Hanieh Khalili

PEGylation and its impact on the design of new protein-based medicines

PEGylation is the covalent conjugation of PEG to therapeutic molecules. Protein PEGylation is a clinically proven approach for extending the circulation half-life and reducing the immunogenicity of protein therapeutics. Most clinically used PEGylated proteins are heterogeneous mixtures of PEG positional isomers conjugated to different residues on the protein main chain. Current research is focused to reduce product heterogeneity and to preserve bioactivity. Recent advances and possible future directions in PEGylation are described in this review. So far protein PEGylation has yielded more than 10 marketed products and in view of the lack of equally successful alternatives to extend the circulation half-life of proteins, PEGylation will still play a major role in drug delivery for many years to come.

Fab-PEG-Fab as a potential antibody mimetic.

IgG antibodies have evolved to be flexible so that they can bind to epitopes located over a wide spatial range. The two Fabs in an IgG antibody are linked together as if each Fab is at the end of a linear, flexible molecule. PEG was used as a scaffold molecule to link two Fabs together to give Fab-PEG-Fab molecules, or FpFs. Preparation of FpFs was achieved with reagents that undergo site-specific conjugation at each PEG terminus by bis-alkylation with the two cysteine thiols from a disulfide bond. This allowed each Fab to be conjugated to the PEG scaffold in essentially the same region that each Fab is linked in an IgG. Fabs were sourced directly (e.g., ranibizumab) or monoclonal IgG antibodies were proteolytically digested to obtain the Fabs. This allowed the resulting FpFs to be directly compared to parent IgGs. PEG scaffolds of 6, 10, and 20 kDa were used to make the corresponding FpFs. Dynamic light scatting data suggested the resulting FpFs were similar in size to an IgG antibody and about half the size of a 20 kDa PEGylated-Fab. The solution size of PEG-conjugated proteins is known to be dominated by the extended solution structure of PEG, so it is thought that the smaller size of the FpFs is due to interactions between the two Fabs. Anti-VEGF and anti-Her2 FpFs were prepared and evaluated. The FpFs displayed similar apparent affinities to their parent IgGs. Slower dissociation rates were observed for the anti-VEGF FpFs compared to bevacizumab. The anti-VEGF FpFs also displayed in vitro anti-angiogenic properties comparable to or better than bevacizumab. These first studies indicate that FpFs warrant further examination in a therapeutic indication where the presence of the Fc may not be required.

Storage stability of bevacizumab in polycarbonate and polypropylene syringes

PurposeTo compare and examine the storage stability of compounded bevacizumab in polycarbonate (PC) and polypropylene (PP) syringes over a 6-month period. PC syringes have been used in a recent clinical study and bevacizumab stability has not been reported for this type of syringe.MethodsRepackaged bevacizumab was obtained from Moorfields Pharmaceuticals in PC and PP syringes. Bevacizumab from the stored syringes was analysed at monthly time points for a 6-month period and compared with bevacizumab from a freshly opened vial at each time point. SDS-PAGE electrophoresis and size-exclusion chromatography (SEC) was used to observe aggregation and degradation. Dynamic light scattering (DLS) provided information about the hydrodynamic size and particle size distribution of bevacizumab in solution. VEGF binding and the active concentration of bevacizumab was determined by surface plasmon resonance (SPR) using Biacore.ResultsSDS-PAGE and SEC analysis did not show any changes in the presence of higher molecular weight species (HMWS) or degradation products in PC and PP syringes from T0 to T6 compared with bevacizumab sampled from a freshly opened vial. The hydrodynamic diameter of bevacizumab in the PC syringe after 6 months of storage was not significantly different to bevacizumab taken from a freshly opened vial. Using SPR, the VEGF binding activity of bevacizumab in the PC syringe was comparable to bevacizumab taken from a freshly opened vial.ConclusionNo significant difference over a 6-month period was observed in the quality of bevacizumab repackaged into prefilled polycarbonate and polypropylene syringes when compared with bevacizumab that is supplied from the vial.

An anti-TNF-α antibody mimetic to treat ocular inflammation

Infliximab is an antibody that neutralizes TNF-α and is used principally by systemic administration to treat many inflammatory disorders. We prepared the antibody mimetic Fab-PEG-Fab (FpFinfliximab) for direct intravitreal injection to assess whether such formulations have biological activity and potential utility for ocular use. FpFinfliximab was designed to address side effects caused by antibody degradation and the presence of the Fc region. Surface plasmon resonance analysis indicated that infliximab and FpFinfliximab maintained binding affinity for both human and murine recombinant TNF-α. No Fc mediated RPE cellular uptake was observed for FpFinfliximab. Both Infliximab and FpFinfliximab suppressed ocular inflammation by reducing the number of CD45+ infiltrate cells in the EAU mice after a single intravitreal injection at the onset of peak disease. These results offer an opportunity to develop and formulate for ocular use, FpF molecules designed for single and potentially multiple targets using bi-specific FpFs.

Comparative thermodynamic analysis in solution of a next generation antibody mimetic to VEGF

An antibody mimetic known as Fab–PEG–Fab (FpF) is a stable bivalent molecule that may have some potential therapeutic advantages over IgG antibodies due to differences in their binding kinetics as determined by surface plasmon resonance. Here we describe the thermodynamic binding properties to vascular endothelial growth factor (VEGF) of the FpF antibody mimetics derived from bevacizumab and ranibizumab. Bevacizumab is an IgG antibody and ranibizumab is an antibody fragment (Fab). Both are used clinically to target VEGF to inhibit angiogenesis. FpFbeva displayed comparable binding affinity (KD) and binding thermodynamics (ΔH = −25.7 kcal mole−1 and ΔS = 14 kcal mole−1) to bevacizumab (ΔH = −25 kcal mole−1, ΔS = 13.3 kcal mole−1). FpFrani interactions with VEGF were characterised by large favourable enthalpy (ΔH = −42 kcal mole−1) and unfavourable entropy (ΔS = 31 kcal mole−1) changes compared to ranibizumab (ΔH = −18.5 kcal mole−1 and ΔS = 6.7 kcal mole−1), which being a Fab, is mono-valent. A large negative entropy change resulting in binding of bivalent FpF to homodimer VEGF might be due to the conformational change of the flexible regions of the FpF upon ligand binding. Mono-valent Fab (i.e. ranibizumab or the Fab derived from bevacizumab) displayed a larger degree of freedom (smaller unfavourable entropy) upon binding to homodimer VEGF. Our report describes the first comprehensive enthalpy and entropy compensation analysis for FpF antibody mimetics. While the FpFs displayed similar thermodynamics and binding affinity to the full IgG (i.e. bevacizumab), their enhanced protein stability, slower dissociation rate and lack of Fc effector functions could make FpF a potential next-generation therapy for local tissue-targeted indications.