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In addition to their use as therapeutics and because of their enhanced properties, PEGylated proteins have potential application in fields such as bioprocessing. However, the use of PEGylated conjugates to improve the performance of bioprocess has not been widely explored. This limited additional industrial use of PEG-protein conjugates can be attributed to the fact that PEGylation reactions, separation of the products, and final characterization of the structure and activity of the resulting species are not trivial tasks. The development of bioprocessing operations based on PEGylated proteins relies heavily in the use of analytical tools that must sometimes be adapted from the strategies used in pharmaceutical conjugate development. For instance, to evaluate conjugate performance in bioprocessing operations, both chromatographic and non-chromatographic steps must be used to separate and quantify the resulting reaction species. Characterization of the conjugates by mass spectrometry, circular dichroism, and specific activity assays, among other adapted techniques, is then required to evaluate the feasibility of using the conjugates in any operation. Correct selection of the technical and analytical methods in each of the steps from design of the PEGylation reaction to its final engineering application will ensure success in implementing a "PEGylaided" process. In this context, the objective of this review is to describe technological and analytical trends in developing successful applications of PEGylated conjugates in bioprocesses and to describe potential fields in which these proteins can be exploited.

In recent years, the effective purification of PEGylated therapeutic proteins from reaction media has received particular attention. Although several techniques have been used, affinity-based strategies have been scarcely explored despite the fact that, after PEGylation, marked changes in the molecular affinity parameters of the modified molecules are observed. With this in mind, future contributions in the bioseparation of these polymer-protein conjugates are expected to exploit affinity in chromatographic and nonchromatographic techniques which will surely derive in the integration of different operations. However, this will only occur as novel ligands which are simultaneously found. As it will be mentioned, these novel ligands may be screened or designed. In both cases, computer-aided tools will support their identification or development. Additionally, ligand discovery by high-throughput screening (HTS) is believed to become a fast, economic, and informative technology that will aid in the mass production of ligands along with genetic engineering and related technologies. Therefore, besides analyzing the state of the art in affinity separation strategies for PEGylated molecules, this review proposes a basic guideline for the selection of adequate ligands to provide information and prospective on the future of affinity operations in solving this particular bioengineering problem. [ABSTRACT FROM AUTHOR]

Antibodies for therapeutic use are being continuously approved and their demand has been steadily growing. As known, the golden standard for monoclonal antibody (mAb) purification is Protein A affinity chromatography, a technology that has gained high interest because of its great performance and capabilities. The main concerns are the elevated resins costs and their limited lifetime compared to other resins (e.g. ion exchange chromatography). Great efforts have been carried out to improve purification conditions, such as resin characterization and designing alkali/acid stable resins with a longer lifetime. Modification of Protein A ligands and alternative formats such as monoliths membranes and microshperes have been tested to increase the purification performance. New technology has been proposed to improve the large‐scale separation; in addition, alternative ligands have been suggested to capture mAbs instead of Protein A ligand; however, most of the information is locked by pharmaceutical companies. This mini review summarizes and describes the advances, results, and impact on the Protein A chromatography purification processing. [ABSTRACT FROM AUTHOR]

Ribonuclease A (RNase A) from bovine pancreas and its PEGylated conjugates has proven to have potential therapeutic applications. Aqueous Two-Phase Systems (ATPS) is a promising primary recovery strategy for the fractionation of proteins and their PEGylated conjugates. However, in order to characterize the partition behavior of these molecules in ATPS, an easy-to-implement method is needed to estimate protein concentration in each phase. This paper presents a novel methodology based on UV absorbance to quantify RNase A and its PEGylated conjugates on polymer (polyethylene glycol) and salt (potassium phosphate) rich environments, simulating conditions found on polymer-salt ATPS. [ABSTRACT FROM AUTHOR]

Background: PEGylation, defined as the covalent attachment of polyethylene glycol, allows the synthesis of PEGylated therapeutic proteins with enhanced physicochemical properties. Traditional alkylating Nterminal PEGylation reactions on amine groups involve the use of modified linear mono-methoxy polyethylene glycol (mPEG) molecules looking for the synthesis of mono-PEGylated products. However, this approach requires different purification steps since inevitably undesired cross-linked products are synthesized. Herein, we propose the use of reactive aqueous two-phase systems (ATPS) to produce and purify PEGylated therapeutic conjugates using Ribonuclease A (RNase A) as a model protein. Results: Selected linear 5 kDa and 20 kDa mPEG - potassium phosphate systems were produced according to equilibrium data obtained from constructed binodal curves. All reactive systems were able to generate biphasic systems and to PEGylate RNase A. Two 5 kDa and two 20 kDa systems were selected based on the reaction yield percentage and the feasibility of purifying the mono-PEGylated RNase A from the di- PEGylated and native RNase A by contrasting the differences in their partition behaviors. The remnant biological activity was of 94% and of 100% for the mono-PEGylated RNase A purified from the 5 kDa and 20 kDa mPEG systems when compared to the mono-PEGylated conjugate obtained by standard procurement methods. Conclusions: This novel approach using reactive ATPS shows that it is feasible to simultaneously produce and purify PEGylated therapeutic proteins with conserved biological activity and presents another example where reactive ATPS can be successfully implemented. [ABSTRACT FROM AUTHOR]