Physical Characterization and Innate Immunogenicity of Aggregated Intravenous Immunoglobulin (IGIV) in an In Vitro Cell-Based Model.

PURPOSE: To investigate in vitro the innate immune response to accelerated stress-induced aggregates of intravenous immunoglobulin (IGIV) using a well-defined human cell-line model, and to correlate the innate response to physical properties of the aggregates. METHODS: IGIV aggregates were prepared by applying various accelerated stress methods, and particle size, count and structure were characterized. Immune cell activation as tracked by inflammatory cytokines released in response to aggregates was evaluated in vitro using peripheral blood mononuclear cells (PBMC), primary monocytes and immortalized human monocyte-like cell lines. RESULTS: IGIV aggregates produced by mechanical stress induced higher cytokine release by PBMC and primary monocytes than aggregates formed by other stresses. Results with the monocytic cell line THP-1 paralleled trends in PBMC and primary monocytes. Effects were dose-dependent, enhanced by complement opsonization, and partially inhibited by blocking toll-like receptors (TLR2 and TLR4) and to a lesser extent by blocking Fc gamma receptors (FcγRs). CONCLUSIONS: Stress-induced IGIV aggregates stimulate a dose-dependent cytokine response in human monocytes and THP-1 cells, mediated in part by TLRs, FcγRs and complement opsonization. THP-1 cells resemble primary monocytes in many respects with regard to tracking the innate response to IgG aggregates. Accordingly, the measurement of inflammatory cytokines released by THP-1 cells provides a readily accessible assay system to screen for the potential innate immunogenicity of IgG aggregates. The results also highlight the role of aggregate structure in interacting with the different receptors mediating innate immunity.

Use of glass transitions in carbohydrate excipient design for lyophilized protein formulations.

This work describes an effort to apply methods from process systems engineering to a pharmaceutical product design problem, with a novel application of statistical approaches to comparing solutions. A computational molecular design framework was employed to design carbohydrate molecules with high glass transition temperatures and low water content in the maximally freeze-concentrated matrix, with the objective of stabilizing lyophilized protein formulations. Quantitative structure-property relationships were developed for glass transition temperature of the anhydrous solute, glass transition temperature of the maximally concentrated solute, melting point of ice and Gordon-Taylor constant for carbohydrates. An optimization problem was formulated to design an excipient with optimal property values. Use of a stochastic optimization algorithm, Tabu search, provided several carbohydrate excipient candidates with statistically similar property values, as indicated by prediction intervals calculated for each property.

A computational molecular design framework for crosslinked polymer networks.

Crosslinked polymers are important in a very wide range of applications including dental restorative materials. However, currently used polymeric materials experience limited durability in the clinical oral environment. Researchers in the dental polymer field have generally used a time-consuming experimental trial-and-error approach to the design of new materials. The application of computational molecular design (CMD) to crosslinked polymer networks has the potential to facilitate development of improved polymethacrylate dental materials. CMD uses quantitative structure property relations (QSPRs) and optimization techniques to design molecules possessing desired properties. This paper describes a mathematical framework which provides tools necessary for the application of CMD to crosslinked polymer systems. The novel parts of the system include the data structures used, which allow for simple calculation of structural descriptors, and the formulation of the optimization problem. A heuristic optimization method, Tabu Search, is used to determine candidate monomers. Use of a heuristic optimization algorithm makes the system more independent of the types of QSPRs used, and more efficient when applied to combinatorial problems. A software package has been created which provides polymer researchers access to the design framework. A complete example of the methodology is provided for polymethacrylate dental materials.

Chemical degradation of peptides and proteins in PLGA: a review of reactions and mechanisms.

Biodegradable poly(lactide-co-glycolide) (PLGA) polymers have been studied extensively for the controlled release of peptide and protein drugs. In addition to polymer biodegradation, chemical degradation of the incorporated peptide/protein has also been reported in PLGA devices, and the role of the polymer in promoting these reactions has been debated. This review summarizes the peptide/protein chemical degradation reactions that have been reported in PLGA systems and their mechanisms. Reported methods for stabilizing peptides and proteins in PLGA devices are also discussed.

Effect of excipients on PLGA film degradation and the stability of an incorporated peptide.

The effect of pH modifying excipients on the chemical stability of a model peptide (VYPNGA) and the degradation of poly(dl-lactide-co-glycolide)(PLGA) was studied in PLGA films under accelerated storage conditions. pH modifiers included a basic amine (proton sponge), a basic salt (magnesium hydroxide) and two pH buffers (ammonium acetate and magnesium acetate). Changes in film pH were monitored using (13)C NMR, peptide degradation products were quantified by LC/MS/MS and PLGA degradation was analyzed by TGA, DSC and SEC. Inclusion of pH modifiers had little impact on PLGA degradation. The proton sponge affected an initial decrease in pH but reduced peptide deamidation and chain cleavage relative to an unbuffered control. Magnesium hydroxide produced an initial increase in pH but also showed increased peptide deamidation. Ammonium acetate decreased pH and increased peptide chain cleavage, presumably due to increased PLGA hydrolysis. Magnesium acetate buffer increased the initial pH but resulted in increased peptide loss. The extent of peptide acylation increased in all formulations, most notably in the proton sponge modified films. The effectiveness of pH modifiers in PLGA formulations under storage conditions is dependant on both the mechanism of pH alteration and the peptide degradation reaction of interest.

Deamidation, acylation and proteolysis of a model peptide in PLGA films.

The relative rates of deamidation, acylation and proteolysis (i.e. amide bond cleavage) were determined for a model peptide (VYPNGA) in poly (dl-lactide-co-glycolide) films. Films were stored at 70 degrees C and either 95%, 75%, 60%, 45%, 28%, or approximately 0% relative humidity and at 37 degrees C and 95% relative humidity. Peptide degradation products were identified by ESI+MS/MS and quantitated by LC/MS/MS. Extensive overlap of degradation mechanisms occurred, producing a complex mixture of products. Acylation was the dominant peptide degradation reaction (10-20% of total peptide) at early stages of PLGA hydrolysis and at intermediate relative humidity (60-45% RH). Deamidation and proteolysis were dominant (25-50% and 20-40% of total peptide, respectively) at later stages and at high relative humidity (95-75% RH). Understanding the relative rates of each peptide degradation reaction will allow for improved design of PLGA formulations that preserve the stability of peptide and protein drugs.

Secondary structure of a dynamic type I'beta-hairpin peptide.

A spontaneously folding beta-hairpin peptide (Lys-Lys-Tyr-Thr-Val-Ser-Ile-Asn-Gly-Lys-Lys-Ile-Thr-Val-Ser-Ile) and related cyclic (cyclo-Gly-Lys-Tyr-Ile-Asn-Gly-Lys-Ile-Ile-Asn) and linear (Ser-Ile-Asn-Gly-Lys) controls were studied to determine the effects of various factors on secondary structure. Secondary structure was evaluated using circular dichroism (CD) and 1D and 2D (1)H nuclear magnetic resonance (NMR). The effects of chemical modifications in the peptide and various solution conditions were investigated to determine their impact on peptide structure. The beta-hairpin peptide displayed a CD minimum at 216 nm and a TOCSY i + 1 - i + 2 and i + 2 -i + 3 interaction, confirming the expected structure. Using NMR alpha-proton (H(alpha)) chemical shifts, the extents of folding of the beta-hairpin and linear control were estimated to be 51 and 25% of the cyclic control (pH 4, 37 degrees C), which was taken to be maximally folded. Substitution of iso-aspartic acid for Asn reduced the secondary structure dramatically; substitution of aspartic acid for Asn also disrupted the structure. This result suggests that deamidation in unconstrained beta-turns may have adverse effects on secondary structure. N-terminal acetylation and extreme pH conditions also reduced structure, while the addition of methanol increased structure.

Effect of viscosity on the deamidation rate of a model Asn-hexapeptide.

The effect of viscosity on the deamidation rate of a model Asn-containing hexapeptide (l-Val-l-Tyr-Pro-l-Asn-Gly-l-Ala) was assessed in aqueous solution and in solids containing varying amounts of poly(vinyl pyrrolidone) (PVP) and water. Stability studies were conducted at 0.1 mg/mL peptide and 0-50% PVP (w/w) in aqueous solution, and at 5% (w/w) peptide and different relative humidities (31.6, 53.1, 74.4 and 96%) in the solid state. The parent peptide and its deamidation products were analysed by reverse-phase high-performance liquid chromatography. Deamidation rates decreased with increasing solvent viscosity in a manner described by a semi-empirical mathematical model developed to describe this relationship. The results suggest that the motion of the Asn side-chain along the reaction coordinate is a function of the macroscopic solvent viscosity. However, the apparent energy barrier for the diffusive movement of the side-chain appears to be less than the energy barrier for that associated with macroscopic viscosity. The dependence of the deamidation rate on viscosity in both viscous solution and hydrated solids further demonstrates the importance of mobility in peptide deamidation.

Formaldehyde production by Tris buffer in peptide formulations at elevated temperature.

This technical note provides evidence for the degradation of Tris buffer in a peptide formulation stored at elevated temperature (70 degrees C). The buffer degrades to liberate formaldehyde, which is shown to react with the peptide tyrosine residue. Those involved in peptide/protein formulation should be aware of the possible instability in this common biological buffer.

Effect of 'pH' on the rate of asparagine deamidation in polymeric formulations: 'pH'-rate profile.

The rate of Asn deamidation of a model hexapeptide (L-Val-L-Tyr-L-Pro-L-Asn-Gly-L-Ala) was measured as a function of effective pH ('pH') in glassy and rubbery polymeric solids containing poly(vinyl pyrrolidone) (PVP) and in solution controls at 70 degrees C. The reaction exhibited pseudo-first-order kinetics in all samples over a wide 'pH' range (0.5 < 'pH' < 12); the formation of similar products suggests that the reaction mechanism is unaffected by matrix type. Rates of deamidation were comparable for the polymeric and solution samples in the acidic range ('pH' < 4). Solution-state rates were faster than those in polymeric solids at neutral 'pH' (6 < 'pH' < 8), increasing to a > 10,000-fold difference in the basic range ('pH' > 8). Specific base catalysis was observed in solution and in the polymeric solids under neutral conditions (6 < 'pH' < 8). In solution, the reaction exhibited general base catalysis for 'pH' > 8, whereas the reaction was 'pH'-independent in the polymeric solids in this range. The 'pH'-rate profile and supporting buffer catalysis data are consistent with a change in the rate-determining step in the basic range from 'pH'-dependent attack of the deprotonated backbone amide nitrogen on the Asn side chain in solution to 'pH'-independent ammonia expulsion in the polymeric solids. The results suggest that polymer matrix incorporation not only affects the magnitude of the deamidation rate constant but also the 'pH' dependency of the reaction and the rate-determining step in the basic 'pH' range.

Effects of solution polarity and viscosity on peptide deamidation.

Deamidation kinetics were measured for a model hexapeptide (L-Val-L-Tyr-L-Pro-L-Asn-Gly-L-Ala, 0.02 mg/mL) in aqueous solutions containing glycerol (0-50% w/w) and poly(vinyl pyrrolidone) (PVP, 0-20% w/w) at 37 degrees C and pH 10 to determine the effects of solution polarity and viscosity on reactivity. The observed pseudo-first order deamidation rate constants, k(obs), decreased markedly when the viscosity increased from 0.7 to 13 cp, but showed no significant change at viscosities >13 cp. Values of k(obs) also increased with increasing dielectric constant and decreasing refractive index. Molecular dynamics simulations indicated that the free energy associated with Asn side-chain motion is insensitive to changes in dielectric constant, suggesting that the observed dielectric constant dependence is instead related primarily to the height of the transition state energy barrier. An empirical model was proposed to describe the effects of the viscosity, refractive index and dielectric constant on k(obs). Analysis of the regression coefficients suggested that both permanent and induced dipoles of the medium affect the deamidation rate constant, but that solution viscosity is relatively unimportant in the range studied.

Reactivity toward deamidation of asparagine residues in beta-turn structures.

Mimetics of beta-turn structures in proteins have been used to calibrate the relative reactivities toward deamidation of asparagine residues in the two central positions of a beta-turn and in a random coil. N-Acetyl-Asn-Gly-6-aminocaproic acid, an acyclic analog of a beta-turn mimic undergoes deamidation of the asparaginyl residue through a succinimide intermediate to generate N-acetyl-Asp-N-Gly-6-aminocaproic acid (6-aminocaproic acid, hereafter Aca) and N-acetyl-L-iso-aspartyl (isoAsp)-Gly-Aca (pH 8.8, 37 degrees C) approximately 3-fold faster than does the cyclic beta-turn mimic cyclo-[L-Asn-Gly-Aca] with asparagine at position 2 of the beta-turn. The latter compound, in turn, undergoes deamidation approximately 30-fold faster than its positional isomer cyclo-[Gly-Asn-Aca] with asparagine at position 3 of the beta-turn. Both cyclic peptides assume predominantly beta-turn structures in solution, as demonstrated by NMR and circular dichroism characterization. The open-chain compound and its isomer N-acetyl-Gly-Asn-Aca assume predominantly random coil structures. The latter isomer undergoes deamidation 2-fold slower than the former. Thus the order of reactivity toward deamidation is: asparagine in a random coil approximately 3x(asparagine) in position 2 of a beta-turn approximately 30x (asparagine) in position 3 of a beta-turn.

Deamidation of a model hexapeptide in poly(vinyl alcohol) hydrogels and xerogels.

Polymeric controlled release systems have been proposed to prolong the half-lives of protein and peptide drugs in vivo and to deliver active drug at a controlled rate. These systems are ineffective, however, if the drug is not stable during storage and release. This study addresses the effect of poly(vinyl alcohol) on the stability and release of an incorporated hexapeptide, VYPNGA, which undergoes deamidation. Two types of peptide-loaded poly(vinyl alcohol) matrices were formed, a semisolid hydrogel and a lower water content 'xerogel', and stored at 50 degrees C for up to 122 days. The hexapeptide was less stable in both poly(vinyl alcohol) matrices than in aqueous buffer or lyophilized polymer-free powders. The type of poly(vinyl alcohol) matrix appeared to influence the degradation mechanism, since the product distributions differ in the hydrogel and the xerogel. The results suggest that, rather than stabilizing this peptide, incorporation in poly(vinyl alcohol) matrices reduces stability relative to solution and lyophilized controls.

Chemical stability of peptides in polymers. 1. Effect of water on peptide deamidation in poly(vinyl alcohol) and poly(vinyl pyrrolidone) matrixes.

This paper examines the effect of water content, water activity, and glass transition temperature (T(g)) on the deamidation of an asparagine-containing hexapeptide (VYPNGA; Asn-hexapeptide) in lyophilized poly(vinyl alcohol) (PVA) and poly(vinyl pyrrolidone) (PVP) at 50 degrees C. The rate of Asn-hexapeptide deamidation increases with increasing water content or water activity and, hence, decreasing T(g). The rate of deamidation is more sensitive to changes in these parameters in PVA than in PVP. Deamidation is clearly evident in the glassy state in both formulations. In the glassy state, the peptide is more stable in PVA than in PVP formulations but is less stable in the rubbery state. No single variable (water content, water activity, or T(g)) could account for the variation in deamidation rates in PVA and PVP formulations. Deamidation rates were correlated with the degree of plasticization by water (distance of T(g) from the dry intrinsic glass transition temperature); coincident curves for the two polymers were obtained with this correlation. Deamidation in PVA and PVP was closely correlated with the extent of water-induced plasticization experienced by the formulation relative to its glass transition at 50 degrees C, suggesting that the physical state of formulations could be used to predict chemical stability.

Chemical stability of peptides in polymers. 2. Discriminating between solvent and plasticizing effects of water on peptide deamidation in poly(vinylpyrrolidone).

The mechanistic role of water in the deamidation of a model asparagine-containing hexapeptide (Val-Tyr-Pro-Asn-Gly-Ala) in lyophilized formulations containing poly(vinylpyrrolidone) (PVP) and glycerol was investigated. Glycerol was used as a plasticizer to vary formulation glass transition temperature (T(g)) without significantly changing water content or activity. Increases in moisture and glycerol contents increased the rate of peptide deamidation. This increase was strongly correlated with T(g) at constant water content and activity, suggesting that increased matrix mobility facilitates deamidation. In rubbery systems (T > T(g)), deamidation rates appeared to be independent of water content and activity in formulations with similar T(g)s. However, in glassy formulations with similar T(g)s, deamidation increased with water content, suggesting a solvent/medium effect of water on reactivity in this regime. An increase in water content also affected the degradation product distribution; less of the cyclic imide intermediate and more of the hydrolytic products, isoAsp- and Asp-hexapeptides, were observed as water content increased. Thus, residual water appears to facilitate deamidation in these solid PVP formulations both by enhancing molecular mobility and by solvent/medium effects, and also participates as a chemical reactant in the subsequent breakdown of the cyclic imide.

Solid-state chemical stability of proteins and peptides.

Peptide and protein drugs are often formulated in the solid-state to provide stabilization during storage. However, reactions can occur in the solid-state, leading to degradation and inactivation of these agents. This review summarizes the major chemical reactions affecting proteins and peptides in the solid-state: deamidation, peptide bond cleavage, oxidation, the Maillard reaction, beta-elimination, and dimerization/aggregation. Physical and chemical factors influencing these reactions are also discussed. These include temperature, moisture content, excipients, and the physical state of the formulation (amorphous vs crystalline). The review is intended to serve as an aid for those involved in formulation, and to stimulate further research on the determinants of peptide and protein reactivity in the solid-state.

Antibody transport in cultured tumor cell layers.

This review summarizes our recent in vitro studies of the factors affecting the tumor penetration of immunoconjugates. The studies were designed to probe the mechanisms of diffusion and convection, using a cultured layer of mouse melanoma cells as a model tumor cell layer and an antibody to the murine transferrin receptor as a model ligand. Transport of the binding antibody was observed to be slower than that of a non-binding control, a result that is consistent with the "binding site barrier" hypothesis (Fujimori et al., J. Nucl. Med., 31: 1191-1198, 1990). Internalization of the antibody/receptor complex was necessary for this effect to be observed, implying that intracellular trafficking is a determinant of net tumor transport rates. Convective fluid flow exhibited a dependence on cell density that is consistent with a Poiseuille flow model, suggesting that convective transport occurs as laminar flow in tortuous channels. Implications for immunoconjugate therapy, limitations of the approach, and future directions of the research program are discussed.

Development of a cell culture system to study antibody convection in tumors.

The convective transport of fluid and of a binding antibody through a cultured tumor cell layer was investigated with a mouse melanoma cell line (B16F10) grown on a microporous polycarbonate filter (Snapwell inserts). The inserts were precoated with Matrigel or collagen, or were uncoated. The cell layers were exposed to nominal pressure gradients from 5 to 25 cm H2O, and the volume flux was measured by collecting the effluent volume over time. The rate of convective transport of a binding monoclonal antibody that recognizes the murina transferrin receptor (a-TfR) was investigated at a nominal pressure gradient of 15 cm H2O and compared with that of an isotype matched, nonbinding control. The resistance, R, of the cell layer to fluid flow was quantified as the hydraulic conductivity, Lp (= 1/R); the ability of the cell layer to retard antibody transport was quantified as the reflection coefficient, sigma. The resulting Lp values decreased with increasing cell density, in a manner consistent with Poiseuille flow. Collagen or Matrigel precoating also decreased Lp values, with cells grown on Matrigel providing the greatest resistance. The sigma values were 0.67 (+/-0.08) for the a-TfR antibody and 0.51 (+/-0.06) for the control, indicating that the cell layer acts as a semipermeable barrier to convective transport of antibody that is less permeable to the binding antibody.

Application of benzyl hyaluronate membranes as potential wound dressings: evaluation of water vapour and gas permeabilities.

Membranes of 75% and 100% benzyl hyaluronate esters (percentage of total carboxylate groups esterified) were prepared and their water vapour, oxygen and carbon dioxide transmission rates determined. The values of these properties were compared with the values obtained for several commercial wound dressings under the same conditions. The benzyl hyaluronate membranes showed water vapour transmission rates (2157-2327 gm-2 per day) comparable to those from commercial skin dressings (426-2047 gm-2 per day). In the dry state, the benzyl hyaluronate membranes showed lower oxygen and carbon dioxide transmission rates. Taking into account the biocompatibility of the hyaluronic acid esters, and the possibility that therapeutic agents could be incorporated into these membranes, the results indicate that the benzyl hyaluronate membranes have potential wound dressing applications.