Polymer- and Lipid-Based Systems for Parenteral Drug Delivery
4.3 Manufacturing and Formulation Approaches for PEGylated Biotherapeutics
4.3.2 Drug Substance Manufacturing Issues
4.3.2.1 Starting Materials
The quality of the starting material is critical to developing a reproducible, scalable GMP manufacturing process. Impurities in the PEG reagent and/or the biotherapeu-tic can result in heterogeneities in the subsequent PEGylated product which are extremely diffi cult to overcome (Seely et al. 2005 ). Although in recent years high quality low polydispersity PEG reagents have become available from an increasing number of manufacturers, ensuring adequate sourcing and availability of the required quantities of quality PEG reagents for manufacturing PEGylated biothera-peutics remains a key issue.
Concerns for PEG reagents include trace process impurities, PEG-related impu-rities, PEGylation reactivity, and stability of both the PEG chain and the linker between PEG and the reactive functional group (Gaberc-Porekar et al. 2008 ; Kumar and Kalonia 2006 ; McGary 1960 ; Seely et al. 2005 ). Susceptibility of PEG chains to oxidation and the reactive groups to hydrolysis and other degradative pathways necessitates detailed impurity profi ling and “use” testing to monitor the PEGylation performance of reagent batches.
4.3.2.2 PEGylation Reaction Process Control
GMP manufacture of PEGylated biotherapeutics requires a well-defi ned PEGylation reaction process that is capable of consistently yielding products of comparable strength, composition and impurity profi le, batch after batch. Ideally, the conjuga-tion reacconjuga-tion would yield the desired PEGylated molecule with minimal residual un-reacted species and product-related impurities. A fully optimized reaction pro-cess can simplify downstream steps and ultimately lead to increased yields and decreased cost. Kinetic modeling and “design of experiment” DOE studies are rec-ommended to determine optimized conditions for successful API manufacture (Buckley et al. 2008 ; Fee and Van Alstine 2006 ).
Conditions such as PEG/biomolecule reactant stoichiometries, concentrations, and reagent order-of-addition schemes; reaction temperatures and times; solution buffers and pH; reaction vessel sizes and geometries; and reagent, intermediate, and
product hold times need to be well defi ned and optimized (Payne et al. 2011 ).
Understanding reaction kinetics is critical especially in cases where relatively non-selective PEG reagents are being utilized to reproducibly generate comparable batches of a PEG-biotherapeutic. This includes an understanding and control of the kinetics of the possible deactivation via hydrolysis or oxidation of the functional group on the PEG reagent during the reaction. Additionally, side reactions with buffer constituents or reagent impurities must be identifi ed and controlled. And fi nally, in order to understand these issues, critical in-process control assays must be developed and in place (Buckley et al. 2008 ; Seely et al. 2005 ; Fee and Van Alstine 2006 ).
In cases where novel process steps are required to complete the PEGylation, optimization of those methodologies must also be thoroughly evaluated. For thiol PEGylations, limited reduction steps must be optimized in order to reduce disul-fi des or to maintain reactive sulfhydryl groups while limiting protein unfolding, disulfi de shuffl ing, or dimer formation (Doherty et al. 2005 ). PEGylation of par-tially buried cysteine thiols may require transient denaturing conditions (Veronese et al. 2007 ) or use of a two-step protocol involving initial glycation of the buried cysteine followed by PEGylation of the oxidized glycosides (Salmaso et al. 2008 ).
For reductive alkylation reactions, choice and concentration of reducing agents such as sodium cyanoborohydride or pyridine borane can be important and the safety implications should be well understood (Cabacungan et al. 1982 ). PEG reactions such as those catalyzed by enzymes (Peschke et al. 2007 ; DeFrees et al. 2006 ; Sato 2002 ) or those with chemical modifi cations such as periodate oxidation of polysac-charides prior to the actual PEGylation step (Wolfe and Hage 1995 ), may require additional in-process monitoring. Lastly, most PEGylation reactions will require a quenching step for stopping the reaction and inactivating residual reactive moieties.
Most PEGylation reactions are carried out in batch reactors where the extent of PEGylation is mainly controlled by fi xed reaction conditions such as temperature, time, mixing speed, reagent stoichiometries, and concentrations. Another method to control the reaction is in the manner of addition. Adding the PEG reagent in a single portion, in separate multiple aliquots or a slow continuous feed can have an effect on the fi nal reaction outcome (Fee and Van Alstine 2006 ). Several alterna-tive approaches to batch PEG reactions have been reported. Size exclusion reac-tion chromatography (SERC) has been described in which PEGylareac-tion occurs in the mobile phase of a size exclusion column. This method utilizes size separation to control the degree of PEGylation and separate the reaction products in a single step (Fee 2003 ). Packed-bed or “on-column PEGylation” has also been reported where one reactant, either PEG reagent or biomolecule, is anchored to a surface through a covalent linkage with other reagents free in solution. Immobilization of biomolecules in this fashion may result in some control of regiospecifi city depend-ing on the orientation of bound biomolecules. Also, the resultant PEGylated bio-molecule is attached to the surface which may facilitate its separation from the other components in solution (Monkarsh et al. 1997 ; Lee and Lee 2004 ; Baran et al. 2003 ).
4.3.2.3 Purifi cation Considerations
Even a well-controlled PEGylation process will most likely yield a reaction mixture containing the desired product along with un-reacted PEG and biomolecule as well as reaction product impurities (e.g., PEGylated positional isomers, unwanted multi- PEGylated products, and aggregates). Techniques used for purifi cation of unmodi-fi ed proteins, peptides, and nucleic acids have been attempted for the puriunmodi-fi cation of PEGylated biomolecules. Reaction mixtures are generally purifi ed through subse-quent chromatography and ultrafi ltration/diafi ltration (UF/DF). However, the per-formance of a particular PEGylated protein in chromatography and other downstream processes will be quite different from that of the unmodifi ed protein (Fee and Van Alstine 2004 ; Buckley et al. 2008 ). Ion exchange chromatography (IEX) is often the fi rst choice in commercial manufacture as conditions can generally be found where the protein-related species will bind, while residual PEG reagent does not.
Subsequently, elution conditions can often be readily developed for separation of the protein species relative to the number of PEGs attached (Piquet et al. 2002 ; Seely et al. 2005 ; Kusterle et al. 2008 ; Chapman et al. 1999 ; Yun et al. 2005 ). PEG proteins have also been successfully purifi ed by other chromatographic methods such as size exclusion chromatography (SEC) (Fee and Van Alstine 2004 ) and hydrophobic interaction chromatography (HIC) (Fee and Van Alstine 2006 ) or through combinations of columns (Clark et al. 1996 ). A comprehensive discussion on PEGylated protein purifi cation considerations can be found in the review by Fee and Van Alstine 2006 .
4.3.2.4 Polydispersity, Hydrodynamic Size, and Viscosity Considerations The polydispersity, large hydrodynamic volume, viscosity, and other characteristics of PEG can make downstream scale-up considerations for PEG-biomolecules a challenge (Payne et al. 2011 ). The large hydrodynamic size of the PEGs typically used often interferes with the protein–resin interactions such that separation is dom-inated by the PEG physical properties and not those of the specifi c protein. Thus, PEGylated positional isomers become very diffi cult to purify. Column loading capacities can be greatly decreased through masking of charged residues, either indirectly, through steric interference of proximally located PEG, or directly, due to linkage at amines or carboxyl groups (Pabst et al. 2007 ). PEG polydispersity can broaden peaks and lower resolution and also complicate process analytics (Veronese and Pasut 2005 ). Commercial scale downstream processes (e.g., buffer exchange, column loading, and fi nal formulation) often require steps at relatively high protein concentrations. The level of hydration on PEG can lead to PEGylated protein solu-tions that become very viscous upon concentration. This becomes important with PEGylated peptides where PEG:peptide weight ratios are 5–10:1. High viscosities in PEGylated protein solutions can have deleterious effects on chromatography such as decreased fl ow rates and increased pressures. These viscous PEG solutions may also slow or completely stop fl ux rates during UF/DF steps through membrane fouling (Fee and Van Alstine 2006 ).
4.3.2.5 Analytical Characterization
Process scale manufacture of a PEGylated protein requires well-defi ned analytical procedures for control of each step. Methods must be developed for PEGylation reaction product characterization and downstream fraction analysis, as well as fi nal drug substance and drug product release.
Similar to purifi cation approaches, traditional protein analytical techniques have been adapted for characterization of PEGylated proteins. For example, the extent and location of PEGylation and impurity profi les can be monitored by such tech-niques as sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Ramon et al. 2005 ), size exclusion (SEC)-HPLC (Fee and Van Alstine 2004 ; McGoff et al. 1988 ), IEX-HPLC (Pabst et al. 2007 ; Zhang et al. 2007 ; Ramon et al.
2005 ), reverse phase (RP)-HPLC (Park et al. 2009 ), HIC (Snider et al. 1992 ), mass spectrometry (MS), proteolytic digests (Kinstler et al. 2002 ; Schneiderheinze et al.
2009 ), and capillary electrophoresis. PEG hydrodynamic volume and polydispersity generally necessitates some modifi cation to the technique and subsequent data anal-ysis and often requires a combination of orthogonal approaches (Seely et al. 2005 ).
Since PEG and proteins of similar molecular weights greatly differ in actual size, multi-angle laser light scattering (MALLS) is often used along with SEC to deter-mine the molecular mass and hydrodynamic radius of a PEGylated protein (Kendrick et al. 2001 ; Koumenis et al. 2000 ; Fee 2007 ). Specifi c detection techniques for PEG such as iodine staining for SDS-PAGE (Kurfürst 1992 ), or in-line detectors on HPLC such as refractive index (RI) detection (Trathnigg and Ahmed 2011 ), corona charged aerosol (CAD) detection (Kou et al. 2009 ), or evaporative light scattering (ELS) detection (Trathnigg and Ahmed 2011 ) are commonly used in conjunction with stan-dard protein detection methods. Due to the polydispersity of large molecular weight PEG, electrospray ionization mass spectrometry (ESI-MS) techniques of intact PEGylated proteins are usually not a viable option; alternatively, matrix- assisted laser desorption ionization mass spectrometry (MALDI-MS) has been used (Cindric et al. 2007 ). Identifi cation of specifi c PEGylation sites generally requires proteolytic digests and mapping with HPLC; however, site conjugation is often confi rmed through disappearance of PEGylated peaks as PEGylated fragments may be diffi cult to resolve and/or characterize (Kinstler et al. 2002 ; Schneiderheinze et al. 2009 ).