Fluorescence based real time monitoring of fouling in process chromatography

Pathak, Manisha; Lintern, Katherine; Chopda, Viki R.; Bracewell, Daniel G.; Rathore, Anurag S.

doi:10.1038/srep45640

Cited by 13 publications

(19 citation statements)

References 11 publications

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“…On the other hand, the foulants (host cell proteins and mAb) have tryptophan together with tyrosine and phenylalanine (detected using LC–MS/MS). Thus, we would expect the fluorescence intensity at 340 nm to be due to foulants present on the resin and the fluorescence intensity at 303 nm to be due to protein A ligand present on the resin …”

Section: Resultsmentioning

confidence: 99%

“…Fluorescence analysis of resin samples used for evaluation of cleaning buffers was carried out in 96 well plates using Spectra Max M2e Multi‐Mode Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) in Greiner Cellstar 96‐well black bottom plate . This analysis was performed in at‐line mode.…”

Section: Methodsmentioning

confidence: 99%

“…Specifically, the fluorescence intensity was recorded every cycle and the deposition of foulants was estimated by subtracting the reading obtained for the freshly packed column using fresh chromatographic resin. The increase in the amount of fluorescence intensity at the end of respective cycles estimated the deposition of foulants on the resin over reuse …”

Section: Methodsmentioning

confidence: 99%

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Implementation of a fluorescence based PAT control for fouling of protein A chromatography resin

Pathak

Rathore

2017

J of Chemical Tech & Biotech

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BACKGROUND Protein A chromatography fouling is accompanied by two major events, one is the loss of protein A ligands and other is fouling due to non‐specific, irreversible interactions of foulants with resin particles. This paper presents implementation of process analytical technology based control for fouling of protein A chromatography resin using a novel, fluorescence based approach. This approach enables direct, in situ measurement of protein A ligand density as well as monitoring of resin fouling during resin reuse. RESULTS A novel, fluorescence based process analytical technology (PAT) tool has been designed and used for screening a variety of cleaning protocols. A two‐step cleaning protocol was created using this methodology. The above mentioned fluorescence based approach was successfully used to monitor fouling and to take a decision on when to initiate cleaning. This resulted in effective maintenance of dynamic binding capacity and step yield at 50 cycles (DBC: 97% of the original value vs 65% with conventional protocols and yield: 93% of the original value vs 73% with conventional protocols) and at 200 cycles (> 90% of the original value). CONCLUSIONS The proposed fluorescence based approach has been effectively used for monitoring and control of resin fouling upon reuse, thereby resulting in a substantial increase in resin lifetime. © 2017 Society of Chemical Industry

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Implementation of a fluorescence based PAT control for fouling of protein A chromatography resin

Pathak

Rathore

2017

J of Chemical Tech & Biotech

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“…The resin samples were monitored for foulant (HCP and residual mAb) deposition and ligand degradation by recording excitation and emission spectra at 250–500 nm. Direct real‐time measurement of foulant accumulation on the resin could facilitate implementation of appropriate cleaning conditions to clear the deposited foulants . Recombinant protein A contains tyrosine and phenylalanine residues but lacks tryptophan.…”

Section: Methodsmentioning

confidence: 99%

“…Consequently, a fluorescence intensity at 340 nm (due to the presence of tryptophan) can be associated with the foulants present on the resin, while the fluorescence intensity at 303 nm can be attributed to the protein A ligand present on the resin. We have evaluated and validated that the emission lambda maxima for protein A ligand bound to porous agarose resin was 303 nm while the mAb bound to protein A agarose resin was ∼340 nm …”

Section: Methodsmentioning

confidence: 99%

Protein A chromatography resin lifetime—impact of feed composition

Pathak

Rathore

Lintern

et al. 2018

Biotechnology Progress

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Adsorbent lifetime during protein A chromatography is not readily predicted or understood, representing a key challenge to be addressed for biopharmaceutical manufacturers. This article focuses on the impact of feed composition on the performance of a typical agarose-based protein A resin across a lifetime of 50 cycles. Cycling studies were performed using three different feed materials with varying levels of feed components including proteases, histones, DNA, and nonhistone proteins. Changes in the process and quality attributes were measured. The DBCs were not seen to vary between conditions although there was a reduction in particle porosity in all cases. Fluorescence spectroscopy and LC-MS/MS were used to identify the contribution and extent of fouling to the observed capacity loss. Residual protein A ligand density and deposition of foulants (HCP, residual mAb, and DNA) varied between the three feed materials. Resins cycled in feed materials containing high concentrations of HCP and histones were seen to have greater extents of capacity loss. The mode of performance loss, capacity loss, or impact on product quality was seen to vary depending on the feed material. The results indicate that feed material composition may be correlated to the rate and mode of resin aging as a basis for improved process understanding. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:412-419, 2018.

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Analytical tools for monitoring changes in physical and chemical properties of chromatography resin upon reuse

et al. 2019

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Protein A resins are often reused for multiple cycles to improve process economy during mAb purification. Significant reduction in binding capacity and product recovery are typically observed due to the presence of unwanted materials (foulants) deposited on the resin upon reuse. In this paper, we have used a wide spectrum of qualitative and quantitative analytical tools (particle size analysis, HPLC, fluorescence, SEM, MS, and FTIR) to compare the strengths and shortcomings of different analytical tools in terms of their capability to detect the fouling of the resin and relate it to chromatographic cycle performance. While each tool offers an insight into this complex phenomena, fluorescence is the only one that can be used for real‐time monitoring of resin fouling. A correlation could be established between fluorescence intensity and the process performance attributes (like yield or binding capacity) impacted upon resin reuse. This demonstration of the application of fluorescence for real‐time monitoring correlated empirically with process performance attributes and the results support its use as a PAT tool as part of a process control strategy. While the focus of this paper is on fouling of protein A chromatography resin, the approach and strategy are pertinent to other modes of chromatography as well.

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Fluorescence based real time monitoring of fouling in process chromatography

Cited by 13 publications

References 11 publications

Implementation of a fluorescence based PAT control for fouling of protein A chromatography resin

Implementation of a fluorescence based PAT control for fouling of protein A chromatography resin

Protein A chromatography resin lifetime—impact of feed composition

Analytical tools for monitoring changes in physical and chemical properties of chromatography resin upon reuse

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