Additional Studies in the Separation of PEGylated Proteins by Reversed Phase Chromatography

Vita Knudson, Tivadar Farkas, and Michael McGinley Phenomenex Inc.

Additional studies were undertaken to better understand the chromatographic behavior of PEGylated proteins in an effort to improve purification and characterization techniques of such proteins. Proteins were PEGylated using larger (20 KDa and 40 KDa) PEGylation reagents that are commonly used in pharmaceutical drug development. Generated PEGylated proteins were separated from unmodified proteins using different reversed phase medias (Jupiter® C4 and Jupiter® C18). In these studies it was found that the Jupiter C18 media provided the best separation of PEGylated proteins from their unmodified counterparts. Such results further clarify good method starting points for developing analytical and preparative separations of PEGylated proteins.

The benefits of covalently conjugated PEG molecules have been well studied (1, 2.) Depending on the desired site of modification, different attachment chemistries can be used to attach single or multiple PEG moieties to a protein; most commonly used are chemistries that attach to primary amines on proteins (either the N-terminus or any Lysine residue) or to a free sulfhydral (Cysteine residues) moiety.

A previous study (3) using low molecular weight PEGs focused on improving reversed phase chromatography conditions for separating different sites of PEGylation on various proteins. Numerous parameters including temperature, gradients, and mobile phase composition were evaluated. These studies were initiated to determine if similar chromatography is observed when larger PEG forms were used. Such PEGs (20 KDa and 40 KDa PEGs with varying chemistries) are more typically used in pharmaceutical drug development and thus might give more relevant information for those developing PEGylated protein therapeutics.

Materials and Methods

Analyses were performed using a HP 1100 LC system (Agilent Technologies, Palo Alto, California) equipped with a DAD detector. Various HPLC columns were used for evaluations including Jupiter® 300 5 μm C4, Jupiter® 300 3 μm C18 and 5 μm C18 (all 150 × 4.6 mm, Phenomenex, Torrance, California). Native proteins were purchased from Sigma Chemicals (St. Louis, Missouri) and PEGylation reagents to modify proteins were obtained from Jenkem Technology (Beijing, China). Solvents were purchased from Fisher Scientific (Fairlawn, New Jersey).

Proteins PEGylation was performed using two PEG N-hydroxysuccinimide (NHS) esters derivatives including Methoxy PEG Suiccinimidyl Carboxy Methyl Ester, MW 20 kDa (M-SCM-20K) and Methyl-PEO12-NHS Ester; Methoxy PEG Propionaldehyde, MW 20 kDa (M-ALD-20K), and Y-shape PEG Aldehyde, MW 40 kDa (Y-ALD-40K). Native proteins were dissolved in phosphate buffer pH 7.8, M-SCM-20K were dissolved in dry water-miscible DMSO; protein PEGylation reaction was done with 6-fold molar excess of M-SCM-20K for protein solution concentration of 40 mg/ml. Another set of native proteins and PEG substances (M-ALD-20K and Y-ALD-40K) were dissolved all in phosphate buffer pH 6.5 with 20mM of sodium cyanoborohydrate; the reaction was done with 8-fold molar excess of M-SCM-20K for protein solution concentration of 40 mg/ml.

The reaction mixture is incubated in an ice bucket for up to 2 h (different time-points were taken for some experiments). Reaction mixture is quenched with an equal volume of 50 mM Tris/ 1% TFA (pH~2). Diluted in mobile phase, aliquots of 10–15 μg of protein (4 μL) were injected in HPLC for analysis.

Aqueous mobile phase used in reversed phase experiments was 0.1% TFA and 2% ACN in water and gradient from 20% to 65% B of organic mobile phase, 90% acetonitrile/ 0.085% TFA in water in 25 min was used. After each run the column had a 5 min flush with 90% B followed by reequilibration at 20% B. The column temperature used was 45°C. Flow rate for all analyses was 1 mL/min and UV monitored protein elution was at 220 nm (such conditions were indicated to give the best results for PEGylated proteins based on the previous study).

Results and discussions

Previous results had shown that reversed phase chromatography (RP) provided equal or superior separation of PEGylated proteins when compared to gel filtration chromatography (GFC). Unlike GFC which can only separate PEGylated proteins based on degrees of polymerization, RP chromatography can also separate PEGylated species based on site of PEG attachment. Method development starting parameters such as mobile phase composition (Water/ TFA and Acetonitrile), temperature (45°C), gradient (20–65%) and RP media (Jupiter 5μm C4) were determined. This study was initiated to see if such parameters applied to larger PEG species as well as determine if a new wide pore media (Jupiter 3μm C18) was useful for separation of PEGylated proteins.

Various PEG derivatives with different PEG chain length, shape, and molecular weight were chosen to monitor heterogeneity and complexity of PEGylated proteins/peptides. Two N-terminal PEGylation reagents were chosen, Methoxy PEG aldehyde, MW 20 kDa (PEG3) and Y-shape 40 kDa PEG aldehyde (PEG4), which undergo reductive amination reactions with primary amines in presence of cyanoborohydride pH 6.5 [to take advantage of the lower pKa of the N-terminal amine (pKa~8) compared to amino acid side chains, such conditions result in selective modification of the N-terminus] (4).

Two other amine PEG reagents, Methoxy PEG NHS-ester, MW 20 kDa (PEG1) and 12-mer Methoxy PEG NHS-ester, MW 1 kDa (PEG2) reacts efficiently with primary amino groups (-NH2) at higher 7.8 pH forming amide bonds at each lysine (K) residue as well as the N-terminal amine.

Reversed phase separation data completed in this study demonstrate a distribution of PEG-protein conjugations. Figure 1 shows chromatographic differences in the PEGylation reaction of alpha-Chymotrypsinogen A protein using the two different 20 KDa PEGylation reagents. As it was described, one PEGylation reagent reacts with both the Lysine side chain and N-terminus of Chymotrypsinogen (PEG 1, Figure 1A); the other PEGylation reagent reacts only with N-terminus (PEG3, Figure 1B). From the results it appears that the reaction occurs at different rates and generates different species that can be separated chromatographically. As expected, PEG1 quickly modifies multiple sites that can be separated chromatographically despite claims of specific modification; it appears that PEG3 reacts with side chains, albeit at a lower reaction rate than PEG1.

Figure 1

PEGylation of Substance P peptide with 1 kDa PEG molecule Methoxy PEG NHS-ester, presumably reacts efficiently with primary amino groups (-NH2) at higher 7.8 pH, separating as one peak on RP media (PEG2, Figure 2).

Figure 2

In addition to polar interaction between PEGylated molecules and reversed phase media, their size, shape, and/or hydrophobicity will also affect separation performance.

To consider the complexity of a PEGylated products separation, several reversed phase columns were selected for this test and several protein sample fractions at different PEGylation reaction time-points were analyzed (Figure 3). Comparing data on Jupiter 300 C18 3μm, Jupiter 300 C18 5μm, and Jupiter 300 C4 5μm for separation of large 20 kDa (Figure 3a and b) and 40 kDa (Figure 3c) PEG molecules conjugated with proteins, the C18 delivers better resolution compared to Jupiter C4. However, for separation of PEGylated proteins and peptides with small PEG molecules less than 1 kDa, the C4 media gives better resolution performance (3) (data not shown here). Furthermore, separation on a C18 3μm column gives a clearly observed advantage in separating big PEGylated molecules compared to a C18 5μm, based on the separation of some selected fractions of PEGylated alpha-chymotrypsinogen A and beta-lactoglobulin A proteins.

Figure 3

Conclusion

This paper shows that the evaluated Jupiter RP media is a good analytical tool to analyze and characterize protein PEGylation processes. Additional useful applications for RP chromatography include defining sufficient excess of PEG moiety for reaction with proteins of interest, monitoring PEG conjugation process for native bio-molecules, and to purify their PEGylated products. We have shown further improvements and advantages for separation of large PEGylated conjugates and unreacted molecules using smaller particles reversed phase Jupiter 300 3 μm C18 media versus Jupiter 300 5 μm C18, and the advantages of separation of smaller PEGylated conjugates on reversed phase Jupiter 300 5 μm C4 media.

References

(1) Veronese, F.M., Harris, J.M.; "Introduction and overview of peptide and protein PEGylation"; Adv. Drug Devil. Rev. 54: 453–456 (2002).

(2) Bailon, P., Palleroni, A., Schaffer, C. A.; "Rational design of a potent, long-lasting form of interferon: a 40kDa-branched polyethylene glycol-conjugated interferon alpha-2a for the treatment of hepatitis"; Bioconj. Chem. 12: 195–202 (2001).

(3) Knudson, V., Farkas, T., McGinley, M.; "Investigation into improving the separation of PEGylated proteins"; Phenomenex: Technical Note TN-1034 (2006).

(4) Na, D.H., Deluca, P.P.; "PEGylation of octreotide: I. Separation of positional isomers and stability against acylation by poly (D,L-lactide-co-glycolide)"; Pharma. Res. 22(5): 736–742 (2005).

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