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

Pathak, Manisha; Rathore, Anurag S.

doi:10.1002/jctb.5358

Cited by 9 publications

(7 citation statements)

References 37 publications

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“…A non‐invasive, fluorescence‐based technique developed by Pathak and Rathore allowed monitoring of fouling in real time using PreDictor™ plates without additional protein tags . The basis of this approach relied on differential amounts of tyrosine and phenylalanine residues in Protein A resin that absorbed at 303 nm, compared with the higher concentrations of tryptophan in foulants, which caused a shift in the absorbance spectra to 340 nm.…”

Section: Introductionmentioning

confidence: 99%

Chromatography process development aided by a dye‐based assay

Jasulaityte

Johansson²,

Bracewell

2019

J of Chemical Tech & Biotech

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BACKGROUND: The lifetime of chromatography resins typically averages between 10 and 300 cycles for the manufacture of a therapeutic protein. Developing and establishing the robustness of the method for each separation process represents a significant challenge and is subject to extensive regulatory oversight. This paper presents a novel fluorescence-based assay for residual aggregated proteins to aid the evaluation of the extent of resin regeneration. RESULTS: The versatility of this method was demonstrated by using strong anion and cation exchange agarose resins PraestoQ and SP in conjunction with bovine serum albumin and monoclonal antibody feed materials. The assay entails applying a molecular rotor dye to a sample of free resin and measuring the fluorescence intensity using a plate reader or visualizing under a confocal laser scanning microscope to gain a more detailed characterization. Following five consecutive chromatography cycles, both methods revealed a 10-fold increase in fluorescence intensity along with a proportional reduction in dynamic binding capacity. Furthermore, the use of the assay suggested that fouling was dependent on spatial bead position in the column, bead channel structure and cleaning conditions. CONCLUSION: This work presents a simple assay suitable for use in resin lifetime studies to enhance process understanding.

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Section: Introductionmentioning

confidence: 99%

Chromatography process development aided by a dye‐based assay

Jasulaityte

Johansson²,

Bracewell

2019

J of Chemical Tech & Biotech

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“…Among the different tools, fluorescence is the only one that can be used for online monitoring of resin fouling. In a previous publications and , we have described the various studies performed to understand the mechanism of the fouling process (also include studies to understand the impact of different feed materials on resin fouling ), monitoring the foulant deposition with number of cycles together with the strategy to control the fouling process. When performing numerous subsequent cycles in column mode, there can be a gradual buildup of contaminants on the chromatography resin, causing fouling.…”

Section: Resultsmentioning

confidence: 99%

“…Figure D represents the predicted and experimental yield data for different cycles, with the error in prediction being <3%. In a prior publication , we have used this finding to monitor resin fouling and thereby take a decision on the cleaning regimen. With this monitoring and control strategy, the resin cycles performance (yield and DBC) were found to be stable till 200 cycles.…”

Section: Resultsmentioning

confidence: 99%

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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“…The use of appropriate cleaning buffers with controlled cleaning strategy would tackle the performance loss caused due to histones and deposition of the HCP on the resin surface over repeated reuse. Our previous publication has demonstrated the use of two step cleaning procedure (reducing agent followed by NaOH) using online monitoring tool results in an efficient cleaning and removal of histones and nonhistone proteins …”

Section: Resultsmentioning

confidence: 99%

“…Our previous publication has demonstrated the use of two step cleaning procedure (reducing agent followed by NaOH) using online monitoring tool results in an efficient cleaning and removal of histones and nonhistone proteins. 21…”

Section: Mode Of Capacity Lossmentioning

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

Cited by 9 publications

References 37 publications

Chromatography process development aided by a dye‐based assay

Chromatography process development aided by a dye‐based assay

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

Protein A chromatography resin lifetime—impact of feed composition

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