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Proteins, which have inherent biorecognition properties, have long been used as therapeutic agents for the treatment of a wide variety of clinical indications. Protein modification through covalent attachment to different moieties improves the therapeutic’s pharmacokinetic properties, affinity, stability, confers protection against proteolytic degradation, and increases circulation half-life. Nowadays, several modified therapeutic proteins, including PEGylated, Fc-fused, lipidated, albumin-fused, and glycosylated proteins have obtained regulatory approval for commercialization. During its manufacturing, the purification steps of the therapeutic agent are decisive to ensure the quality, effectiveness, potency, and safety of the final product. Due to the robustness, selectivity, and high resolution of chromatographic methods, these are recognized as the gold standard in the downstream processing of therapeutic proteins. Moreover, depending on the modification strategy, the protein will suffer different physicochemical changes, which must be considered to define a purification approach. This review aims to deeply analyze the purification methods employed for modified therapeutic proteins that are currently available on the market, to understand why the selected strategies were successful. Emphasis is placed on chromatographic methods since they govern the purification processes within the pharmaceutical industry. Furthermore, to discuss how the modification type strongly influences the purification strategy, the purification processes of three different modified versions of coagulation factor IX are contrasted.

BACKGROUND Protein refolding represents the limiting step in the recovery of these molecules from inclusion bodies and from denaturing operations. This raises the need for establishing different methodologies to efficiently recover and refold proteins. Aqueous two‐phase systems (ATPS) besides being a primary recovery operation provide good and affordable physicochemical conditions to refold these molecules. RESULTS 48 different PEG–potassium phosphate ATPS were tested for the partition and refolding of native and denatured invertase. Invertase in both forms has a partition preference for the bottom salt‐rich phase where the polymer molecular weight and the tie‐line length (TLL) selected for the system play a preponderant role. Refolding percentages of up to 100% can be obtained in the polymer‐rich phase and between 50% and 75% in the bottom phase. CONCLUSIONS This study reports the characterization of how the different ATPS design parameters such as polymer molecular weight, tie‐line length (TLL) and volume ratio (VR), influence protein partition and refolding using invertase as the protein model. A PEG 3350 g mol−1, TLL 25.0% w/w and VR = 1.00 system was selected as the best option for denatured invertase recovery and refolding with an enzyme recovery of 60.8% and a refolding percentage of 59.7% in the bottom phase of the system. These findings represent advancements en route to establishing an ATPS operation for the potential in situ recovery and refolding of denatured protein based‐products. © 2015 Society of Chemical Industry

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BACKGROUND: Purification of PEGylated proteins from reactions is still a challenge due to the formation of isomers. In this work, the recovery of PEGylated lysozyme was studied in a novel integrative approach called in situ aqueous two-phase systems (ATPS). The excess of PEG in the lysozyme PEGylation reaction (LPR) was used as part of the phase forming chemicals with different polymer (UCON, ficoll, dextran) and salt (sodium phosphate, potassium sulphate, sodium sulphate, ammonium sulphate, sodium carbonate) solutions. RESULTS: The best option for the in situ ATPS formation was the addition of a 4 mol L−1 ammonium sulphate in 20 mmol L −1 Tris-HCl (pH 7.0) solution to the PEGylation reaction. PEGylated conjugates of lysozyme exhibited a preferential partition to the top (PEG-rich) phase while native protein was partitioned to the bottom (salt-rich) phase. Di-PEGylated lysozyme partitioned entirely to the top phase (recovery of 100%), while the mono-PEGylated protein presented a recovery yield of 56% in the top phase. On its part, 97.7% of the native lysozyme was concentrated in the bottom phase. CONCLUSION: The proof-of-concept presented in this work allows the recovery of PEGylated proteins directly from a PEGylation reaction. It is the first time that PEGylated proteins have been fractionated using a PEG-ammonium sulphate system taking advantage of the unreacted PEG left in a PEGylation reaction. Based on reaction engineering, in situ ATPS are a potential mechanism for PEGylation control, a good alternative for 'packed-bed' or on-column PEGylation processes and a pioneer strategy in the development of phase transfer catalysis in PEGylation.

PEGylated or polyethylene glycol-modified proteins have been used as therapeutic agents in different diseases. However, the major drawback in their procurement is the purification process to separate unreacted proteins and the PEGylated species. Several efforts have been done to separate PEGylation reactions by chromatography using different stationary phases and modified supports. In this context, this study presents the use of chromatographic monoliths modified with polyethylene glycol (PEG) to separate PEGylated Ribonuclease A (RNase A). To do this, Convective Interaction Media (CIM) Ethylenediamine (EDA) monolithic disks were PEGylated using three PEG molecular weights (1, 10, and 20 kDa). The PEGylated monoliths were used to separate PEGylated RNase A modified, as well, with three PEG molecular weights (5, 20, and 40 kDa) by hydrophobic interaction chromatography. Performance results showed that Bovine Serum Albumin (BSA) can bind to PEGylated monoliths and the amount of bound BSA increases when ammonium sulfate concentration and flow rate increase. Furthermore, when PEGylated RNase A was loaded into the PEGylated monoliths, PEG-PEG interactions predominated in the separation of the different PEGylated species (i.e., mono and di-PEGylated). It was also observed that the molecular weight of grafted PEG chains to the monolith impacts strongly in the operation resolution. Interestingly, it was possible to separate, for the first time, isomers of 40 kDa PEGylated RNase A by hydrophobic interaction chromatography. This technology, based on PEGylated monoliths, represents a new methodology to efficiently separate proteins and PEGylated proteins. Besides, it could be used to separate other PEGylated molecules of biopharmaceutical or biotechnological interest.