning-in-place of immunoaffinity resins monitored by in situ ATR-FTIR spectroscopy.

Publications

Cleaning-in-place of immunoaffinity resins monitored by in situ ATR-FTIR spectroscopy.

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