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

Boulet-Audet M, Byrne B, Kazarian SG - Anal Bioanal Chem (2015)

Bottom Line: ATR-FTIR spectroscopy also showed that Protein A proteolysis does not seem to occur under typical CIP conditions (below 1 M NaOH).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Imperial College London, South Kensington Campus, London, SW7 2AZ, UK.

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.

No MeSH data available.


Related in: MedlinePlus

Amide II band centre of gravity (COG) as a function of time for a typical rProtein A Sepharose resin and MabSelect resin under 420 mM NaOH CIP. See ESM Fig. S4 for a close-up of the amide I and II region for different samples under a 200-g load
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Fig5: Amide II band centre of gravity (COG) as a function of time for a typical rProtein A Sepharose resin and MabSelect resin under 420 mM NaOH CIP. See ESM Fig. S4 for a close-up of the amide I and II region for different samples under a 200-g load

Mentions: Figure 5 shows that ATR-FTIR spectroscopy can detect subtle Protein A ligand conformation changes during CIP in situ. For both rProtein A Sepharose and MabSelect resin exposed to 420 mM NaOH, the amide II COG shifted to a higher wavenumber as a function of time. The COG shifted faster at the beginning of the incubation than after ~4 h, particularly for the Sepharose resin. The amide II band still continued to rise, albeit slowly, after exposing the resin to NaOH for 18 h suggesting that unfolding is not complete even after prolonged exposure times. Under such CIP conditions, the resin would be expected to have lost substantial binding capacity and be rendered unusable [16]. ATR-FTIR spectroscopy could thus follow the ligand’s structure during CIP steps without the need to interfere with the production pipeline or rely on mAb binding interactions.Fig. 5


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

Boulet-Audet M, Byrne B, Kazarian SG - Anal Bioanal Chem (2015)

Amide II band centre of gravity (COG) as a function of time for a typical rProtein A Sepharose resin and MabSelect resin under 420 mM NaOH CIP. See ESM Fig. S4 for a close-up of the amide I and II region for different samples under a 200-g load
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4551555&req=5

Fig5: Amide II band centre of gravity (COG) as a function of time for a typical rProtein A Sepharose resin and MabSelect resin under 420 mM NaOH CIP. See ESM Fig. S4 for a close-up of the amide I and II region for different samples under a 200-g load
Mentions: Figure 5 shows that ATR-FTIR spectroscopy can detect subtle Protein A ligand conformation changes during CIP in situ. For both rProtein A Sepharose and MabSelect resin exposed to 420 mM NaOH, the amide II COG shifted to a higher wavenumber as a function of time. The COG shifted faster at the beginning of the incubation than after ~4 h, particularly for the Sepharose resin. The amide II band still continued to rise, albeit slowly, after exposing the resin to NaOH for 18 h suggesting that unfolding is not complete even after prolonged exposure times. Under such CIP conditions, the resin would be expected to have lost substantial binding capacity and be rendered unusable [16]. ATR-FTIR spectroscopy could thus follow the ligand’s structure during CIP steps without the need to interfere with the production pipeline or rely on mAb binding interactions.Fig. 5

Bottom Line: ATR-FTIR spectroscopy also showed that Protein A proteolysis does not seem to occur under typical CIP conditions (below 1 M NaOH).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Imperial College London, South Kensington Campus, London, SW7 2AZ, UK.

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.

No MeSH data available.


Related in: MedlinePlus