Tuesday, April 24, 2012

FORMULATION - Proteins and Peptides | PEGylation Hurdles Prompt Innovative Approaches

Tim Donald
PEGylation Hurdles Prompt Innovative Approaches

Reversible and noncovalent methods enhancing protein and peptide performance

PEGylation—the covalent attachment of polyethylene glycol groups to proteins and peptides—is a strategy commonly used to improve the performance of these therapeutic molecules in vivo. However, successful drug development using PEGylation can pose a number of challenges, not least of which is that the process of attaching PEG covalently can alter the activity of the protein or peptide itself.
In response to these challenges, a number of novel variations on PEGylation have been developed in recent years, including reversible PEGylation and noncovalent PEGylation. There has also been the realization that there is “nothing magical” about PEG, in the words of one researcher, and that other biomolecules, such as starches, can be used to change pharmacokinetic qualities.

Challenges of Formulation

While PEGylation can extend the in vivo circulatory half-life and medicinal effect of proteins or peptides, any such chemical modification can also alter these molecules’ physical properties. These changes introduce challenges in creating a drug, said Mark C. Manning, PhD, chief scientific officer of Legacy BioDesign, a contract formulation service in Johnstown, Colo.
At least eight PEGylated protein and peptide therapeutics have been developed and reached the market, beginning with Adagen in 1990.
“If you take a protein and chemically modify it, whether it’s by PEGylation or something else, then you’ve introduced new chemistry, and that then affects the manufacturing, the approval process, and so on. It complicates the analytical methods you have to use, it changes the evaluation of [active pharmaceutical ingredient] purity, it affects formulation, and so on,” he said.
Dr. Manning and colleagues recently reviewed the challenges to drug development with PEGylated proteins.1 They noted that the chemistry of attaching PEG to the protein is the critical issue affecting product development. In addition, the quality of the starting materials and the coupling process must be carefully controlled, and these issues become exacerbated as the scale is increased and the chemistry must be performed under current good manufacturing practice (cGMP) conditions. Further, a suitable purification method must be selected for the potentially complex mixture of multiple isomers of the PEGylated compound. Once the purified drug substance is obtained, proper analytical methods must be chosen to characterize the material.
“These are all things that drug developers should think about as they embark along these lines,” Dr. Manning said. “We say this technology is well-established and can provide benefit, which is true, but it comes at a cost, a price in terms of the time required to characterize these materials.”
Despite these challenges, at least eight PEGylated protein and peptide therapeutics have been developed and reached the market, beginning with Adagen (pegademase bovine, Enzon) in 1990, and more than two dozen PEGylated peptides and proteins of interest have been described in the literature.1 A recently introduced PEGylated product, Cimzia (certolizumab pegol, UCB), is discussed in the case study.
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Case Study: PEGylation Increases Half-Life of TNF Blocker

Despite the challenges of using PEGylation to create useful therapeutic entities, at least eight PEGylated therapeutic products have reached the market, and many more molecules of interest have been described in the literature. One of the most recently approved PEGylated therapeutics is Cimzia (certolizumab pegol, UCB), a tumor necrosis factor blocker indicated for treatment of Crohn’s disease and rheumatoid arthritis.1
Certolizumab pegol is a recombinant, humanized antibody Fab’ fragment with specificity for human TNF-alpha, conjugated to an approximately 40 kDa polyethylene glycol. The Fab’ fragment is manufactured in E. coli and subsequently subjected to purification and conjugation to the PEG to generate certolizumab pegol. The molecular weight of certolizumab pegol is approximately 91 kDa.1
PEGylation is an essential factor in the formulation of certolizumab pegol because of the instability of the active molecule, said Mark C. Manning, PhD.
“Without PEGylation, the active ingredient would be cleared in minutes, in vivo. It’s a very unstable molecule,” he said. “Once it is PEGylated, it can reside in the body for hours.”
Even though many PEGylated proteins exhibit compromised efficacy—a possible side effect of PEGylation—this could be an acceptable tradeoff in return for longer in vivo stability, Dr. Manning said.
“Even if you compromise the intrinsic activity a bit, you’re willing to pay that price because the overall effectiveness for the patient is going to increase significantly,” he said. —TD


  1. UCB, Inc. Cimzia [package insert]. Available at: http://cimzia.com/?v=GOOG&WT.srch=1&gclid=COurlfa35a4CFWYJRQodISNRvQ. Accessed March 13, 2012.

Reversible PEGylation

One of the potential drawbacks of protein or peptide PEGylation is that the attachment of the PEG polymer may block the active sites of these therapeutic molecules. Linking a PEG or another polymer to protein drugs often yields derivatives that are largely or completely devoid of biologic potency, and therefore pharmacologically ineffective, said Matityahu Fridkin, PhD, the Lester B. Pearson Professorial Chair of Protein Research in the department of organic chemistry at the Weizmann Institute of Science in Rehovot, Israel.
“Classical PEGylation of peptides and proteins often leads to prevention of binding and activation of certain specific receptors, as for instance is known to occur with interferon,” Dr. Fridkin said.
In order to overcome this phenomenon, Dr. Fridkin and colleagues have used a process called reversible PEGylation, in which chain-like spacers are used to link PEG to the protein drug. This attachment turns the short-acting protein or peptide drug into a long-acting prodrug, which maintains circulating levels for extended periods after administration. The reversible chemical bonds dissolve slowly under physiologic conditions, releasing the active drug slowly over time, Dr. Fridkin and colleagues have shown.
“Reversible PEGylation leads to the slow release of the intact parent drug with full bioactivity,” Dr. Fridkin said.
He noted that the group’s work with insulin has demonstrated the potential advantages of reversible PEGylation. In a 2008 publication, the researchers engineered a long-acting prodrug of insulin that released biologically active insulin with a half-life of 30 hours under physiologic conditions.2 By contrast, conventional PEGylation of insulin led to inactivation of the hormone.
In addition to PEG, other molecules can be used to improve the PK of proteins and peptides in vivo. A number of biomolecules are being investigated and used in this way, including polyglycine and, increasingly, several types of starches.
The preparation of large-scale amounts of reversibly PEGylated conjugates remains beyond the researchers’ current capability, Dr. Fridkin said. “This will be the major direction in our near future research,” he said. “We are attempting to simplify the current synthetic methodology to achieve this goal.”

Noncovalent PEGylation

Another approach to obtaining stable biologically active compounds in vivo is to use noncovalent bonds to attach PEG to proteins and peptides.
Researchers at the University of Kansas, led by Cory J. Berkland, PhD, recently used noncovalent PEGylation by polyanion complexation to improve the in vivo stability of keratinocyte growth factor-2.3
“Normally, we use a covalent chemical bond to conjugate the PEG onto the protein, and that can affect the activity of the protein. We thought that if we can use a noncovalent bond to put the PEG on the protein, when the formulation goes into the body the protein can be released from the PEG quite easily, and that would not interfere with the protein activity. That was the starting point of our work,” said Supang Khondee, PhD, first author of the paper on noncovalent PEGylation of KGF-2.3 Now a research fellow in internal medicine at the University of Michigan Medical School, she was a graduate student at the University of Kansas under Dr. Berkland at the time of this research.
Using the polyanions pentosan polysulfate and dextran sulfate, the researchers attached PEG noncovalently to KGF-2, a heparin-binding protein with regenerative properties. This increased the melting temperature and improved the stability of the compound. The researchers suggested that this approach can be used with other heparin-binding proteins.

Related Technologies

In addition to PEG, other molecules can be used to improve the PK of proteins and peptides in vivo, Dr. Manning noted.
“There’s nothing magical about PEG,” he said. “In general, if you attach another polymer to a protein, you will increase its circulating half-life. So we’re seeing a lot of people exploring whether other biomolecules that are safe can chemically attach to proteins and therefore change their PK properties.”
A number of biomolecules are being investigated and used in this way, including polyglycine and, increasingly, several types of starches, Dr. Manning said.
The starch derivative hydroxyethyl starch has been used in this manner in a proprietary process called HESylation. HESylation allows targeted modification of drugs and their characteristics by site-specific coupling to HES molecules, according to Boehringer Ingelheim, of Ingelheim, Germany.4
In a collaboration between Fresenius Kabi, of Bad Homburg, Germany, and Boehringer Ingelheim, HES was coupled to a therapeutic protein, and the resulting HESylated pharmaceutical was produced at industrial scale with quality and yield comparable to product made in the laboratory, a press release from Boehringer Ingelheim stated. The press release did not name the compound, but it stated that the two companies will continue to pursue their collaboration.


  1. Payne RW, Murphy BM, Manning MC. Product development issues for PEGylated proteins. Pharm Dev Technol. 2011;16(5):423-440.
  2. Shechter Y, Mironchik M, Rubinraut S, et al. Reversible pegylation of insulin facilitates its prolonged action in vivo. Eur J Pharm Biopharm. 2008;70(1):19-28.
  3. Khondee S, Olsen CM, Zeng Y, Middaugh CR, Berkland C. Noncovalent PEGylation by polyanion complexation as a means to stabilize keratinocyte growth factor-2 (KGF-2). Biomacromolecules. 2011;12(11):3880-3894.
  4. Boehringer Ingelheim. Boehringer Ingelheim RCV and Fresenius Kabi successfully coupled HES to a therapeutic protein in an industrial scale applying Fresenius Kabi’s HESylation Technology [press release]. Boehringer Ingelheim website. November 11, 2010. Available at: www.boehringer-ingelheim.com/news/news_releases/press_releases/2010/11_november_2010fresenius.html. Accessed March 13, 2012.

Editor’s Choice

  1. Gokarn YR, McLean M, Laue TM. Effect of PEGylation on protein hydrodynamics [published online ahead of print Feb. 21, 2012]. Mol Pharm.
  2. Vasudev SS, Ahmad S, Parveen R, et al. Formulation of PEG-ylated L-asparaginase loaded poly (lactide-co-glycolide) nanoparticles: influence of pegylation on enzyme loading, activity and in vitro release. Pharmazie. 2011;66(12):956-960.
  3. Mehmet Saka O, Bozkir A. Formulation and in vitro characterization of PEGylated chitosan and polyethylene imine polymers with thrombospondin-I gene bearing pDNA [published online ahead of print Jan. 25, 2012]. J Biomed Mater Res B Appl Biomater.

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