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PEGylation
-An
innovAtive
ApproAch
for
protein delivery
Keyur VasaVa
CONTENTS Introduction PEGylation: DEFINITION PEG - Chemical properties Merits & demerits Chemistry Processes Mechanism of action How can we get site specific PEGylation? Factors affecting performance of peptides PEGylated
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PEGylated product Purification and Analysis PEGylation Process optimization PEGylated pharmaceuticals on the market References
1. INTRODUCTION
1.1 PROTEIN PHARMACEUTICALS AN OVERVIEW
The biotechnology revolution has produced novel peptides and proteins as they hold great promise as therapeutic agents. More than 200 FDA approved protein drugs are in the market and 500 more are undergoing clinical trials. About one-third of drug candidates in clinical trials are polypeptides.
Although occasionally successful, these methods have limitations. For example: Liposomes besides delivering drugs to diseases tissues, also rapidly enters liver, spleen, kidneys and reticulo-endothelial systems and leak drugs while in circulation. Liposomes also activate complement systems, which causes pseudo allergic reactions that damage heart and liver cells. So, an alternative solution required for efficient protein delivery
Approaches for oral delivery Enzyme inhibitors and absorption enhancers to overcome the major hurdles caused by enzymes. Modifying protein structures by chemical synthesis to stable analogs Targeting the specific absorption site and dosage form modification Formulation vehicles like polyethylene glycol, mucoadhesive polymers and so on. Among these the PEGylation technique using polyethylene glycol as formulation vehicle gained much importance.
2. PEGylation
2.1 DEFINITION:
PEGylation is the process of covalent attachment of polyethylene glycol polymer chains to another molecule, normally a drug or therapeutic protein. PEGylation is routinely achieved by incubation of a reactive derivative of PEG with the target macromolecule.
PEG, in its methoxy form, is so far the ideal polymer: It possesses only one potentially reactive group. It is FDA approved for human use. Furthermore, PEG has: High water and organic solvent solubility Flexible and hydrated chain (each PEG monomer binds 2-3 water molecules that increase the PEG hydrodynamic volume and finally decrease its kidney filtration). Low polydispersity i.e. Molecular weight distribution is narrow (1.01 1.1) Allows an easy and clean chemistry of activation Currently, there are two main types of commercially available PEG: Linear and Branched Inert, non-toxic and non-immunogenic Highly soluble, amphiphilic polyether diol Does not harm active protein or cells
Enhanced membrane penetration, sustained clinical response with minimal dosing leading to improved quality of life and reduced treatment cost.
PEGylation DEMERITS
Proteins as well as the PEG agents are expensive. Losses during the PEGylation process of 25% in late stage/commercial production to 5075% in early clinical production are not uncommon. Maximizing the benefits require Yield improvement, but usually not performed due to time constraints.
In addition to the attachment of PEG via primary amino groups which often produces heterogenous PEG conjugates, site-directed pegylation of proteins can be achieved via other functional groups on the protein surface. These include, free cysteines, oligosaccharides and alcoholic groups. PEG reagents used for site-specific pegylations are: PEG-Vinyl sulphone (via free cysteine); PEG-Hydrazide (via oligosaccharide); PEG-Isocyanate (via alcohol or amino group).
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Techniques:
It is classified into two types: Early PEGylation technology (First generation PEGylation) Advanced PEGylation technology (Second generation PEGylation)
Early PEGylation was performed with methoxy-PEG (m-PEG), which was contaminated with PEG diol and which resulted in the cross-linking of proteins to form inactive aggregates. Diol contamination can reach upto 10-15 %. Despite these limitations, several first generation PEGylated drugs receive regulatory approval.
Examples: Still in use today are Pegademase (Adagen), a PEGylated form of the enzyme adenosine de-aminase for the treatment of Severe Combined Immuno-Deficiency (SCID) and Pegaspargase (Oncaspar), a PEGylated form of enzyme asparginase for the treatment of Leukemia.
It includes several techniques as follows: 1) Array of chemistries to improve PEG derivatives and their linkages to drug For example: Generating Carboxylic acid intermediates of PEG permits upto 97 % of diol impurities to be removed by ion-exchange chromatography before PEG attachment to polypeptide drugs 2) PEGylation at the SH (thiol) groups of Cysteine of polypeptides PEGylating site specifically can minimize the loss of biological activity and reduce immunogenecity. -SH groups are ideal site for PEGylation compared to lysine as it causes reduction in immunogenecity, but the problem is that there are far fewer cysteine residues than lysine groups on polypeptides.
For this, cysteines can be added to polypeptides precisely where they are desired by genetic engineering
3) Incorporation of the degradable linkages to release drug at the targeted sites 4) Hetero-bifunctional PEGs Hetero-bifunctional PEGs are the one bearing dissimilar terminal groups. Such hetero-bifunctional PEGs bearing appropriate functional groups may be used to link two entities where a hydrophilic, flexible, and biocompatible spacer is needed. Hetero-bifunctional PEGs can be used in a variety of ways that includes linking macromolecules to surfaces (for immunoassays, biosensors or various probe applications), targeting of drugs, liposomes and viruses to specific tissues, liquid phase peptide synthesis and many others. Preferred end groups for hetero-bifunctional PEGs are NHS esters, maleimide, vinyl sulfone, pyridyl disulfide, amine, and carboxylic acids. 5) Use of Branched structures Second generation PEGylation uses branched structures of PEG, in contrast to the solely linear structures found in first generation PEGs. Branched PEGs of greatly increased molecular masses upto 60 kDa or more, as compared with the 12 kDa or less found in the first generation PEGs have been prepared. A branched PEG acts as if it were much larger than a corresponding linear PEG of the same molecular mass. Branched PEGs are also better at cloaking the attached polypeptide drug from the immune system and proteolytic enzymes, thereby reducing its antigenecity and likelihood of destruction.
Example: A competing treatment for chronic hepatitis C utilizes IFN (Interferon) 2a coupled to PEG. The first formulation in 1999 used a first generation linear PEG of 5 kDa. In the first clinical trials, this PEGylated drug was administered to patients with chronic hepatitis C once weekly and compared with its un-PEGylated counterpart administered three times a week. The PEGylated IFN 2a produced no clinical advantages
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A second generation, branched PEG of 40 kDa, was then coupled to IFN 2a. This version (Pegasys) under clinical investigation, showed nearly constant blood concentration of IFN 2a and renal clearance is reduce to 100-fold related to unPEGylated IFN 2a. PEGylated also increased the half-life of IFN 2a from 9 to 77 hours. It is illustrated in the following graphs:
2) We can change the amino acid residues accessibility By reversible protection of groups involved in activity e.g; Insulin. By changing the protein conformation with solvents e.g; GRF 3) Branched PEG presents reduced access to enzyme active site The branched PEG structure plays also a positive role in the biological behavior of conjugates Examples: Pegasys, Cimzia, Macugen 4) Take advantage of genetic engineering: to introduce a residue suitable for specific reaction (i.e. Cysteine), Cysteines are seldom present in proteins and may easily replace Ala, Thr, etc. without changing the conformation. (NB. There are specific thiol reagents, i.e. PEG-maleimide, -vinylsulphone, pyridin disulphide) e.g; interferon 5) Take advantage of enzymes as biocatalysts to link PEG to residues that cannot be conjugated by chemical methods. Enzymatic PEGylation of Gln by Transglutaminase e.g; G-CSF
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Chemistry used to attach PEG to peptide, purity of raw materials, intermediates and final products It is very important factor. Peptide and linker must be very pure and very stable to yield pure conjugate with high efficiency. Speed of conjugation reaction can be hours or days to completion and critical parameters must be optimized and monitored to maximize yield and purity.
Size-exclusion chromatography (SEC) can separate un-PEGylated from PEGylated proteins but not the PEG dimer from the PEG monomer.
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Hydrophobic interaction chromatography generally works poorly for this application (depending on the hydro-phobicity of the protein relative to the PEG) Ion-exchange chromatography seems to be the method of choice for purification of PEGylated product. Here, the separation of the PEG dimer from the other components in the reaction mixture appeared to be based on the PEG-to-protein ratio.
ANALYSIS OF THE PEGylated PROTEIN PRODUCT: Characterization of PEGylated proteins is difficult due to the fact that the PEG molecule is more polydisperse than the protein and imparts size heterogeneity to the conjugated protein. Size-exclusion chromatography (SEC) has often been used to characterize these conjugates. This tool is limited by the extra peak-broadening due to the polydispersity of the PEG conjugate. SEC also will not detect "PEGylation site isomers" in which the protein is PEGylated at different residues. Cation-exchange HPLC is a powerful tool for resolving these PEGylation site isomers.
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Synthetic: PVP (Kaneda et al, Osaka Univ.) Poly(metacryloyl phosphorylcholine),(Polytherics Ltd.) Poly(oxazoline) (POZ) ( Serina Therapeutics, inc.)
REFERENCES:
Pegylation of Proteins: an Updated Review by Francesco M. Veronese, Deptartment of Pharmaceutical Sciences Padua University-Italy Chemistry for peptide and protein PEGylation Advanced Drug Delivery Reviews Volume 54, Issue 4, 17 June 2002, Pages 459-476 PEGylation: Concepts and Applications Miss Debora Freitas University of So Paulo PEGylation --en.wikipedia.org/wiki/PEGylation http://www.pharmainfo.net/pharma-student-magazine/oral-route-protein-and-peptidedrug-delivery-systems-pegylation-mechanism http://www.scribd.com/doc/21588754/PEGylation-and-Its-Application-insPharmaceuticals
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