Protein PEGylation: An overview of chemistry and process considerations

Bioconjugation and polymer therapeutics

The rapid growth in biotechnology and molecular biology during the last two decades has contributed to a substantial increase in the number of biological products such as proteins, peptides, hormones and enzymes for pharmaceutical applications^1,2. The pharmaceutical significance of these biomolecules can be mainly attributed to their high specificity, rapid onset of action and requirement for relatively small doses compared to conventional synthetic molecules³. Unfortunately, most biomolecules are characterised by short circulating lives, low stability due to proteolytic and enzymatic degradation in vivo and rapid clearance from the body via glomerular filtration^4,5.

To overcome these drawbacks, biomolecules can be protected through covalent binding with another molecule, or bioconjugation6. Many polymers, from both biological and synthetic origins, are used to protect biomolecules. The resulting polymer bioconjugates are characterised by improved properties such as reduced immunogenicity, decreased antibody recognition, increased in vivo residence time, increased drug targeting specificity and bioavailability, and improved pharmacokinetics^6-10. The concept of polymer bioconjugation has been extended to small, high-value prodrugs, leading to a new era of polymeric drug delivery systems and polymer therapeutics8. Commonly used polymers in drug delivery applications include poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA), poly(oligoethylene glycol methyl ether methacrylate) (POEGMA), poly(D,L-lactic-co-glycolic acid) (PLGA), poly(glutamic acid) (PGA), poly(N-isopropyl acrylamide) (PNIPAM), poly(N,N’- diethyl acrylamide) (PDEAM), polystyrene and poly(ethylene glycol) (PEG).

PEG – the polymer of choice

PEG is biocompatible, lacks immunogenicity, antigenicity and toxicity, is soluble in water and other organic solvents, is readily cleared from the body and has high mobility in solution, making this the polymer of choice for bioconjugation^11-13. The conjugation of a biomolecule with PEG will result in the modification of its physiochemical properties, particularly size, and increase the systemic retention of the therapeutic agent in the body. It may also enable the moiety to cross the cell membrane by endocytosis to reach particular intracellular targets8. Moreover, PEG is one of a small number of synthetic polymers generally regarded as safe by the US FDA for internal administration¹⁴.

Protein PEGylation and its pharmaceutical significance

In the late 1970s, Professor Frank Davis and his colleagues covalently linked methoxy PEG (mPEG) to bovine serum albumin¹⁵ and bovine liver catalase¹⁶, using cyanuric chloride as an activating agent. Their studies showed that “hanging a bit of PEG onto a protein” markedly improved the overall properties and stability of the protein^17,18. This technique is now well established and is known as “PEGylation”. The applications of PEGylation can be extended to peptides, enzymes, antibody fragments, nucleotides and even small organic molecules^19,20.

PEGylation can impart several significant and distinct pharmacological advantages over the unmodified form, including improved drug solubility, reduced dosage frequency, toxicity and rate of kidney clearance, an extended circulating life, increased drug stability, enhanced protection from proteolytic degradation, decreased immunogenicity and antigenicity, and minimal loss of biological activity^4,21-23.

The reduced kidney clearance of PEGylated proteins can be attributed to an apparent shielding of protein surface charges and an increased hydrodynamic volume of the conjugated product24 due to the ability of PEG molecules to coordinate with two to three water molecules per monomer unit25. Furthermore, these hydrated polymer chains provide a protecting mask for the protein, becoming more effective with an increase in the number and molecular weights of the attached PEGs, reducing the phagocytic uptake by parenchimal cells, preventing opsonisation and increasing residence time in systemic circulation^24,26.

In addition to these pharmacological advantages, PEGylation can substantially alter the physicochemical properties of the parent protein, including electrostatic and hydrophobic properties²³. PEGylation significantly influences the elimination pathway of the molecule, by shifting from a renal to a hepatic pathway. The tissue-organ distribution profile of the molecule is also greatly influenced by PEGylation, wherein PEGylated proteins preferably follow a peripheral distribution^24,26. The pharmaceutical value of PEGylation is now well accepted, with many FDA approved drugs already launched in the market and many in clinical trials. Table 1 shows some examples of approved PEGylated therapeutics.

Table 1 – Examples of FDA approved PEGylated drugs

The chemistry of protein conjugation

PEGylation conjugation strategy has developed from non-specific random conjugations, known as “first generation PEGylation,” to the more recent site-specific conjugation methods known as “second generation PEGylation”²⁷, although both methods continue to be used. The increase in PEGylation specificity can be mainly credited to the availability of more specific functionalisation of PEG molecules capable of reacting to particular functional moieties in the protein. The result is controlled, well-defined conjugated products with improved product profiles over those obtained through non-specific random conjugations.

Amine conjugation

Because of the availability of a number of accessible primary amino groups on the surface of a protein, conjugation through this functional group is the most extensively used method. Lysine, ornithine and N-terminal amino groups are the most commonly exploited²⁸. Early developments in PEGylation mainly targeted the N-terminal amino groups of lysine. The first reactions reported by Davis and his colleagues involved the reaction of cyanuric chloride activated PEG with the primary amine groups of bovine serum albumin¹⁵ and bovine liver catalase¹⁶, through alkylation of their respective amine terminals (see Figure 1a). Thereafter, PEG-tresylate was also developed for protein conjugation through alkylation29 but all these reactions resulted in non-specific, multiply conjugated products.

Figure 1 – PEGylation process using amine conjugation chemistries

A greater specificity and selectivity in N-alkyl conjugation strategy was developed after the introduction of PEG aldehyde derivatives, particularly mPEG-propionaldehyde³⁰, capable of forming a stable secondary amine linkage with amino groups through reductive alkylation using sodium cyanoborohydride (see Figure 1b). Because the reactivity of aldehyde groups depends on the nucleophilicity of amine groups, reaction will take place only when the pH of the medium is near or above the pKa of that particular amine terminal. Hence, by controlling the pH of the reaction medium, the heterogeneity of the product profile can be greatly reduced¹². This conjugation strategy was adopted for the development of Neulasta®, a PEGylated granulocyte colony stimulating factor (PEG-G-CSF), where a linear 20 kDa mPEG-aldehyde derivative was selectively attached to an N-terminal methionine residue of filgrastim through reductive alkylation under mild acidic conditions^31,32. Another example using reductive alkylation chemistry is the PEGylation of a recombinant soluble tumour necrosis factor receptor type I (sTNF-RI) from E. coli, for use in treating chronic inflammatory diseases³³.

The acylation of the N-terminal amino acids results in the formation of stable amide and urethane linkages. PEG derivatives activated with succinimidyl succinate (PEG-SS)³⁴, succinimidyl carbonate (PEG-SC)³⁵, benzotriazole carbonate (PEG-BTC)³⁶, phenyl carbonate^37,38, carbonylimidazole³⁹, and thiazolidine-2-thione⁴⁰ were used early in protein conjugation, following the N-terminal acylation pathway. Adagen® (PEG-adenosine deaminase) and Oncaspar® (PEG-asparaginase), the first FDA-approved PEGylated products launched in the market, are prepared based on this chemistry⁴¹ (see Figure 1c, page 19). PEG-Intron® (PEG-α-interferon 2b) is prepared by conjugating interferon with a single chain 12 kDa PEG-SC via a urethane bond (see Figure 1, page 19), where the PEG is mainly conjugated with the histidine residue⁴².

The introduction of monosubstituted propionic and butanoic acid PEG derivatives by Harris and Kozlowski⁴³ and their subsequent activation using succinimide derivatives contributed a significant improvement in amine conjugatio. Pegasys®, another PEGylated interferon, is prepared by mono-PEGylation of interferon-α-2a with an N-hydroxysuccinimide (NHS) activated 40 kDa branched PEG molecule⁴⁴ (see Figure 2). Somavert®, is also prepared by conjugating 4 – 5 NHS activated 5 kDa PEG derivatives with the lysines of human growth hormone antagonist^45,46. Due to the selectivity of NHS active esters towards primary amine terminals, this conjugation technique remains one of the most common conjugation strategies⁴⁷. Even though amine conjugation is widely accepted and clinically proven, the method is associated with some potential disadvantages, including loss of bioactivity due to the inactivation of critical functional groups and the formation of multiply PEGylated products due to the presence of multiple amino groups available for conjugation⁴⁸.

Figure 2 – Conjunation chemistry for Pegasys 44

Thiol conjugation

Selective thiol conjugation with natural or genetically engineered, unpaired cysteine residues provides a site-specific conjugation methodology. Thiol selective derivatives such as PEG-maleimide, vinylsulfone, iodoacetamide, and orthopyridyl disulfide are used for cysteine conjugation through formation of thioether or disulfide linkages¹². Examples using PEG-maleimide include those at the genetically introduced cysteine residue of trichosanthin (TCS) using 5 and 20 kDa⁴⁹, antitumor necrosis factor-α-scFv fragment (anti-TNF-α-scFv) using 5, 20 and 40 kDa⁵⁰ and recombinant staphylokinase (Sak) using 5, 10 and 20 kDa derivatives⁵¹ (see Figure 3).

Figure 3 – Cysteine conjugation using PEG-maleimide

Because of the limited availability of single cysteine residues and the chances of protein dimerisation resulting from the introduction of genetically engineered cysteines, the use of this strategy has been limited. Taking advantage of a higher number of accessible disulfide linkages present with paired cysteines in proteins, Balan et al. reported a thiol specific bis-alkylation PEGylation, with the two sulphur atoms which were generated by the mild reduction of these disulfide bonds^52,53. A selective reduction of the disulfide bridges was performed using dithiothreitol (DTT) in neutral conditions or tris-(2-carboxyethyl) phosphine hydrochloride (TCEP-HCl) in slightly acidic conditions. The reduced protein was then treated with an active ester PEG-monosulfone at an acidic pH (see Figure 4, page 20). Steric shielding by the attached PEG molecule against a second molecule approaching the same reduced site, an important requirement for the bridged structure, prevented heterogeneity of the product profile and resulted predominantly in the mono-PEGylated derivative. Retention of the protein’s tertiary structure and selectivity and reversibility of the thiol conjugation are some of the attractive features of this technique.

Figure 4 – Disulfide bridging PEGylation and structure of PEG-monosulfone

Oxidised carbohydrate or N-terminal conjugation

The enzymatic (e.g., glucose oxidase) or chemical (e.g., sodium periodate) oxidation of carbohydrate groups present in glycoproteins or N-terminal serine or threonine residues generates reactive aldehyde groups, which can be further conjugated with PEG hydrazide or amine derivatives^12,47,54 (see Figure 5).

Figure 5 – Oxidised carbohydrate or N-terminal conjugation

Zalipsky and his colleagues used this methodology for PEGylating immunoglobulin G (IgG), which contains nearly 4% carbohydrate. IgG was first oxidised with periodate and then conjugated with mPEG-hydrazide derivative⁵⁴. Following the same chemistry, periodate oxidised Ricin A-chain (RTA), an excellent immunotoxin source, was successfully conjugated with a 5 kDa mPEG-hydrazide⁵⁵.

Transglutaminase (TGase) mediated enzymatic conjugation

A novel, site specific PEGylation methodology targeting glutamine residues was reported by Sato⁵⁶, using a TGase catalysed acyl transfer reaction between the glutamine (Gln) terminal and PEG primary amino group. Sato developed this distinctive strategy by incorporating a short sequence of Gln residues at the protein terminal, without disturbing its flexibility and conformation, and modified it with primary amine derivatives of PEGs in the presence of TGase (see Figure 6). Compared with other methodologies, TGase mediated conjugations were found to be more site-specific, reliable, reproducible and versatile⁵⁷. TGase catalysed selective PEGylations of apomyoglobin (apoMb), α-lactalbumin (α-LA), human growth hormone (hGH) human granulocyte colony-stimulating factor (hG-CSF) and human interlukin-2 (hIL-2) with PEG amines are some of the proven examples of this new technique^57,58.

Figure 6 – TGase catalysed conjugation

Miscellaneous conjugation chemistries

DeFrees et al⁵⁹ reported a new site specific process known as GlycoPEGylation, using an enzymatic N-acetylgalactosamine (GalNAc) O-glycolization, followed by PEGylation of the introduced O-glycans using a PEG sailic acid derivative. The new click chemistry strategies⁶⁰ may also play an emerging role in PEGylation⁶¹. Deiters et al⁶² reported site specific mono-PEGylation of genetically modified superoxide dismutase (SOD) using a PEG-alkyne derivative to attach to the azide terminal (see Figure 7, page 23). Although it has attracted little attention and has limited direct pharmaceutical relevance, PEGylation of human serum albumin using PEG-phenyl-isothiocyanate⁶³ and PEG-epoxide⁶⁴ may be of scientific interest in developing PEGylation technologies.

Figure 7 – Site specific PEGylation using click chemistry

Reversible PEGylation

The improved physicochemical properties of protein PEGylation are offset in many cases by a substantial reduction in the in vitro protein activity arising from the permanent linkages formed during PEG conjugation. Consequently, a reversible (or releasable) PEGylation concept has been formulated, in which proteins are attached to PEG derivatives through cleavable linkages, which release the protein in vivo at a predetermined kinetic rate65. One example is the release of PEGylated lysozyme using a 1,6-benzyl elimination mechanism^66,67 (see Figure 8). A number of similar reversible PEG derivatives have been reported, using bicin, oligo-lactic acid ester, succinic ester, disulfide and β-alanine ester linkers^68,69. However, even though this technique potentially provides controlled release of proteins in their fully active forms, the chances of undesirable residual tags remaining with the protein and/or the in vivo formation of reaction by-products remain areas of great concern with this technique.

Figure 8 – Reversible PEGylation using 1,6-benzyl elimination

Structure of PEGs

The development of PEGylation is also characterised by a marked improvement and diversity in the nature of the PEGs used for protein conjugation. Early stage PEGylations were performed mainly with linear and low molecular weight PEGs. The earliest PEG derivatives were also characterised by higher diol contents (around 15%) and polydispersity, contributing to an increased product heterogeneity^12,27. The recent commercial availability of pure and low polydisperse PEG derivatives has resulted in much improved product profiles.

The introduction of branched PEG derivatives resulted in an improvement in pharmacological properties, the reasons for which are not yet clear. It has been shown that the net size of linear- and branched-PEG protein conjugates are closely similar⁷⁰, if not identical, so it is likely that more effective local surface masking and slight decreases in PEG chain flexibility better protect the conjugated protein from proteolytic attack, rather than alter glomerular filtration. The first branched PEG derivative for protein conjugation was prepared by Matsushima et al.⁷¹, using the reaction between cyanuric chloride and mPEG, and the resulting species were conjugated with E. coli asparaginase (see Figure 9). Another branched PEG derivative, mPEG2-COOSu, prepared by linking mPEG to both α and ε amino groups of lysine⁷², forms the basic conjugation strategy for manufacturing Pegasys® (see Figure 2, page 20).

Figure 9 – PEGylation using cyanuric chloride activated branched PEG

The introduction of PEG dendrimers resulted in increased protection due to the addition of very high molecular weights. In one such instance, Meireles et al⁷³ introduced a branched PEG derivative having four mPEG branches, with a terminal COOH group available for protein conjugation (see Figure 10, page 24).

Figure 10 – Four-branched mPEG-COOH dendrimer derivative

They successfully conjugated this with a number of therapeutic proteins, including IFN-α2b, recombinant streptokinase (r-SK), erythropoietin (EPO), granulocyte-colony stimulating factor (G-CSF) and epidermal growth factor (EGF), through NHS activation and found improved pharmacological properties for these products compared with those obtained from two branched-structure of similar molecular mass. Recent developments in the synthesis of linear and branched multifunctional PEG derivatives, known as MultiPEGs (see Figure 11, page 24), have contributed a substantial improvement in the pharmacological advantages of PEGylation technology^74-78. However, one disadvantage of very high molecular weight PEGylation is that large, inert PEG molecules are not easily cleared by glomerular filtration and therefore remain in circulation long after the protein moiety has been eliminated through proteolysis. Thus, the effects of long-term PEG retention in vivo should be carefully considered, particularly for therapeutics that require prolonged treatment regimens.

Figure 11 – A MultiPEG derivative (TetraPEG-(OH)10)75

Process considerations

The overall PEGylation processes used to date for protein conjugation can be broadly classified into two types, namely a solution phase batch process and an on-column fed-batch process⁷⁹. The simple and commonly adopted batch process involves the mixing of reagents together in a suitable buffer solution, preferably at a temperature between 4 and 6°C, followed by the separation and purification of the desired product using a suitable technique based on its physicochemical properties, including sixe exclusion chromatography (SEC), ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), membranes or aqueous two phase systems⁸⁰. A detailed discussion of the separation and purification of PEGylated products is beyond the scope of this paper and we restrict our discussion below to techniques that have been used to affect conjugation.

Normally with the batch process, prolonged contact between reacting species and products results in multiple conjugations and gives rise to a number of PEG isomers. Consequently a heterogeneous product mixture results, constituting unreacted starting materials, hydrolysed activating agents and a wide range of PEGylated products with varying degrees of conjugation. A typical SEC elution profile for various PEGylated α-lactalbumin products obtained from a batch process is illustrated in Figure 12 (see page 24. Hence, extensive multistep purifications and downstream processing are required to isolate the desired product⁷⁹, significantly decreasing overall yields. The high cost of the therapeutic proteins, along with the cost of separating the desired PEGylated protein from the reaction mixtures, makes the products extremely expensive.

Figure 12 – SEC elution profile of various PEGylated a-lactalbumin products obtained from batch process 87

A number of on-column PEGylation techniques have been attempted recently, with an emphasis on improving the product profile and specificity of conjugation. The first such approach was reported by Felix et al^81,82, who developed a site-specific solid phase peptide PEGylation, in which the peptide sequence was tethered onto a Rink amide MBHA-resin and was conjugated with a PEG derivative through a side chain lysine or aspartic acid. Finally, the mono-PEGylated peptide was cleaved off from the resin using trifluoroacetic acid (TFA). In a similar way, folate was derivatised with a PEG derivative attached to HMP resin through a peptide linker and the peptide-PEG-folate conjugate was cleaved off the resin using TFA⁸³. Solid-phase synthesis is not practical for large polypeptides (proteins) and the harsh chemicals, such as TFA, required for the release of solid-linked PEGylated products means that direct application of this methodology is not viable with highly sensitive species.

Utilising the ion exchange interactions between protein and ion exchange resins, Monkarsh et al⁸⁴ prepared and separated various positional isomers of PEGylated interferone-α-2a (PEG-INF) using an on-column process. INF was first adsorbed to a strong anion exchange resin and the activated PEG derivative was then circulated through the column. After eluting the unreacted PEGs and by-products, PEG-INF was collected by increasing the salt concentration. Unfortunately, this method resulted in multi-PEGylated products, without any improvement in the product profile.

Similarly, Lee et al⁸⁵ PEGylated recombinant interferon-α-2a (rhIFN-α-2a) by adsorbing on a CM-Sepharose cation exchange resin through ion exchange interactions. After washing out the unbound rhIFN-α-2a, mPEG-aldehyde and sodium cyanoborohydride solutions were passed through the column, resulting in N-terminal PEGylation. Suo et al⁸⁶ PEGylated bovine haemoglobin loaded onto CM-Sepharose using mPEG-succinimidyl carbonate and their studies showed that the selectivity of this method was increased by an increase in the molecular weight of the conjugated PEG. Even though these methods resulted in mono-PEGylated products at up to 75% yield, based on the loaded native protein, consistency in the reaction efficiency remains a challenge, due to the random orientations of adsorbed protein on the ion exchange matrix.

A unique on-column PEGylation methodology, known as size exclusion reaction chromatography (SERC), was introduced by Fee⁸⁷, incorporating the principle of SEC in separating various molecular sized species based on their different linear velocities through a column packed with porous beads. In this method, activated PEG and protein form a transient in-situ moving reaction zone within the column, in which the PEGylated protein, having a larger size than either of the reagents, moves ahead of the reaction zone, thus limiting its residence time in contact with activated PEG and reducing over-PEGylation. This process, however, is unable to control positional isomers.

Finally, some solution-phase⁸⁸ and on-column⁸⁹ attempts have been made to protect the active site of a protein by conjugating in the presence of an affinity partner. In this approach, which appears to be effective in some cases, the inherent steric hindrance of the protein complexed with its binding partner is used to preferentially PEGylate areas away from the active site and thus retain the in vitro activity of the released PEG-protein.

Conclusion

Nearly four decades of development in PEGylation technology has proven its pharmacological advantages and acceptability but the technology still lags in providing a commercially attractive, generic process to produce highly specific PEGylated therapeutic products at high yield. As a multi-million dollar annual business with the growing interest from both emerging biotechnology and established multinational pharmaceutical companies, there is great scientific and commercial interest in improving present methodologies and in introducing innovative process variations.

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About the authors

Vinod Babu Damodaran, University of Canterbury, New Zealand

Vinod Babu Damodaran is a senior PhD student working under Professor Fee on improving the yield and selectivity of protein PEGylation. Before this he was a Senior Research Chemist at the Merck Development Centre, India. His background is organic chemistry and his interests include polymer therapeutics, bioconjugation and controlled drug delivery.

Professor Conan Fee, University of Canterbury, New Zealand

Conan Fee is a Professor of Chemical Engineering and is also Co-Director of the Biomolecular Interactions Centre at the University of Canterbury. He holds a PhD in Chemical Engineering and a Diploma in Strategic Management and Leadership. He has research interests in bioseparations, protein PEGylation, drug delivery and biomolecular interactions and has been a consultant in bioseparation process development for a number of companies internationally.