Protein formulation by PEGylation

Protein Formulation by PEGylation
Pratik Umesh Parikh
F. Y. M. Pharmacy 2nd semester
(pharmaceutical biotechnology)
Roll no.: 08
Email : pratikparik42@gmail.com
Seminar on
Guided by :
Dr. V. P. Patel
Head of Department
Pharmaceutical Biotechnology
Sanjivani College of Pharmaceutical Education and Research,
Kopargaon
01/07/2021 SANJIVANI COLLEGE OF PHARMACEUTICAL EDUCATION & RESEARCH, KOPARGAON 1

Contents :
❖Introduction about PEG
❖What is PEGylation
❖Chemistry of PEGylation
❖Common approaches
❖PEGylation in pharmaceuticals
❖Properties of PEGylation
❖Benefits
❖Marketed products
❖Summary
❖Reference
SANJIVANI COLLEGE OF PHARMACEUTICAL EDUCATION & RESEARCH, KOPARGAON
01/07/2021 2

About PEG
Polyethylene Glycol is produced by Polymerization of Ethylene oxide with Ethylene Glycol.
• The reaction is catalyzed by acidic or basic catalysts
• Exothermic pro
• To prevent coagulation of polymer chains from solution, chelating additives such as dimethylglyoxime are
used They can be synthesized as linear, branched, or forked structures with functional groups at one or
• more termini to enable several conjugation strategies

Varying molecular weight of PEG
• PEG 400
• PEG 500
• PEG 3350
• PEG 4000
• PEG 6000
• PEG 8000
• PEG 5k
• PEG 20k
• PEG 40k
Approved by FDA for human oral, intravenous and
dermal pharmaceutical applications
( https://www.accessdata.fda.gov/scripts/cder/iig/index.cfm?event=BasicSearch.page )
Structure of PEG

What is PEGylation
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➢ PEGylation is the covalent coupling of non-toxic, hydrophilic polyethylene glycol (PEG) to active
pharmaceutical ingredients(API).
➢ The technology was developed in the 1950s and 1960s working on the coupling of polymers to
proteins, Later on in 1970s Frank F. Davis used PEG for protein modifications.
➢ The first PEG-protein company Enzon, founded in 1981, and the first PEG-drug product was
PEG-adenosine deaminase, approved in 1990.
➢ Nowadays, PEGylated proteins represent a significant therapeutic and business importance:
worldwide sales of PEGylated drugs total about $6 billion annually.
➢ Most widely used in delivering anticancer drugs clinically

Protein PEG PEGylated Protein
conjugate

PEGylation of proteins is usually achieved by a chemical reaction between the protein and
suitably activated PEGylation reagents.
There are various chemical groups in the amino acid side chains that could in principle be
exploited for the reaction with PEG moiety, such as –NH2, -NH-, -COOH, -OH, -SH groups as well
as disulfide (-S-S-) bonds.
Consideration : The attachment site on the protein, activation type of the PEG reagent, nature
(permanent or cleavable), length and shape of the linker, length, shape and structure of the PEG
reagent as well as end capping of the PEG chains.
Chemistry of PEGylation

Linkers covalently attach the molecule to the parent drug directly or indirectly.
❖ The choice of linkers enables PEG to be placed in various positions on the molecule.
Placement can fine tune a drug's pharmacokinetic properties and maintain its efficacy
❖Conjugation bonds can be stable or releasable to create entirely new compounds or novel
prodrugs
❖ - Stable PEG linkages create new pharmaceutical entities with respect to such
pharmacokinetic parameters as circulating half life, clearance, absorption, and bioavailability.
❖ PEGs may harm the pharmacodynamics properties of target binding because of steric
hindrance.
❖ The Releasable attachments used in PEG prodrugs enable controlled drug release through the
choice of architecture, attachment sites, and linker molecules
Linkers in PEGylation :

PEG is modified by
functionalization
through the use of
❖ Cyanuric chloride,
❖ PEG-succinimidyl succinate,
and
❖Imidazolyl formate
Common approaches for PEG conjugation with proteins

Protein & peptide
Liposomes
Enzymes
Carbohydrates
Antibody fragments
Small organic molecule
Biologics
PEGylation in pharmaceuticals

Improved solubility Reduced toxicity
Increased pH and
thermal stability
Increased potency
Increased patient
tolerance
Protection against
proteolysis
Longer in-vivo half
life & improved
bioavailability
Increased efficacy
Reduced side
effects
Reduced
immunogenicity
Low volume of
distribution
Lower frequency of
dosing
Successful
transportation
across cell
membrane
Reduced clearance
rate through
kidneys
Sustained
absorption from
injection site
Properties of PEGylation

PEGylated
therapeutics
Increased
hydrodynamic volume
Reduced proteolytic
degradation
Decreased RES
uptake
Reduced
immunogenicity
Increased solubility / reduced kidney clearance / increased
stability and serum half-life
Enhanced therapeutic efficacy
Benefits of PEGylation

Neulasta (PEGfilgrastim), Amgen, USA
To produce pegfilgrastim, a20 kD monomethoxypolyethylene glycol molecule
residue of filgrastim (using site-directed reductive alkylation). The average
molecular weight of pegfilgrastim is approximately 39 kD.
• PEGfilgrastim is a long-acting form of filgrastim.
• PEGylation increases the size of filgrastim & it becomes too large for renal
clearance.
•Half life :
✓Filgrastim : 3.5 to 3.8 hrs
✓PEGfilgrastim : 42 hrs
Marketed products
Filgrastim is water soluble protein with 175
amino acids with mol. Wt. 19 kD

Pegasys (PEGinterferon alfa-2a), Roche
The PEG moiety is linked at a single site to the interferon alfa moiety via
stable amide bond to lysine. PEGinterferon alfa-2a has an approximate
molecular weight of 60 kD.
•Enhanced half-life relative to the standard INF-a
•Lower rate of absorption following subcutaneous(SC) injection
•Reduces renal clearance
•Decreases immunogenicity
Marketed products
Recombinant interferon alfa-2a with mol. Wt. 20 kD

Somavert (PEGvisomant), Pfizer
PEG attachment sites Lysines & N-terminal site (Phe1, Lys38, Lys 41, Lys
70, Lys 115, Lys 120, Lys 140, Lys 145, Lys148)
•Slow rate of absorption from injection site(SC)
•Bound PEG polymers reduce clearance rate
•Mean half life : 74 – 172 hours
Marketed products
Analogue of growth hormone with 191 amino acids
Molecular weight : 22kD

PEG-Irinotecan
PEG-Irinotecan is a prodrug of irinotecan which uses a new architecture of four arms PEG as
carrier.
In preclinical models, accumulation of PEG-irinotecan in tumor achieved a 300 fold increase
compared to naïve.
Due to large molecular weight it penetrates the leaky vasculature within the tumor tissue
more readily than normal vasculature.
Marketed products

Fig. Passive targeting with acid sensitive PEG-prodrugs that clave in
extracellular space
Fig. Active targeting : Receptor mediated internalization and endosomal /
lysosomal degradation of the prodrug during active targeting

Fig. Active targeting : with acid sensitive PEG – prodrugs which cleaves in the extracellular space

PEGylation offers a great advantage for bioactive molecules in pharmaceutical and biological
applications by way of reducing protein immunogenicity and increased serum half-life.
The process can also be combined effectively with active targeting and stimuli-responsive
targeted therapies for the development of new methodologies for the treatment of cancer.
Molecular weight of PEG chain and its structural modification carries strategic importance for
conjugation with drug molecule for effective PEGylation.
Summary

Reference :
1. Protein Purification – principles, high resolution methods & Applications edited by Jan- Christer
janson published by A Johnson Willey & sons INC. Publication, third edition, 339 -360.
2. Banerjee, S. S., Aher, N., Patil, R., & Khandare, J. (2012). Poly(ethylene glycol)-Prodrug Conjugates:
Concept, Design, and Applications. Journal of Drug Delivery, 2012, 1–17.Held, J. R. (1983).
Appropriate animal models. Annals of the New York Academy of Sciences.
3. K. D. Hinds, “Protein conjugation, cross-linking, and PEGylation,” in biomaterials for Delivery and
Targeting of Proteins and Nucleic Acids, R. I. Mahato, Ed., pp. 119–185, CRC Press, Boca Raton,
Fla, USA, 2005
4. A. H. Sehon, “Suppression of antibody responses by conjugates of antigens and
monomethoxypoly(ethylene glycol),” Advanced Drug Delivery Reviews, vol. 6, no. 2, pp. 203–217,
1991
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Reference :
5. R. Webster, E. Didier, P. Harris et al., “PEGylated proteins: evaluation of their safety in the
absence of definitive metabolism studies,” Drug Metabolism and Disposition, vol. 35, no. 1,
pp. 9–16, 2007
6. J. Khandare and T. Minko, “Polymer-drug conjugates: progress in polymeric prodrugs,”
Progress in Polymer Science, vol. 31, no. 4, pp. 359–397, 2006.
7. https://www.slideshare.net/sultanwardag/pegylation-of-protiens-drugs
8. https://www.slideshare.net/Biochempeg/the-applications-of-pegylation-reagents
9. https://pharmawiki.in/wp-content/uploads/2014/01/PEGylation-Technique-in-Drug-
delivery.ppt
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