Enhancing Protein and Peptide Drugs with PEGylation

PEG modification has widespread applications in improving the performance of protein and peptide drugs. It involves covalently combining PEG with drugs to form complexes, offering benefits such as controlled drug release, extended half-life, reduced immunogenicity, lowered toxicity, enhanced drug targeting, and minimized dosage and administration frequency. PEGylation significantly enhances the pharmacokinetic and pharmacodynamic properties of various drugs.

 

Protein drugs, encompassing cytokines, enzymes, antibodies, and hormones, face challenges like short half-life, immunogenicity, low solubility, high toxicity, and susceptibility to degradation by proteases, thereby diminishing their clinical efficacy.

 

Peptide drugs, employed for disease prevention, treatment, and diagnosis, cover a range of types, including peptide vaccines, anti-tumor peptides, anti-viral peptides, peptide-directed drugs, cytokine mimetic peptides, antibacterial active peptides, and diagnostic peptides.

 

 

Mechanism of PEGylation

PEG modification of proteins and peptides extends drug half-life and reduces immunogenicity while preserving biological activity. The process involves chemically modifying the protein with PEG, causing an increase in molecular weight and a change in spatial structure. PEG acts as a protective barrier, preventing degradation by proteases and minimizing antibody production. Modified proteins, exceeding the glomerular filtration threshold, can evade filtration in the kidneys, resulting in prolonged blood circulation.

 

 

Main Modification Site

Polar amino acid residues, including cysteine, lysine, and terminal nitrogen acids, are common targets for PEG modification. The nucleophilic activity generally decreases in the order: thiol > α-amino > ɛ-amino > carboxyl > hydroxyl.

 

Reactions between PEG and proteins or peptides involve acylation, alkylation, redox reactions, aromatic ring substitution, and more. Key modification pathways include amino modification (N-terminal acylation, lysine side chain acylation, N-terminal alkylation), carboxyl modification, sulfhydryl modification, and other techniques such as pH control, metal ion or enzyme catalysis, and site-directed modification of glycosyl groups in glycoproteins.

 

 

1）Amino Modification

Commonly used amino modifiers include PEG-SS, PEG-SC, PEG-SPA, PEG-NHS, PEG-BTC, PEG-CHO, PEG-ALD, PEG-tresylate, and PEG-epoxide.

 

To investigate the effective modification of Human Leucocyte Antigen (HLA) using mPEG, a study involved the modification of lymphocyte surface HLA-I class antigen with mPEG-BTC. Results demonstrated a negative microlymphocytotoxicity test, indicating complete blocking of the specific immune response between HLA-I antigens and their corresponding antibodies after modification with mPEG [1].

 

 

2）Octreotide Modification:

Octreotide underwent chemical modification using SPA-mPEG (MW 2000) or ALD-mPEG (MW 2000/5000). The biological activity detection revealed that SPA-mPEG modification resulted in reduced activity, while ALD-mPEG, specifically modified at the N-terminus of octreotide, maintained biological activity. Furthermore, ALD-mPEG 5K significantly improved the pharmacokinetic properties [2].

 

 

3）Thiol Modification:

Sulfhydryl groups are present only in cysteine within protein-neutralizing peptides. PEG modifiers, leveraging the high nucleophilicity of sulfhydryl groups, can be selectively chosen for directional modification of proteins and peptides. For proteins lacking cysteine, sulfhydryl reaction sites can be introduced through genetic engineering or Traut’s reagent (2-imino hydrochlorothiol). Commonly used thiol modifiers include PEG-Mal, PEG-OPSS, PEG-VS, PEG-IA, PEG-DAQ, and PEG-Se. A case study involving the site-specific modification of human ciliary neuronotrophic factor (hCNTF) using mPEG-MAL (MW 40kDa) demonstrated a modification rate exceeding 90%, resulting in a 95% purity single modification product. This led to a remarkable 30.3 times increase in in vivo circulating half-life compared to the original protein [3].

 

 

4）Carboxyl Modification:

Carboxyl modification sites include aspartic acid, glutamic acid, and terminal carboxyl groups. The activation of the carboxyl group involves converting PEG's carboxyl group into an amino group, followed by combination with the protein's carboxyl group in the presence of DCCI or EDC. Commonly used carboxyl modifiers are PEG-HZ or PEG-NH2 [4].

 

While the terminal ends of single-chain polyethylene glycol have limitations in modifying new conjugated drugs, leading to low drug loading capacity, advancements in branched polyethylene glycol offer diverse conjugation possibilities. Branched structures, such as branched PEG, forked PEG, and multi-arm PEG with a dendritic structure at the end, significantly enhance drug loading capacity and provide varied conjugation properties.

 

 

PEGylation Agents List

货号	品名	描述
ECS4133A	mPEG-NHS, MW 350	mPEG-Succinimidyl ester
AJL0190A	mPEG-SC, MW 1K
ACS0112B	mPEG-SC, MW 2K
ECS4382A	mPEG-SC, MW 3.4K
AJL0190B	mPEG-SC, MW 5K
ACS0115B	mPEG-SC, MW 10K
ECS4219A	mPEG-SPA, MW 1K	mPEG-Succinimidyl Propionate
ECS4356A	mPEG-SPA, MW 2K
ECS4275A	mPEG-SPA, MW 5K
ECS4094A	mPEG-SPA, MW 20K
ECS4284A	mPEG-SS, MW 5K	mPEG-Succinimidyl Succinate ester
ECS2001A	mPEG-SCM, MW 20K	mPEG-Succinimidyl Carboxymethyl Ester
ECS2103A	mPEG-SCM, MW 40K
ECS4171A	mPEG-SG, MW 2K	mPEG-Succinimidyl Glutarate ester
ECS4334A	mPEG-SVA, MW 5K	mPEG-Succinimidyl valerate
ECS4154A	mPEG-Mal, MW 350	mPEG-Maleimide
ECS4155A	mPEG-Mal, MW 500
ECS4156A	mPEG-Mal, MW 1K
DCS0133A	mPEG-Mal, MW 2K
ECS4168A	mPEG-Mal, MW 4K
ACS0135B	mPEG-Mal, MW 5K
ACS0136A	mPEG-Mal, MW 10K
ACS0137A	mPEG-Mal, MW 20K
ECS4150A	mPEG-ALD, MW 1K	mPEG-Aldehyde
ECS4194A	mPEG-CHO, MW 2K
ACS0121A	mPEG-CHO, MW 5K
ECS4212A	mPEG-EPO, MW 550	mPEG-Epoxide
ECS4198A	mPEG-EPO, MW 2K
ECS4182A	mPEG-EPO, MW 10K
ECS4317A	mPEG-HZ, MW 350	mPEG-Hydrazide
ECS4278A	mPEG-HZ, MW 2K
ECS4157A	mPEG-NH2, MW 350	mPEG-Amine
ECS4244A	mPEG-NH2, MW 550
ECS2165A	mPEG-NH2, MW 1K
ACS0105B	mPEG-NH2, MW 2K
ACS0106B	mPEG-NH2, MW 3.4K
ECS4296A	mPEG-NH2, MW 4K
ACS0107B	mPEG-NH2, MW 5K
ECS2180A	mPEG-NH2, MW 10K
ACS0109B	mPEG-NH2, MW 20K
ECS2069C	Mal-PEG-SCM, MW 2K	Maleimide-PEG-Succinimidyl Carboxymethyl Ester
ECS2069B	Mal-PEG-SCM, MW 5K
ECS2069A	Mal-PEG-SCM, MW 10K
ECS4208A	Mal-PEG-NHS, MW 200	Maleimide-PEG-NHS
ACS1695B	Mal-PEG-NHS, MW 2K
ECS2072A	Mal-PEG-NHS, MW 3.4K
ECS2065A	Mal-PEG-NHS, MW 5K
ECS4137A	MAL-PEG-SVA, MW 5K	Maleimide-PEG-Succinimidyl valerate
ECS4192A	Mal-PEG-HZ, MW 2K	Maleimide-PEG-Hydrazide
ECS2005A	NHS-PEG-SH,MW 200	Thiol-PEG- Succinimidyl ester
ECS2006A	NHS-PEG-SH,MW 400
ECS2007A	NHS-PEG-SH,MW 500
ECS2017A	SH-PEG-NHS, MW 600
ECS2009A	NHS-PEG-SH,MW 800
ECS2010A	NHS-PEG-SH,MW 1K
ECS4307A	SH-PEG-NHS, MW 2K
ECS4407A	SH-PEG-NHS, MW 5K
ECS4205A	SH-PEG-NH2, MW 600	Thiol-PEG-Amine
ECS4095A	SH-PEG-NH2, MW 2K
ECS1978B	SH-PEG-NH2, MW 3.4K
DCS2049A	SH-PEG-NH2, MW 5K
ECS4142A	SH-PEG-NH2, MW 10K
ECS2096A	SH-PEG-COOH, MW 400	Thiol-PEG-Acid
ECS4245A	SH-PEG-COOH, MW 1K
ECS1991B	SH-PEG-COOH, MW 2K
ECS2021A	SH-PEG-COOH, MW 3.4K
ECS2118A	SH-PEG-COOH, MW 5K
ECS2182A	SH-PEG-CHO, MW 5K	Thiol-PEG- Aldehyde
ECS4174A	SH-PEG-SH, MW 400	Thiol-PEG-Thiol
ECS4173A	SH-PEG-SH, MW 600
ECS2084A	SH-PEG-SH, MW 2K
ECS4143A	SH-PEG-HZ, MW 10K	Thiol-PEG-Hydrazide
ECS4383A	SH-PEG-Biotin, MW 200	Thiol-PEG-Biotin
ECS4412A	SH-PEG-Biotin, MW 5K
ECS4292A	SC-PEG-SC, MW 400	Succinimidyl ester-PEG-Succinimidyl ester
ECS2117B	SC-PEG-SC, MW 2K
ECS4233A	SC-PEG-SC, MW 3.4K
ECS4264A	SC-PEG-SC, MW 5K
ECS4311A	SC-PEG-SC, MW 10K
ECS4255A	NHS-PEG-COOH, MW 3.4K	NHS-PEG-Acid
ECS2071A	NHS-PEG-VS, MW 3.4K	NHS-PEG-Vinylsulfone
ECS4135A	NHS-PEG-OH, MW 2K	NHS-PEG-Hydroxy
ECS4354A	SS-PEG-SS, MW 2K	Succinimidyl Succinate ester-PEG-Succinimidyl Succinate ester
ECS4172A	SG-PEG-SG, MW 2K	Succinimidyl Glutarate ester-PEG-Succinimidyl Glutarate ester
ECS4231A	SG-PEG-SG, MW 3.4K
ECS2129A	SPA-PEG-SPA, MW 3.4K	Succinimidyl Propionate-PEG-Succinimidyl Propionate
ECS4290A	SCM-PEG-SCM, MW 2K	Succinimidyl Carboxymethyl Ester-PEG-Succinimidyl Carboxymethyl Ester
ECS4195A	SCM-PEG-SCM, MW 5K
ECS4291A	SCM-PEG-SCM, MW 20K
ECS4298A	OPSS-PEG-NHS, MW 5K	Ortho-Pyridyldisulfide-PEG-NHS
ECS4082A	OPSS-PEG-SCM, MW 5K	Ortho-Pyridyldisulfide-PEG-Succinimidyl Carboxymethyl Ester
ECS4199A	AC-PEG-SC, MW 2K	Acrylate-PEG-NHS
ECS2051A	AC -PEG-SVA, MW 3.4K	Acrylate-PEG-Succinimidyl valerate
ECS2098A	AC-PEG-SCM, MW 2K	Acrylate-PEG-SCM
ECS4247A	Biotin-PEG-SCM, MW 600	Biotin-PEG-Succinimidyl Carboxymethyl Ester
ECS4343A	Biotin-PEG-SC, MW 2K	Biotin-PEG- Succinimidyl ester
EYD0528B	Biotin-PEG-SC, MW 5K
ECS4248A	Biotin-PEG-SS, MW 600	Biotin-PEG-Succinimidyl Succinate ester
ECS4249A	Biotin-PEG-SG, MW 600	Biotin-PEG-Succinimidyl Glutarate ester
ECS4246A	Biotin-PEG-SVA, MW 600	Biotin-PEG-Succinimidyl valerate
ECS4162A	CHO-PEG-OH, MW 2K	Aldehyde-PEG-Hydroxy
ECS2174A	CHO-PEG-CHO, MW 2K	Aldehyde-PEG-Aldehyde

 

 

References:

[1] Dong Hee Na, Kang Choon Lee, Patrick P DeLuca. PEGylation of octreotide: II. Effect of N-terminal mono-PEGylation on biological activity and pharmacokinetics. Pharm Res, 2005, 22(5): 743-9.

[2] Kynclova E, Elsner E, K?pf A, et al. Novel method for coupling of poly(ethyleneglycol) to carboxylic acid moieties of proteins. Journal of Molecular Recognition, 1996, 9(5-6):644-651.