PEGylated Protein Purification Techniques

PEGylation is a procedure of growing interest for enhancing the therapeutic and biotechnological potential of peptides and proteins. However, PEGylation process usually leads to complex mixture of unreacted proteins, unreacted PEGs, hydrolysis fragments and undesired PEGamers (proteins with varying number of attached PEG molecules). Moreover, various positional isomers of PEGylated conjugates may also be formed, with different number of grafted chains, length and attaching sites. Therefore, effective purification strategies are always necessary to process these complex mixtures for commercial approval.

Fig. 1 Distribution of the publications reported in recent 10 years involving chromatographic and non-chromatographic techniques for purification of PEGylated proteins. (Brazilian Journal of Pharmaceutical Sciences 2018, 54 (SPE))

Chromatographic Fractionation Platforms

• Size Exclusion Chromatography (SEC)

Size exclusion chromatography (SEC) is a chromatographic technique for separation according to the size of samples. As well known, PEGylation of proteins always leads to the increase of hydrodynamic radius. SEC, therefore, is one of the first methods used for PEGylated protein purification. This technique is very efficient in removing low molecular weight by-products (PEG hydrolysis byproducts or buffer components), unreacted PEG and native proteins from the reaction mixture.

• Ion Exchange Chromatography (IEX)

Ion exchange chromatography (IEX) is the most commonly used technique for protein purification, and it also plays a role in the purification of PEGylated proteins. Because of PEG chains could shield the charges at the surface of proteins, thus altering the superficial charge density and the pH value of some residues. This can be exploited to separate positional isomers of the same degree of PEGylation, since the site of PEGylation affects differently the binding properties of the protein.

• Hydrophobic Interaction Chromatography (HIC)

Hydrophobic interaction chromatography (HIC) is an elution hierarchy method for separating and purifying protein-based biomacromolecules based on the difference of weak hydrophobic interaction between proteins and hydrophobic adsorbents. Although HIC is not so comprehensively as IEC or SEC, it is also used to purify PEGylated proteins. HIC can act as a great supplementary tool to IEC, realizing purification of proteins which are difficult to be purified by IEC. However, HIC has relative low capacity and poor resolution between adjacent peaks.

• Reverse Phase Chromatography (RP-HPLC)

Reverse phase chromatography (RPC, or RP-HPLC) refers to elution chromatography that uses a non-polar reversed-phase medium as the stationary phase and an aqueous solution of a polar organic solvent as the mobile phase to realize separation and purification according to the difference in solute polarity (hydrophobicity). RPC is widely used for the purification of peptides and small proteins. The separation of PEGylated conjugates using RPC can also be performed on analytical scale, involving identification of PEGylation sites and separation of positional isomers.

Non-Chromatographic Fractionation Platforms

• Capillary Electrophoresis (CE)

Capillary electrophoresis (CE) is a new type of liquid phase separation technology with capillary as the separation channel and high-voltage direct current electric field as the driving force. It combines multiple separation techniques by taking advantage of differences in charge density, size, shape, and surface properties for separating the PEGylated proteins. Therefore, it has gradually become a powerful technology for the high-resolution separation of different PEGylated products, especially for analysis and small-scale purification of PEGylated proteins.

• Membrane Separation Techniques

Membrane separation processes are the simplest methods among all non-chromatographic techniques currently used and are based on the molecular weight and hydrodynamic radius of the proteins, which have three main types: ultrafiltration, diafiltration and dialysis. PEGylated species can be efficiently separated and recovered using these membrane separation techniques. Although they are not able to separate isomers of PEGylated conjugates, they are most cost-effective.

• Aqueous Two-Phase System (ATPS)

When two polymers, one polymer and one lyophilic salt or two salts (one is a discrete salt and the other is a lyophilic salt) are mixed at an appropriate concentration or at a specific temperature, a two-phase system is formed. Compared with the water-organic solvent system, aqueous two-phase system (ATPS) provides more mild operating conditions that do not damage or denature unstable bioproducts. In addition, the polymer layer stabilizes the extracted proteins, which is beneficial to increase the targeted protein concentration to achieve a more effective extraction effect.

• Microfluidic Devices

To overcome the main drawbacks of nonchromatographic downstream processes, the use of microfluidic devices is emerging as a promising strategy. The microfluidic device integrates the basic operation units such as sample preparation, reaction, separation, and purification in the biological, chemical, and medical analysis process into a micron-scale chip to automatically complete the entire development process. We wish to give our customers a one-step operation with high throughput to realize rapid separation, concentration, and recovery of PEGylated proteins.

References

1. Pfister, D.; Morbidelli, M., Process for protein PEGylation. Journal of Controlled Release 2014, 180, 134-149.
2. Santos, J. H. P. M.; Torres-Obreque, K. M.; et al. Protein PEGylation for the design of biobetters: from reaction to purification processes. Brazilian Journal of Pharmaceutical Sciences 2018, 54 (SPE).