Practical approach to protein recovery by countercurrent distribution in aqueous two‐phase systems

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BACKGROUND The implementation of efficient processes for the development, recovery and purification of biological products is a main challenge for the bioengineering field. PEGylation can enhance the therapeutic properties of the protein, and often results in heterogeneous population of conjugated species and unmodified protein that presents a protein separations challenge. Non‐chromatography strategies, such as countercurrent distribution in aqueous two‐phase systems (CCD‐ATPS) and ultrafiltration (UF) for the fractionation of PEGylated RNase A conjugates represent attractive alternative worth of consideration. RESULTS In this work the potential application of CCD‐ATPS for the separation of closely related proteins (i.e. mono‐PEGylated and di‐PEGylated RNase A) was performed under selected conditions. Process conditions involved PEG 8000 13% (w/w), phosphate 10.4% (w/w), at pH 7.0 and volume ratio of 1.0 resulted, after 30 transfers in CCD, in distinguishable peaks that can be attributed to the mono‐PEGylated protein (61%) and di‐PEGylated (58%) form of RNase A. Alternatively, an ultrafiltration‐based strategy resulted in the partial separation of mono‐PEGylated and di‐PEGylated protein, 38% and 60%, respectively, in the concentrate with a 30 kDa membrane. Using a 50 kDa membrane the mono‐PEGylated RNase A accumulates in the filtrate (43%) and the di‐PEGylated in the concentrate (45%). CONCLUSIONS The results reported here can be considered for the separation of PEGylated proteins, as a first step in the implementation of a purification process. Copyright © 2012 Society of Chemical Industry

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

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Practical non‐chromatography strategies for the potential separation of PEGylated RNase A conjugates

Galindo-López

Rito‐Palomares

2012

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Bioseparation: The limiting step in bioprocess development

Rito‐Palomares

2008

Editorial Bioseparation: The limiting step in bioprocess developmentBioseparation can be defined as the set of sequential unit operations which, in a bioprocess, result in the recovery and purification of biological products -including proteins, enzymes, vaccines, colorants or antibodies. These are usually commercialized according to their biological function, and it is easy to anticipate that the bioseparation processes required to obtain, for example, industrial enzymes and oral or injected delivery products will differ greatly. It has been generally recognized that the recovery and purification stages (i.e. bioseparation), because the purity needed varies, represent the limiting steps in process development -but despite recent investigations, in particular from the industry, extensive research in this area has generally been neglected. Although several explanations for this trend can be given, we would like to highlight three major factors: (i) historical effect, (ii) process complexity and (iii) regulatory and validation issues. The historical effect is attributed to the need to develop the production stage of bioprocesses first. Thus, the bioprocesses initially developed to bring biological products to the market were strongly oriented towards the optimization of the fermentation step -neglecting the bioseparation part. This tendency was further accentuated by process complexity issues as fermentation, representing only one unit operation in comparison with typically more than ten unit operations for the bioseparation part of the process, was more thoroughly investigated. Finally, it is well-known that the implementation of advances in bioseparation processes has been hindered by validation and cost constraints. Regulatory and validation considerations have promoted a reluctance to embrace an entirely new technology, in this and other fields of biochemical engineering and biotechnology.In this Special Issue, JCTB presents a particular approach to trends and challenges in bioseparation. We have collected a Mini-Review, a Perspective, three Reviews and seven original research papers to outline the evolution of the type of problems, as well as the technologies, encountered in the field of product recovery and purification.The past, present and future challenges in efficient separations of recombinant proteins are nicely addressed by Juan Asenjo and Barbara Andrews in the first Mini-Review 1 . It raises how the challenges faced by bioseparation have changed over the past few years. The three main issues addressed are the increase in product concentration, the change of paradigm regarding the use of protein A approach and the need for complex and sophisticated mathematical models -key issue that will require particular attention from both academics and industrialists. The future application and industrial implementation of in-situ product removal (ISPR) techniques are addressed in the Perspective by John Woodley and co-workers 2 . The findings reported there are the results of a round-table discussion held at the...

Aqueous two‐phase systems in Latin America: perspective and future trends

Picó

Lima

Mussagy

et al. 2021

Aqueous two‐phase systems (ATPS) have been demonstrated to be a simple and robust worldwide applied bioprocessing technology. From high value microscopic to macroscopic biological molecules obtained from a wide broad of matrices, ATPS have shown a high efficiency capacity for their concentration, enrichment, recovery, and purification. Particularly in Latin America, four relevant countries have paved the way and played important leading roles in this transformation history. This perspective article aims to present a critical analysis of the practical evolution of the main implementations of ATPS in Argentina, Brazil, Chile, and Mexico. Special attention is given to novel applications of this biphasic bioprocess that are being explored to accomplish visionary therapeutic tasks for the healthcare, biotechnology, and pharmaceutical sectors, as well as other environmental purposes. © 2021 Society of Chemical Industry (SCI).