A Seminar on

PEGylation

-An

innovAtive

ApproAch

for

protein delivery
Keyur VasaVa…

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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

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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.

1.2 PROTEIN PHARMACEUTICALS – PROBLEMS
  Isolation and purification of Protein Pharmaceuticals is very difficult. But these issues are solved with the advances in genetic engineering Storage and hence the issue of stability is again a major problem of protein pharmaceuticals. It could be achieved by appropriate formulation and applying proper storage conditions viz. refrigeration, etc. Delivery to the target site is one of the major problems which the proteins are facing till today, and many different approaches were tried but no technology has sufficed to full extent.

1.3 PROTEIN DELIVERY TO THE TARGET SITE – SHORTCOMINGS
Protein drugs are:  Susceptible to destruction by proteolytic enzymes  Short circulating half-life  Short shelf life  Low solubility  Rapid kidney clearance  Propensity to generate antibodies

1.4 ATTEMPTS TO IMPROVE CLINICAL PROPERTIES ….
Scientists have searched for years to overcome the problems associated with protein delivery. Initial approaches tried were:  Alteration of amino-acid sequences to reduce degradation by enzymes and antigenic sideeffects  Fusing them to immuno-globulins or albumin to improve half-life  Polymer-entrapment in insoluble matrices  Glycosylation  Incorporating them into the drug delivery vehicles such as Liposomes to prolong action
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Immobilization onto polymer resins.

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

1.5 ROUTES OF ADMINISTRATION OF PROTEIN THERAPEUTICS:
      Parenteral route, the most common route Pulmonary delivery Nasal drug delivery Transdermal delivery And finally comes the least accepted oral route, as it has several limitations

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.

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2.2 PEG - CHEMICAL PROPERTIES
   General formula: HO-(CH2CH2O)n-CH2CH2OH Molecular weight: Few hundred to approximately 20,000 PEG is a viscous liquid at molecular weights lower than 1000 and solid at higher molecular weights.

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

2.3 PEGylation – MERITS
      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). Increase the hydrodynamic size (size in solution) of the agent which prolongs its circulatory time by reducing renal clearance LEADING to improved bioavailability. Avoidance of reticulo-endothelial (RES) clearance. Improved bioavailability via reduced losses at subcutaneous injection sites PEGylation can also provide water solubility to hydrophobic drugs and proteins. A low volume of distribution and sustained absorption from the injection site useful for slow release depot administration strategies i.e. Optimized pharmacokinetics resulting in sustained duration. Protection against proteolysis by forming shell around the protein Reduced dosage frequency, without diminished efficacy with potentially reduced toxicity Altered biodistribution; Altered biological properties.
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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.

2.4 PEGylation – CHEMISTRY
 To couple PEG to a molecule (i.e. polypeptides, polysaccharides, polynucleotides and small organic molecules) it is necessary to activate the PEG by preparing a derivative of the PEG having a functional group at one or both termini. The functional group is chosen 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, N-terminal amino group and the C-terminal carboxylic acid. In the case of glycoproteins, vicinal hydroxyl groups can be oxidized with periodate to form two reactive formyl moieties. The amino groups (e.g. lysine) of proteins are the best residues for PEG-polymer conjugation by alkylation or acylation. Alkylation maintains a positively charged amine (aldehyde, epoxide PEGs) whereas acylation yields a neutral amide [PEGs terminating with a carboxylic group reactive with N-hydroxysuccinimide (NHS)]. PEGs activated as carbonates are also frequently used, although they yield less stable urethane linkage with the protein. When multiple lysines have been modified, a heterogeneous mixture is produced that is composed of a population of several polyethylene glycol molecules attached per protein molecule (‘PEGmers’).

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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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2.5 PEGylation -- PROCESSES
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 Solution phase  PEG reagents such as N-hydroxysuccinimide esters, the final protein- to-reagent molar ratio used is usually 1:3–5. The reaction pH (7–8), the temperature (4–8ºC) and the reaction is terminated after 8–16 h by adjusting the pH to 4.5 with glacial acetic acid.  The reaction mixture is then diluted with water and adsorbed onto a cation exchange column. Excess reagent and reaction byproducts are washed away. PEGylated protein is then separated from the unmodified protein by using increasing concentrations of salt in the buffer. On-column fed-batch process (Solid-phase pegylation)  In solid-phase PEGylation, the protein to be pegylated is adsorbed onto an anion exchange column and the PEG reagent is circulated through the column for a predetermined period of time. Excess reagent and other reaction byproducts are removed from the column by washing with buffer. The salt concentration of the elution buffer is chosen so that only the pegylated protein elutes, while the unmodified protein remains on the column.  In the solid-phase pegylation process, reaction, separation and purification are performed on the same column and can be repeated as many times as required.

 Techniques:
It is classified into two types:  Early PEGylation technology (First generation PEGylation)  Advanced PEGylation technology (Second generation PEGylation)

2.5.1 FIRST GENERATION PEGylation
 With first generation PEGylation, the PEG polymer was generally attached to ε amino group of lysine, and gave mixtures of PEG isomers with different molecular masses. The existence of these isomers makes it difficult to reproduce drug batches, and can contribute to the antigenecity of the drug and poor clinical outcomes. In addition, first generation methods mainly used linear PEG polymers of 12 kDa or less. Unstable bonds between the drug and PEG were also sometimes used, which leads to degradation of PEG-drug conjugate during manufacturing and injection

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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.

2.5.2 SECOND GENERATION PEGylation
  Second generation PEGylation strives to avoid pitfalls associated with the first generation PEGylation. Overall goal of this technology is to create larger PEG polymers to improve the Pharmacokinetics and Pharmacodynamic effects seen with lower molecular mass PEGs

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.

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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) – α2a 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 – α2a produced no clinical advantages
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A second generation, branched PEG of 40 kDa, was then coupled to IFN – α2a. This version (Pegasys) under clinical investigation, showed nearly constant blood concentration of IFN – α2a and renal clearance is reduce to 100-fold related to unPEGylated IFN – α2a. PEGylated also increased the half-life of IFN – α2a from 9 to 77 hours. It is illustrated in the following graphs:

2.6 PEGylation – MECHANISM OF ACTION
 After administration, when PEG comes in contact of aqueous environment, ethylene glycol sub-unit gets tightly attached to the water molecule. This binding to water renders them high mobility and hydration. Hydration and rapid motion causes PEGylated protein to function, as it causes PEG to sweep out a large volume which acts like a shield to protect the attached drug from enzymatic degradation and interaction with cell surface proteins. This increased size also helps to prevent rapid renal filtration and clearance sustaining the drug bioavailability

2.7 HOW CAN WE GET SITE SPECIFIC PEGylation?
1) We can take advantage of differences in pKa of the amino groups ε-amino = 9.3-9.5 α-amino = 7.6-8.0 histidine= intermediate pKa e.g; Neulasta, PEG-Intron  We can conjugate in the presence of inhibitors: Immobilized inhibitor e.g; Tripsin Soluble inhibitors e.g; Uricase
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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

2.7 FACTORS AFFECTING PERFORMANCE OF PEGylated PEPTIDES
Molecular Weight and Structure  Molecular weight < 1000 Da: broken down into sub-units, and have some toxicity.  Molecular weight > 1000 Da: does not demonstrate any toxicity in vivo.  Molecular Weight upto 40,000 – 50,000 Da: used in clinical and approved pharmaceutical application.  The molecular weight of PEG has a direct impact on the activity; Higher molecular weight PEGs tends to have lower in-vitro activity but have higher invivo activity due to the improved pharmacokinetic profile Number of PEG chains  Two or more lower molecular weight chains can be added to increase total molecular weight of PEG complex Specific location of PEG site of attachment to the peptide  It should be carefully engineered to retain the highest possible binding efficiency and activity of the peptide ligand

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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.

Pharmacokinetic increase and activity loss following PEGylation

2.8 PEGylated PRODUCT – PURIFICATION AND ANALYSIS
 PURIFICATION OF THE PEGylated PROTEIN PRODUCT: The principles of the following example of the purification of PEGylated protein can be applied to other PEGylated products. The product in this example is a PEG-linked homodimer (PEG dimer fig.1) made from 20kDa PEG-bis vinylsulfone (fig.2). A number of reaction components and side products must be removed, most notably PEG monomer and unPEGylated protein.

Figure 1. Structure 20 kDa PEG-linked Protein Dimer

Figure 2. Structure of 20 kDa PEG Bis-vinylsulfone

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.

2.9 PEGylation – PROCESS OPTIMIZATION
The “design of experiments” approach to optimization of PEGylation conditions can be very useful.  For thiol specific PEGylation reactions, parameters to consider include:  Protein concentration,  PEG-to-protein ratio (on a molar basis),  Temperature,  pH,  Reaction time, And in some instances, the exclusion of oxygen (oxygen can contribute to intermolecular disulfide formation by the protein, which will reduce the yield of the PEGylated product.) The same factors should be considered (with the exception of oxygen) for amine –specific modification except that pH may be even more critical, particularly when targeting the Nterminal amino group.  Objectives should be set before optimization of the process parameters such that the process should:  Reduce cycle time  Minimize impurities  Maximize yield (> 85 %)

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2.10 PEGylated PHARMACEUTICALS ON THE MARKET
ADAGEN (PEG- bovine adenosine deaminase) 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. Some of the successful examples are:

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New polymers are under study as alternative to PEG, among these:
    Natural: Polysialic acids (Gregoriadis et al. London Univ.) Dextrins (Duncan et al. Cardiff Univ.)etc. Polypeptides (Amunix technology)

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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 São 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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