Customized PEGylation Services

The concentration of protein in the reaction mixture is important in determining the final yield of PEGylation. The best yields are normally obtained at protein concentrations of 1–5 mg/mL. Therefore, it is important to know the protein concentration before the coupling procedure. This can be determined by amino acid analysis, UV, or classic colorimetric tests (1). PEGylation conversion yields also depend on PEG molecular weight, reactivity of the PEG reagent, the functional group on the protein and its surface exposition.

Most PEG reagents react rapidly with their targets so that the risk of protein degradation is low even if the reaction is performed at room temperature. In most cases, the reaction is completed within several hours at room temperature. Coupling can also be performed at 4°C. In such a case, one has to keep in mind that any reaction is significantly slower at this temperature. As a rule of thumb, the reaction rate doubles when the temperature is increased by 10°C.

Handling caution of activated PEG reagents

PEG is hygroscopic, which means it will absorb moisture from the air. If not stored under dry conditions, for example, activated PEG-carbonates and activated PEG-esters are slowly hydrolyzed during storage to form non-reactive PEGs. To minimize the levels of deactivation due to hydrolysis, store activated PEG reagents below -15°C under nitrogen and warm the package to room temperature before opening. Do not prepare the stock solution of these PEGs in water, keep them as solid powder.

Primary amino groups are good nucleophiles and are the residues most commonly used as a target in PEGylation development. Lysines are relatively abundant and exposed on protein surface so easily accessible to reactive PEG reagents (e.g., activated PEG-carbonates and activated PEG-esters). These activated PEGs are deactivated in water (see Table at page 12), therefore an excess of activated PEG, with respect to the protein, is usually needed for these reaction strategy, especially when conducted in basic buffers (pH 8.0–9.5) as solvent. A primary amine is also present at the N-terminus of protein chains, commonly referred to as α-amine group, when this residue is not masked by posttranslational modification it can also react with PEGylating agents targeting the amino group of lysine.

Random PEGylation at amino groups typically yields mixtures of different isomers and different degrees of modification (i.e. different number of PEG chains per protein unit). Up to day, several marketed PEGylated protein products have been produced by the Random PEGylation.

Protocol of Random PEGylation at amino groups of protein

1. Prepare the protein solution (1–5 mg/mL) in 0.1 M borate buffer, pH 8.0–9.0 (lower pH values can be eventually used to decrease the rate of PEG conjugation if needed).
2. Determine the exact concentration of the protein by a suitable method of choice (e.g. UV absorption using its molar extinction coefficient (1)).
3. Add the activated PEG-carbonate or activated PEG-ester to the protein solution under gentle stirring. An excess of activated PEG or each protein amino group is usually required. The optimum molar ratio of PEG to each protein amino group may range from 1 to 10, depending on the particular PEG and the reactivity of the amino groups on the protein. There are some examples of protein conjugation experiments that have been reported in the literature (2,3,4,5).
4. Incubate the reaction mixture at room temperature for 1–5 h, Nitrogen or Argon atmosphere can be used if needed to avoid protein oxidation. Monitor the reaction by HPLC.
5. Quench the reaction with a glycine solution (the molar ratio of glycine to PEG is above 100) and stir for 1 h.
6. Dialyze the resulting solution to eliminate low-molecular weight products and to exchange the buffers as eventually required by the chosen purification technique. As an alternative to dialysis, ultrafiltration/diafiltration can also be used.
7. Purify the PEG–protein conjugate by chromatographic techniques, such as Ion Exchange Chromatography and Size-Exclusion Chromatography.

In order to obtain a highly homogeneous product, it is possible to direct selectively the PEG coupling reaction at the N-terminus of a protein. This selectivity is possible by taking advantage of the different pKa values between the ε-amine of lysines and the α-amine of the N-terminus. By lowering the pH of the reaction mixture to 4.5–6, the ε-amine of lysines will tend to be protonated, and therefore not reactive, whereas the α−amine at N-terminus will still be partially present as a free base, available for coupling with activated PEG molecules. This method generally gives optimal results when activated PEG-aldehydes are used. In these reactions, the unstable Schiff base, initially formed, is reduced to yield a stable secondary amine. Several papers describe the modification of primary amines with PEG-propionaldehyde (6, 7). This conjugation method has been successfully exploited for the preparation of several PEG–protein conjugates; among these, Neulasta®, an N-terminal mono-PEGylated granulocyte colonystimulating factor (G-CSF), has demonstrated therapeutic and marketing success (8).

Protocol of Selective PEGylation at N-terminus of protein

1. Prepare a protein solution at 1–5 mg/mL in 0.1M buffer with pH 4.5–6.0 (acetate or phosphate buffer etc.).
2. Determine the exact concentration of the protein by a suitable method of choice (e.g. UV absorption using its molar extinction coefficient (1)).
3. Add PEG-aldehyde to the protein solution at the desired molar ratio. Depending on the protein properties, an excess of PEGaldehyde may be needed. It is advisable to test several different PEG/protein molar ratios to optimize the reaction (e.g., 5–25 eq. with respect to the amount of protein molecules).
4. Incubate the reaction mixture for 1 h, and then add NaCNBH3 solution (20 mM) (add over 100 eq. of NaCNBH3 with respect to the amount of protein molecules).
5. Incubate the reaction mixture at 4°C for 24 h under gentle stirring and under nitrogen flushing. Monitor the reaction by HPLC.
6. Quench the reaction with glycine solution (the molar ratio of glycine to PEG is above 100) and stir for 1 h.
7. Dialyze the resulting solution to eliminate low-molecular weight products and to exchange the buffers as eventually required by the chosen purification technique. As an alternative to dialysis, ultrafiltration/diafiltration can also be used.
8. Purify the PEG–protein conjugate by chromatographic techniques, such as Ion Exchange Chromatography and Size-Exclusion Chromatography.

Cysteine residues, when present, are valuable targets for achieving site-specific modification of proteins or peptides, but free cysteines have a low natural abundance level compared to the oxidized cysteine species involved in the disulfide bridges. Nevertheless, cysteines – if present – are often found partially or fully buried within the structure of proteins, eventually in hydrophobic tasks with limited accessibility for chemical reagents especially polymers (9). Under appropriate conditions, cysteine residues can be modified selectively, rapidly, quantitatively, and either in a reversible or irreversible fashion (10). Furthermore, thanks to its relatively facile coupling chemistry, there are several examples of the insertion of cysteines by genetic engineering at desired positions in a protein sequence for site-specific conjugation (11). If the target is cysteine residue derived from disulfide bridge, it is necessary to reduce the disulfide bond of the protein in advance of PEGylation using reductive reagents (12).

PEG-maleimide (PEG-Mal), PEG-iodoacetamide (PEG-IA) derivatives have been used to obtain thioether bonds between polymers and proteins. Maleimide group is hydrolyzed at basic condition and changes to maleamic acid (see Table at page 14). When PEGMal is used, pH conditions above 7.5 should be avoided. PEG-IA is less reactive and infrequently used, whereas PEG-Mal yields quantitative protein modification.

Protocol of Site-specific PEGylation at free thiol groups of protein

1. Prepare a protein solution of 1–5 mg/mL in 0.1 M buffer with pH 6.0-7.2 (phosphate buffer containing 10 mM EDTA etc.).
2. Determine the exact concentration of the protein by a suitable method of choice (e.g. UV absorption using its molar extinction coefficient (1)).
3. Add PEG-Mal to the protein solution at a molar ratio 1-2 to 1 with respect to the amount of free thiol present. If PEG-IA is used, an excess of 2–10 eq. is recommended.
4. Incubate the reaction mixture at 4°C for 4–24 h (depending on the PEG derivatives used) under gentle stirring and inert atmosphere, argon or nitrogen. Monitor the reaction by HPLC.
5. Quench the reaction with cysteine solution (the molar ratio of cysteine to PEG is above 100) and stir for 1 h.
6. Dialyze the resulting solution to eliminate low-molecular weight products and to exchange the buffers as eventually required by the chosen purification technique. As an alternative to dialysis, ultrafiltration/diafiltration can also be used.
7. Purify the PEG–protein conjugate by chromatographic techniques, such as Ion Exchange Chromatography and Size-Exclusion Chromatography.