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.
Protein production can be improved if methods for soluble protein expression are developed. Interferon consensus (IFN-con) is used to treat hepatitis C. IFN-con has superior activity compared to other clinically used interferon ? subtypes. However IFN-con is a challenging protein to produce in a soluble form using an Escherichia coli expression system. Here we describe the expression of soluble and active recombinant IFN-con in E. coli. The IFN-con gene sequence was optimised for expression in E. coli, which was then cloned into the Champion™ pET SUMO expression vector downstream of the SUMO fusion protein and under strong T7lac promoter. The SUMO-IFN-con fusion protein was efficiently expressed using the SHuffle™ E. coli strain and existed in soluble form as 86-88% of the total IFN-con. After removal of the SUMO fusion partner, approximately 50mg of recombinant IFN-con of at least 98% purity (by RP-HPLC) was obtained from a 1L fermentation culture. Using an A549/EMCV antiviral assay, the specific activity of the recombinant IFN-con was determined to be 960×10(6) IU/mg as calculated to NIBSC standard for IFN-con (3×10(5)pfu/mL virus titre). Comparison of the antiviral activity of the produced IFN-con to IFN ?-2a showed that IFN-con displays 2.8 times greater activity, which is in good agreement with what has been reported in the literature for pure protein. IFN-con expression in a soluble form from E. coli allowed us to use a simple, two-step purification process to yield highly pure and active IFN-con which is more efficient than obtaining IFN-con from inclusion bodies.
Many clinically used protein therapeutics are modified to increase their efficacy. Example modifications include the conjugation of cytotoxic drugs to monoclonal antibodies or poly(ethylene glycol) (PEG) to proteins and peptides. Monothiol-specific conjugation can be efficient and is often accomplished using maleimide-based reagents. However, maleimide derived conjugates are known to be susceptible to exchange reactions with endogenous proteins. To address this limitation in stability, we have developed PEG-mono-sulfone 3, which is a latently reactive, monothiol selective conjugation reagent. Comparative reactions with PEG-maleimide and other common thiol-selective PEGylation reagents including vinyl sulfone, acrylate, and halo-acetamides show that PEG-mono-sulfone 3 undergoes more efficient conjugation under mild reaction conditions. Due to the latent reactivity of PEG-mono-sulfone 3, its reactivity can be tailored and, once conjugated, the electron-withdrawing ketone is easily reduced under mild conditions to prevent undesirable deconjugation and exchange reactions from occurring. We describe a comparative stability study demonstrating a PEG-maleimide conjugate to be more labile to deconjugation than the corresponding conjugate obtained using PEG-mono-sulfone 3.
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.
Small tablets for implantation into the subconjunctival space in the eye are being developed to inhibit scarring after glaucoma filtration surgery (GFS). There is a need to evaluate drug dissolution at the molecular level to determine how the chemical structure of the active may correlate with dissolution in the nonsink conditions of the conjunctival space. We conducted molecular dynamics simulations to study the dissolution process of tablets derived from two drugs that can inhibit fibrosis after GFS, 5-fluorouracil (5-FU) and the matrix metalloprotease inhibitor (MMPi), ilomastat. The dissolution was simulated in the presence of simple point charge (SPC) water molecules, and the liquid turnover of the aqueous humor in the subconjunctival space was simulated by removal of the dissolved drug molecules at regular intervals and replacement by new water molecules. At the end of the simulation, the total molecular solvent accessible surface area of 5-FU tablets increased by 60 times more than that of ilomastat as a result of tablet swelling and release of molecules into solution. The tablet dissolution pattern shown in our molecular dynamic simulations tends to correlate with experimental release profiles. This work indicates that a series of molecular dynamic simulations can be used to predict the influence of the molecular properties of a drug on its dissolution profile and could be useful during preformulation where sufficient amounts of the drug are not always available to perform dissolution studies.
The crystal structure of the TLR4-MD-2-LPS complex responsible for triggering powerful pro-inflammatory cytokine responses has recently become available. Central to cell surface complex formation is binding of lipopolysaccharide (LPS) to soluble MD-2. We have previously shown, in biologically based experiments, that a generation 3.5 PAMAM dendrimer with 64 peripheral carboxylic acid groups acts as an antagonist of pro-inflammatory cytokine production after surface modification with 8 glucosamine molecules. We have also shown using molecular modelling approaches that this partially glycosylated dendrimer has the flexibility, cluster density, surface electrostatic charge, and hydrophilicity to make it a therapeutically useful antagonist of complex formation. These studies enabled the computational study of the interactions of the unmodified dendrimer, glucosamine, and of the partially glycosylated dendrimer with TLR4 and MD-2 using molecular docking and molecular dynamics techniques. They demonstrate that dendrimer glucosamine forms co-operative electrostatic interactions with residues lining the entrance to MD-2s hydrophobic pocket. Crucially, dendrimer glucosamine interferes with the electrostatic binding of: (i) the 4phosphate on the di-glucosamine of LPS to Ser118 on MD-2; (ii) LPS to Lys91 on MD-2; (iii) the subsequent binding of TLR4 to Tyr102 on MD-2. This is followed by additional co-operative interactions between several of the dendrimer glucosamines carboxylic acid branches and MD-2. Collectively, these interactions block the entry of the lipid chains of LPS into MD-2s hydrophobic pocket, and also prevent TLR4-MD-2-LPS complex formation. Our studies have therefore defined the first nonlipid-based synthetic MD-2 antagonist using both animal model-based studies of pro-inflammatory cytokine responses and molecular modelling studies of a whole dendrimer with its target protein. Using this approach, it should now be possible to computationally design additional macromolecular dendrimer based antagonists for other Toll Like Receptors. They could be useful for treating a spectrum of infectious, inflammatory and malignant diseases.
The effect of adding a third polymer to immiscible binary solid dispersions was investigated. The model actives griseofulvin (GF), progesterone (PG) and phenindione (PD) were selected because they exemplify a key property of many poorly soluble molecules of having at least one hydrogen bonding acceptor moiety while not having any hydrogen bond donating moieties. Ternary solid dispersions of the drug, PVP (polyvinylpyrrolidone) (proton acceptor) and PHPMA (poly[2-hydroxypropyl methacrylate]) (proton acceptor and donor) were prepared by spray drying. Stability results showed that binary solid dispersions (API and PVP) of GF and PVP crystallized quickly while the amorphous form was not possible to prepare for PG and PD. The amorphous form was prolonged upon the incorporation of PHPMA in the solid dispersion (API, PHPMA and PVP). Based on measuring the melting points, the energy of mixing the drug with the polymer was calculated using the Flory-Huggins theory. The results showed that GF had the lowest free energy followed by PG and finally PD which agreed well with the stability results. These results suggest that the addition of a third polymer to immiscible binary solid dispersions can significantly improve the stability of the amorphous form.
The molecular modeling of hyperbranched molecules is currently constrained by difficulties in model building, due partly to lack of parameterization of their building blocks. We have addressed this problem with specific relevance to a class of hyperbranched macromolecules known as dendrimers by describing a new concept and developing a method that translates monomeric linear sequences into a full atomistic model of a hyperbranched molecule. Such molecular-modeling-based advances will enable modeling studies of important biological interactions between naturally occurring macromolecules and synthetic macromolecules. Our results also suggest that it should be possible to apply this sequence-based methodology to generate hyperbranched structures of other dendrimeric structures and of linear polymers.
The partial modification of carboxylic acid terminated polyamidoamine (PAMAM) dendrimers with glucosamine has been reported to give dendrimer glucosamine conjugates novel immuno-modulatory and anti-angiogenic properties. Experimental analysis of these glycosylated dendrimers showed that, on average, eight glucosamine molecules were covalently bound to each dendrimer. In order to better understand the surface loading and distribution of these glucosamine molecules, molecular reactivity was determined by evaluation of electronic properties using frontier molecular orbital theory (FMOT) and molecular dynamics simulations. It was shown that the surface loading and distribution of zero length amide bond-conjugated glucosamine molecules was determined by both electronic effects and by the different dynamic conformations adopted by the modified dendrimer during the incremental addition of glucosamine. Importantly, the structural features and the dynamic behavior of the partially glycosylated generation 3.5 PAMAM dendrimer showed that its flexibility and polarity changed with the incremental addition of glucosamine. These peripheral glucosamine molecules remained available on the dendrimers surface for interaction with the biological target.
The use of solid dispersions for oral dosage forms can increase the dissolution rate of poorly soluble drugs. Spray drying is one process that can be used to prepare solid dispersions. Spray dried solid dispersions of griseofulvin, poly[N-(2-hydroxypropyl)methacrylate] (PHPMA) and polyvinylpyrrolidone (PVP) were prepared from acetone and water. When methanol was substituted for water, the morphology, stability and dissolution properties of the solid dispersion changed dramatically. The glass transition temperature for the ternary solid dispersion (GF, PHPMA, and PVP) shifted from 83 degrees C (acetone/water) to 103 degrees C for the acetone/methanol system. These differences in the dispersions are thought to derive from conformational variations of the polymers in solution prior to spray drying. Both PHPMA and PVP formed globules in solution of a size range between 16 and 33 nm. The effect of drug and polymer concentration in solution (before spray drying) on the properties of the solid dispersion was studied. It was found that solid dispersions that were prepared using lower concentrations of drug and polymers in solutions resulted in the formation of particles that display a lower relaxation rate. This result supports the hypothesis that the polymer conformation may significantly change the properties of the solid dispersion.
Protein PEGylation is the most clinically validated method to improve the efficacy of protein-based medicines. Antibody fragments such as Fabs display rapid clearance from blood circulation and therefore are good candidates for PEGylation. We have developed PEG-bis-sulfone reagents 1 that can selectively alkylate both sulfurs derived from a native disulfide. Using PEG-bis-sulfone reagents 1, conjugation of PEG specifically targets the disulfide distal to the binding region of the Fab (Scheme 2 ). PEG-bis-sulfone reagents 1 (10-40 kDa) were used to generate the corresponding PEG-mono-sulfones 2 that underwent essentially quantitative conjugation to give the PEG-Fab product 4. Four Fabs were PEGylated: Fab(beva), Fab(beva), Fab(rani), and Fab(trast). Proteolytic digestion of bevacizumab with papain gave Fab(beva), while digestion of bevacizumab with IdeS gave F(ab)(2-beva), which after reaction with DTT and PEG-mono-sulfone 2 gave PEG(2)-Fab(beva). Ranibizumab, which is a clinically used Fab, was directly PEGylated to give PEG-Fab(rani). Trastuzumab was proteolytically digested with papain, and its corresponding Fab was PEGylated to give PEG-Fab(trast). Purification of the PEGylated Fabs was accomplished by a single ion exchange chromatography step to give pure PEG-Fab products as determined by silver-stained SDS-PAGE. No loss of PEG was detected post conjugation. A comparative binding study by SPR using Biacore with low ligand immobilization density was conducted using (i) VEGF(165) for the bevacizumab and ranibizumab derived products or (ii) HER2 for the trastuzumab derived products. VEGF(165) is a dimeric ligand with two binding sites for bevacizumab. HER2 has one domain for the binding of trastuzumab. Binding studies with PEG-Fab(beva) indicated that the apparent affinity was 2-fold less compared to the unPEGylated Fab(beva). Binding properties of the PEG-Fab(beva) products appeared to be independent of PEG molecular weight. Site-specific conjugation of two PEG molecules gave PEG(2×20)-Fab(beva), whose apparent binding affinity was similar to that observed for PEG-Fab(beva) derivatives. The k(d) values were similar to those of the unPEGylated Fab(beva); hence, once bound, PEG-Fab(beva) remained bound to the same degree as Fab(beva). Biacore analysis indicated that both Fab(rani) and PEG(20)-Fab(rani) did not dissociate from the immobilized VEGF at 25 °C, but ELISA using immobilized VEGF showed 2-fold less apparent binding affinity for PEG(20)-Fab(rani) compared to the unPEGylated Fab(rani). Additionally, the apparent binding affinities for trastuzumab and Fab(trast) were comparable by both Biacore and ELISA. Biacore results suggested that trastuzumab had a slower association rate compared to Fab(trast); however, both molecules displayed the same apparent binding affinity. This could have been due to enhanced rebinding effects of trastuzumab, as it is a bivalent molecule. Analogous to PEG-Fab(beva) products, PEG(20)-Fab(trast) displayed 2-fold lower binding compared to Fab(trast) when evaluated by ELISA. The variations in the apparent affinity for the PEGylated Fab variants were all related to the differences in the association rates (k(a)) rather than the dissociation rates (k(d)). We have shown that (i) Fabs are well-matched for site-specific PEGylation with our bis-alkylation PEG reagents, (ii) PEGylated Fabs display only a 2-fold reduction in apparent affinity without any change in the dissociation rate, and (iii) the apparent binding rates and affinities remain constant as the PEG molecular weight is varied.
We are developing tablet dosage forms for implantation directly into the subconjunctival space of the eye. The matrix metalloproteinase inhibitor, ilomastat, has previously been shown to be efficacious at suppressing scarring following glaucoma filtration surgery (GFS). We report on the physical characterisation of ilomastat which is being developed for ocular implantation. Since ilomastat is being considered for implantation it is necessary to examine its polymorphs and their influence on aspects of the in vitro drug release profile. X-ray powder diffraction identified two polymorphs of ilomastat from different commercial batches of the compound. Tablets were prepared from the two different polymorphs. Isothermal perfusion calorimetry was used to show that amorphous content is not increased during tablet formulation. The melting points of the two polymorphs are 188 and 208°C as determined by differential scanning calorimetry. Utilising single crystal X-ray diffraction, the structural conformations and packing arrangements of the different polymorphs were determined. The orthorhombic crystal crystallised as a monohydrate while the second monoclinic crystal form is non-solvated. Ilomastat tablets prepared from the two different solid forms exhibited similar drug release profiles in vitro under conditions mimicking the aqueous composition, volume and flow of the subconjunctival space after GFS. This suggests that a reproducible dose at each time point during release after implantation should be achievable in vivo with ilomastat tablets prepared from the two polymorphs identified.
The efficacy of protein-based medicines can be compromised by their rapid clearance from the blood circulatory system. Achieving optimal pharmacokinetics is a key requirement for the successful development of safe protein-based medicines. Protein PEGylation is a clinically proven strategy to increase the circulation half-life of protein-based medicines. One limitation of PEGylation is that there are few strategies that achieve site-specific conjugation of PEG to the protein. Here, we describe the covalent conjugation of PEG site-specifically to a polyhistidine tag (His-tag) on a protein. His-tag site-specific PEGylation was achieved with a domain antibody (dAb) that had a 6-histidine His-tag on the C-terminus (dAb-His(6)) and interferon ?-2a (IFN) that had an 8-histidine His-tag on the N-terminus (His(8)-IFN). The site of PEGylation at the His-tag for both dAb-His(6)-PEG and PEG-His(8)-IFN was confirmed by digestion, chromatographic, and mass-spectral studies. A methionine was also inserted directly after the N-terminal His-tag in IFN to give His(8)Met-IFN. Cyanogen bromide digestion studies of PEG-His(8)Met-IFN were also consistent with PEGylation at the His-tag. By using increased stoichiometries of the PEGylation reagent, it was possible to conjugate two separate PEG molecules to the His-tag of both the dAb and IFN proteins. Stability studies followed by in vitro evaluation confirmed that these PEGylated proteins retained their biological activity. In vivo PK studies showed that all of the His-tag PEGylated samples displayed extended circulation half-lives. Together, our results indicate that site-specific, covalent PEG conjugation at a His-tag can be achieved and biological activity maintained with therapeutically relevant proteins.
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