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Example of targeted pegylated liposome delivery
Transfection applications and production of short pegylated liposomes
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*Plasmid delivery through PEGylated anionic nanocomplexes containing cationic targeting [[peptide|peptides]] precisely targeted brain tissues in a method developed Tagalakis et al.<ref>{{cite journal |last1= Tagalakis|first1= Aristides|last2= Kenny|first2= Gavin|date= 28 January 2014|title= PEGylation improves the receptor-mediated transfection efficiency of peptide-targeted, self-assembling, anionic nanocomplexes|url= http://www.sciencedirect.com/science/article/pii/S0168365913009036|journal= Journal of Controlled Release|volume= 174|pages= 177-187|doi= https://doi.org/10.1016/j.jconrel.2013.11.014|access-date= 6 September 2017}}</ref>
*Plasmid delivery through PEGylated anionic nanocomplexes containing cationic targeting [[peptide|peptides]] precisely targeted brain tissues in a method developed Tagalakis et al.<ref>{{cite journal |last1= Tagalakis|first1= Aristides|last2= Kenny|first2= Gavin|date= 28 January 2014|title= PEGylation improves the receptor-mediated transfection efficiency of peptide-targeted, self-assembling, anionic nanocomplexes|url= http://www.sciencedirect.com/science/article/pii/S0168365913009036|journal= Journal of Controlled Release|volume= 174|pages= 177-187|doi= https://doi.org/10.1016/j.jconrel.2013.11.014|access-date= 6 September 2017}}</ref>
*PEGylated liposomes used to coat protein complexes and plasmids for transfection. The effectivness of these liposomes is inversely proportional to their size, and short PEGylated liposomes produced by Avanti Polar Lipids<ref>{{cite journal |last1= Hood|first1= Renee|last2= Chenren|first2= Shao|date= June 1 2014|title= Microfluidic Synthesis of PEG- and Folate-Conjugated Liposomes for One-Step Formation of Targeted Stealth Nanocarriers|url= https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3650128/|journal= Pharmaceutical Research|volume= 30|issue= 6|pages= 1597–1607|doi= 10.1007/s11095-013-0998-3|access-date= 6 September 2017}}</ref> and [[Altogen Labs CRO| Altogen Labs]]<ref>{{cite web|url=http://www.biocompare.com/Life-Science-News/77261-PEG-Liposome-SiRNA-In-Vivo-Transfection-Kit-From-Altogen-Biosystems/|title= PEG-Liposome SiRNA In Vivo Transfection Kit From Altogen Biosystems|date= October 21 2010|access-date = 26 August 2017)</ref> are made through a process of [[extrusion]] to break down the lipids into smaller pieces.


==Process==
==Process==

Revision as of 01:29, 7 September 2017

This article is about PEGylation in a pharmaceutical context. For the bulk industrial process, see Ethoxylation.
Polyethylene glycol (in pharmacy called macrogol)

PEGylation (often styled pegylation) is the process of both covalent and non-covalent attachment or amalgamation of polyethylene glycol (PEG, in pharmacy called macrogol) polymer chains to molecules and macrostructures, such as a drug, therapeutic protein or vesicle, which is then described as PEGylated (pegylated). PEGylation is routinely achieved by incubation of a reactive derivative of PEG with the target molecule. The covalent attachment of PEG to a drug or therapeutic protein can "mask" the agent from the host's immune system (reduced immunogenicity and antigenicity), and increase the hydrodynamic size (size in solution) of the agent which prolongs its circulatory time by reducing renal clearance. PEGylation can also provide water solubility to hydrophobic drugs and proteins.

History

Around 1970, Frank F. Davis, a professor of biochemistry at Rutgers University, became interested in developing a process to render usable bioactive proteins of potential medical value. After considerable study, he concluded that the attachment of an inert and hydrophilic polymer might extend blood life and control immunogenicity of the proteins.[1] Polyethylene glycol was chosen as the polymer. A team of Davis, Theodorus Van Es and Nicholas C. Palczuk conducted animal studies and found that PEG attachment greatly extended blood life and controlled immunogenicity of the proteins. A patent application was filed in 1973 and patent issued in 1979.[2] The inventors and Abraham Abuchowski conducted extensive additional PEGylation studies on various enzymes.[3][4] In 1981 Davis and Abuchowski founded Enzon, Inc., which brought three PEGylated drugs to market. Abuchowski later founded and is CEO of Prolong Pharmaceuticals.

Overview

A comparison of uricase and PEG-uricase. PEG-uricase includes 40 polymers of 10kDa PEG. PEGylation improves its solubility at physiological pH, increases serum half-life and reduces immunogenicity without compromising activity. Upper images show the whole tetramer, lower images show one of the lysines that is PEGylated. (uricase from PDB: 1uox​ and PEG-uricase model from reference;[5] only 36 PEG polymers included)

PEGylation is the process of attaching the strands of the polymer PEG to molecules, most typically peptides, proteins, and antibody fragments, that can improve the safety and efficiency of many therapeutics.[6] It produces alterations in the physiochemical properties including changes in conformation, electrostatic binding, hydrophobicity etc. These physical and chemical changes increase systemic retention of the therapeutic agent. Also, it can influence the binding affinity of the therapeutic moiety to the cell receptors and can alter the absorption and distribution patterns.

PEGylation, by increasing the molecular weight of a molecule, can impart several significant pharmacological advantages over the unmodified form, such as:

  • Improved drug solubility
  • Reduced dosage frequency, without diminished efficacy with potentially reduced toxicity
  • Extended circulating life
  • Increased drug stability
  • Enhanced protection from proteolytic degradation

PEGylated drugs also have the following commercial advantages:

  • Opportunities for new delivery formats and dosing regimens
  • Extended patent life of previously approved drugs

PEG is a particularly attractive polymer for conjugation. The specific characteristics of PEG moieties relevant to pharmaceutical applications are:

  • Water solubility
  • High mobility in solution
  • Lack of toxicity and low immunogenicity
  • Ready clearance from the body
  • Altered distribution in the body

PEGylated pharmaceuticals on the market

The clinical value of PEGylation is now well established. ADAGEN (pegademase bovine) manufactured by Enzon Pharmaceuticals, Inc., US was the first PEGylated protein approved by the U.S. Food and Drug Administration (FDA) in March 1990, to enter the market. It is used to treat X-linked severe combined immunogenicity syndrome, as an alternative to bone marrow transplantation and enzyme replacement by gene therapy. Since the introduction of ADAGEN, a large number of PEGylated protein and peptide pharmaceuticals have followed and many others are under clinical trial or under development stages. Sales of the two most successful products, Pegasys and Neulasta, exceeded $5 billion in 2011.[7][8] All commercially available PEGylated pharmaceuticals contain methoxypoly(ethylene glycol) or mPEG. PEGylated pharmaceuticals currently on the market (in reverse chronology by FDA approval year) include:

Uses of PEGylation in Biotechnology

PEGylation has practical uses in biotechnology for protein delivery, cell transfection, and gene editing in non-human cells.[10] In particular, PEGylation of proteins and liposomes greatly contributes to their ability to penetrate cell membranes and escape the innate immune response of cells when delivering plasmids and enzymes. PEGylated proteins have lower immunogenicity than their natural counterparts, allowing them to continue affecting cell output and gene expression well after delivery.[11] PEGylation of lyposomic packages reduces the innate immune response of cells and lowers their cytotoxicity.[12] PEGylated liposomes also tend to spread to diseased or cancerous tissues as a consequence of poor lymphatic drainage in damaged areas [11]. The widespread interest in PEGylated liposomes in the biotechnology industry has stemmed from the search for non-viral delivery mechanisms. Examples of developments in PEGylated liposome delivery include:

  • Plasmid delivery through PEGylated anionic nanocomplexes containing cationic targeting peptides precisely targeted brain tissues in a method developed Tagalakis et al.[13]
  • PEGylated liposomes used to coat protein complexes and plasmids for transfection. The effectivness of these liposomes is inversely proportional to their size, and short PEGylated liposomes produced by Avanti Polar Lipids[14] and Altogen Labs[15] are made through a process of extrusion to break down the lipids into smaller pieces.

Process

The first step of the PEGylation is the suitable functionalization of the PEG polymer at one or both terminals. PEGs that are activated at each terminus with the same reactive moiety are known as "homobifunctional", whereas if the functional groups present are different, then the PEG derivative is referred as "heterobifunctional" or "heterofunctional". The chemically active or activated derivatives of the PEG polymer are prepared to attach the PEG to the desired molecule.

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 process.[16] 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 size exclusion chromatography (SEC), ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC) and membranes or aqueous two phase systems.[17][18]

The choice of the suitable functional group for the PEG derivative is based on the type of available reactive group on the molecule that will be coupled to the PEG. For proteins, typical reactive amino acids include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine. The N-terminal amino group and the C-terminal carboxylic acid can also be used as a site specific site by conjugation with aldehyde functional polymers.[19]

The techniques used to form first generation PEG derivatives are generally reacting the PEG polymer with a group that is reactive with hydroxyl groups, typically anhydrides, acid chlorides, chloroformates and carbonates. In the second generation PEGylation chemistry more efficient functional groups such as aldehyde, esters, amides etc. made available for conjugation.

As applications of PEGylation have become more and more advanced and sophisticated, there has been an increase in need for heterobifunctional PEGs for conjugation. These heterobifunctional PEGs are very useful in linking two entities, where a hydrophilic, flexible and biocompatible spacer is needed. Preferred end groups for heterobifunctional PEGs are maleimide, vinyl sulfones, pyridyl disulfide, amine, carboxylic acids and NHS esters.

Third generation pegylation agents, where the shape of the polymer has been branched, Y shaped or comb shaped are available which show reduced viscosity and lack of organ accumulation.[20]

Limitations

Unpredictability in clearance times for PEGylated compounds may lead to the accumulation of large molecular weight compounds in the liver leading to inclusion bodies with no known toxicologic consequences.[21] Furthermore, alteration in the chain length may lead to unexpected clearance times in vivo.[22]

Future perspectives

Four decades of development in PEGylation technology have proven its pharmacological advantages and acceptability. As a multibillion-dollar annual business with 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.[23]

See also

References

  1. ^ Davis, Frank F. (2002). "The origin of pegnology". Advanced Drug Delivery Reviews. 54 (4): 457–8. doi:10.1016/S0169-409X(02)00021-2. PMID 12052708.
  2. ^ Davis, F. F., Van Es, T., and Palczuk, N. C. (1979) United States Patent US 4179337 . Non-immunogenic polypeptides. Originally filed in 1973
  3. ^ Abuchowski, A; Van Es, T; Palczuk, N. C.; Davis, F. F. (1977). "Alteration of immunological properties of bovine serum albumin by covalent attachment of polyethylene glycol". The Journal of biological chemistry. 252 (11): 3578–81. PMID 405385.
  4. ^ Abuchowski, A; McCoy, J. R.; Palczuk, N. C.; Van Es, T; Davis, F. F. (1977). "Effect of covalent attachment of polyethylene glycol on immunogenicity and circulating life of bovine liver catalase". The Journal of biological chemistry. 252 (11): 3582–6. PMID 16907.
  5. ^ Sherman, MR; Saifer, MG; Perez-Ruiz, F (3 January 2008). "PEG-uricase in the management of treatment-resistant gout and hyperuricemia". Advanced drug delivery reviews. 60 (1): 59–68. doi:10.1016/j.addr.2007.06.011. PMID 17826865.
  6. ^ Veronese, FM; Harris, JM (2002). "Introduction and overview of peptide and protein pegylation". Advanced drug delivery reviews. 54 (4): 453–6. doi:10.1016/S0169-409X(02)00020-0. PMID 12052707.
  7. ^ Klauser, Alexander (Head), Roche Group Media Relations, "Roche in 2011: Strong results and positive outlook," www.roche.com/med-cor-2012-02-01-e.pdf, Feb 1, 2012, p.7
  8. ^ "Amgen 2011 Annual Report and Financial Summary," [1][dead link] 2011 AnnualReport.pdf, Feb 23 2012, p. 71
  9. ^ http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm472643.htm
  10. ^ Balazs, Daniel; Godbey, WT (29 October 2010). "Liposomes for Use in Gene Delivery". Journal of Drug Delivery. 2011: 12. doi:http://dx.doi.org/10.1155/2011/326497. Retrieved 6 September 2017. {{cite journal}}: Check |doi= value (help); External link in |doi= (help)
  11. ^ a b Milla, P; Dosio, F (13 January 2012). "PEGylation of proteins and liposomes: a powerful and flexible strategy to improve the drug delivery". Current Drug Metabolism. 13 (1): 105–119. doi:10.2174/138920012798356934. Retrieved 6 September 2017.
  12. ^ Chan, CL; Majzoub, RN; Shirazi, RS (1 April 2012). "Endosomal escape and transfection efficiency of PEGylated cationic liposome-DNA complexes prepared with an acid-labile PEG-lipid". Biomaterials. 33 (19): 4928–4935. doi:10.1016/j.biomaterials.2012.03.038. Retrieved 26 August 2017.
  13. ^ Tagalakis, Aristides; Kenny, Gavin (28 January 2014). "PEGylation improves the receptor-mediated transfection efficiency of peptide-targeted, self-assembling, anionic nanocomplexes". Journal of Controlled Release. 174: 177–187. doi:https://doi.org/10.1016/j.jconrel.2013.11.014. Retrieved 6 September 2017. {{cite journal}}: Check |doi= value (help); External link in |doi= (help)
  14. ^ Hood, Renee; Chenren, Shao (June 1 2014). "Microfluidic Synthesis of PEG- and Folate-Conjugated Liposomes for One-Step Formation of Targeted Stealth Nanocarriers". Pharmaceutical Research. 30 (6): 1597–1607. doi:10.1007/s11095-013-0998-3. Retrieved 6 September 2017. {{cite journal}}: Check date values in: |date= (help)
  15. ^ {{cite web|url=http://www.biocompare.com/Life-Science-News/77261-PEG-Liposome-SiRNA-In-Vivo-Transfection-Kit-From-Altogen-Biosystems/%7Ctitle= PEG-Liposome SiRNA In Vivo Transfection Kit From Altogen Biosystems|date= October 21 2010|access-date = 26 August 2017)
  16. ^ Fee, Conan J.; Van Alstine, James M. (2006). "PEG-proteins: Reaction engineering and separation issues". Chemical Engineering Science. 61 (3): 924. doi:10.1016/j.ces.2005.04.040.
  17. ^ Veronese, edited by Francesco M. (2009). "Protein conjugates purification and characterization". PEGylated protein drugs basic science and clinical applications (Online-Ausg. ed.). Basel: Birkhäuser. pp. 113–125. ISBN 978-3-7643-8679-5. {{cite book}}: |first1= has generic name (help)
  18. ^ Fee, Conan J. (2003). "Size-exclusion reaction chromatography (SERC): A new technique for protein PEGylation". Biotechnology and Bioengineering. 82 (2): 200–6. doi:10.1002/bit.10561. PMID 12584761.
  19. ^ Fee, Conan J.; Damodaran, Vinod B. (2012). "Biopharmaceutical Production Technology": 199. doi:10.1002/9783527653096.ch7. ISBN 9783527653096. {{cite journal}}: |chapter= ignored (help); Cite journal requires |journal= (help)
  20. ^ Ryan, Sinéad M; Mantovani, Giuseppe; Wang, Xuexuan; Haddleton, David M; Brayden, David J (2008). "Advances in PEGylation of important biotech molecules: Delivery aspects". Expert Opinion on Drug Delivery. 5 (4): 371–83. doi:10.1517/17425247.5.4.371. PMID 18426380.
  21. ^ Kawai, F (2002). "Microbial degradation of polyethers". Applied microbiology and biotechnology. 58 (1): 30–8. doi:10.1007/s00253-001-0850-2. PMID 11831473.
  22. ^ Veronese, F. M. (2001). "Peptide and protein PEGylation: A review of problems and solutions". Biomaterials. 22 (5): 405–17. doi:10.1016/s0142-9612(00)00193-9. PMID 11214751.
  23. ^ Damodaran V. B. ; Fee C. J. (2010). "Protein PEGylation: An overview of chemistry and process considerations". European Pharmaceutical Review. 15 (1): 18–26.{{cite journal}}: CS1 maint: multiple names: authors list (link)
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