A case study of nondelamination glass dissolution resulting in visible particles: implications for neutral pH formulations.

Visible particles were unexpectedly observed in a neutral-pH placebo formulation stored in glass vials but were not observed in the same formulation composition that contained protein. The particles were identified as silica gel (SiO2 ) and polysorbate 20, suggesting dissolution of the glass vial. Time course studies were performed to assess the effect of variables such as pH, excipients, storage temperature, and duration on particle formation. Data suggest that glass dissolution occurred during the storage in the liquid state, as shown by increased Si levels in solution. Upon freezing, the samples underwent freeze concentration and likely became supersaturated, which resulted in the appearance of visible silica particles upon thawing. The glass degradation described here is unique and differs from the more commonly reported delamination, defined by the presence of reflective, shard-like glass flakes in solution that are often termed lamellae. This case study underscores the importance of an early assessment (during formulation development) of potential incompatibility of the formulation with the primary container.

Rational design of lyophilized high concentration protein formulations-mitigating the challenge of slow reconstitution with multidisciplinary strategies.

An increasing number of protein therapies require chronic administration at high doses (>200 mg) by subcutaneous (sc) injection. Due to the injection volume limitation (<1.5 mL) associated with sc administration, high protein concentration formulations at or exceeding 100 mg/mL are required to achieve the dose. Development of a high concentration protein formulation can be challenging due to increased aggregation at higher concentration and/or chemical instability, which necessitates the development of lyophilized formulation for high protein concentration drug products. Unique challenges, such as long reconstitution time for a lyophilized high protein concentration drug product, can limit practical usage and commercial marketability of the product. In this paper, a systematic approach is presented to develop a lyophilized high concentration protein formulation. The focus is on achieving reasonable reconstitution times with multidisciplinary strategies. Many strategies have been shown to provide nominal improvement in reconstitution times, such as adding wetting agents in the diluents, incorporating high annealing steps in the lyophilization cycle and reconstituting under vacuum. The reconstitution strategy of reduced diluent volume, however, has enabled significant decrease in reconstitution time (4-7-fold) of lyophilized high protein concentration formulations.

Elucidation of degradants in acidic peak of cation exchange chromatography in an IgG1 monoclonal antibody formed on long-term storage in a liquid formulation.

PURPOSE: An IgG1 therapeutic monoclonal antibody showed an increase in acidic or pre-peak by cation exchange chromatography (CEX) at elevated temperatures, though stable at 2-8°C long-term storage in a liquid formulation. Characterization effort was undertaken to elucidate the degradants in CEX pre-peak and effect on biological activity. METHODS: Purified CEX fractions were collected and analyzed by peptide mapping, size exclusion, intact and reduced-alkylated reversed phase techniques. Biophysical characterization, isoelectric focusing and Isoquant analysis were also performed to determine nature of degradants. Bioassay and surface plasmon resonance experiments were performed to determine the impact on biological activity of the degradants. RESULTS: No major degradation due to oxidation, clipping or aggregation was detected; conformational differences between purified fractions observed were not significant. Sialic acid, N-terminal glutamine cyclization and glycation differences contributed to the CEX pre-peak in the mAb control sample; increase in CEX pre-peak at 25°C and higher was caused by additive degradation pathways of deamidation, related isomerization and clipping. CONCLUSIONS: The observed CEX pre-peak increase was caused by multiple degradations, especially deamidation and clipping. This elucidation of degradants in CEX peaks may apply to other therapeutic IgG1 monoclonal antibodies.

Silicone oil- and agitation-induced aggregation of a monoclonal antibody in aqueous solution.

Silicone oil, which is used as a lubricant or coating in devices such as syringes, needles and pharmaceutical containers, has been implicated in aggregation and particulation of proteins and antibodies. Aggregation of therapeutic protein products induced by silicone oil can pose a challenge to their development and commercialization. To systematically characterize the role of silicone oil on protein aggregation, the effects of agitation, temperature, pH, and ionic strength on silicone oil-induced loss of monomeric anti-streptavidin IgG 1 antibody were examined. Additionally, the influences of excipients polysorbate 20 and sucrose on protein aggregation were investigated. In the absence of agitation, protein absorbed to silicone oil with approximately monolayer coverage, however silicone oil did not stimulate aggregation during isothermal incubation unless samples were also agitated. A synergistic stimulation of aggregation by a combination of agitation and silicone oil was observed. Solution conditions which reduced colloidal stability of the antibody, as assessed by determination of osmotic second virial coefficients, accelerated aggregation during agitation with silicone oil. Polysorbate 20 completely inhibited silicone oil-induced monomer loss during agitation. A formulation strategy involving optimization of colloidal stability of the antibody as well as incorporation of surfactants such as polysorbate 20 is proposed to reduce silicone oil-induced aggregation of therapeutic protein products.

Human IgG2 antibodies display disulfide-mediated structural isoforms.

In this work, we present studies of the covalent structure of human IgG2 molecules. Detailed analysis showed that recombinant human IgG2 monoclonal antibody could be partially resolved into structurally distinct forms caused by multiple disulfide bond structures. In addition to the presently accepted structure for the human IgG2 subclass, we also found major structures that differ from those documented in the current literature. These novel structural isoforms are defined by the light chain constant domain (C(L)) and the heavy chain C(H)1 domain covalently linked via disulfide bonds to the hinge region of the molecule. Our results demonstrate the presence of three main types of structures within the human IgG2 subclass, and we have named these structures IgG2-A, -B, and -A/B. IgG2-A is the known classic structure for the IgG2 subclass defined by structurally independent Fab domains and hinge region. IgG2-B is a structure defined by a symmetrical arrangement of a (C(H)1-C(L)-hinge)(2) complex with both Fab regions covalently linked to the hinge. IgG2-A/B represents an intermediate form, defined by an asymmetrical arrangement involving one Fab arm covalently linked to the hinge through disulfide bonds. The newly discovered structural isoforms are present in native human IgG2 antibodies isolated from myeloma plasma and from normal serum. Furthermore, the isoforms are present in native human IgG2 with either kappa or lambda light chains, although the ratios differ between the light chain classes. These findings indicate that disulfide structural heterogeneity is a naturally occurring feature of antibodies belonging to the human IgG2 subclass.

Structural and functional characterization of disulfide isoforms of the human IgG2 subclass.

In the accompanying report ( Wypych, J., Li, M., Guo, A., Zhang, Z., Martinez, T., Allen, M. J., Fodor, S., Kelner, D. N., Flynn, G. C., Liu, Y. D., Bondarenko, P. V., Ricci, M. S., Dillon, T. M., and Balland, A. (2008) J. Biol. Chem. 283, 16194-16205 ), we have identified that the human IgG2 subclass exists as an ensemble of distinct isoforms, designated IgG2-A, -B, and -A/B, which differ by the disulfide connectivity at the hinge region. In this report, we studied the structural and functional properties of the IgG2 disulfide isoforms and compared them to IgG1. Human monoclonal IgG1 and IgG2 antibodies were designed with identical antigen binding regions, specific to interleukin-1 cell surface receptor type 1. In vitro biological activity measurements showed an increased activity of the IgG1 relative to the IgG2 in blocking interleukin-1beta ligand from binding to the receptor, suggesting that some of the IgG2 isoforms had lower activity. Under reduction-oxidation conditions, the IgG2 disulfide isoforms converted to IgG2-A when 1 m guanidine was used, whereas IgG2-B was enriched in the absence of guanidine. The relative potency of the antibodies in cell-based assays was: IgG1 > IgG2-A > IgG2 >> IgG2-B. This difference correlated with an increased hydrodynamic radius of IgG2-A relative to IgG2-B, as shown by biophysical characterization. The enrichment of disulfide isoforms and activity studies were extended to additional IgG2 monoclonal antibodies with various antigen targets. All IgG2 antibodies displayed the same disulfide conversion, but only a subset showed activity differences between IgG2-A and IgG2-B. Additionally, the distribution of isoforms was influenced by the light chain type, with IgG2lambda composed mostly of IgG2-A. Based on crystal structure analysis, we propose that IgG2 disulfide exchange is caused by the close proximity of several cysteine residues at the hinge and the reactivity of tandem cysteines within the hinge. Furthermore, the IgG2 isoforms were shown to interconvert in whole blood or a "blood-like" environment, thereby suggesting that the in vivo activity of human IgG2 may be dependent on the distribution of isoforms.

Optimization of a reversed-phase high-performance liquid chromatography/mass spectrometry method for characterizing recombinant antibody heterogeneity and stability.

An enhanced analytical RP-HPLC/MS method was developed for monitoring the stability and production of intact and fragmented monoclonal antibodies (MAbs). The use of high column temperatures (70-80 degrees C), organic solvents with high eluotropic strength coefficients (isopropyl and n-propyl alcohols), and Zorbax StableBond columns, were critical for good recovery and resolution of immunoglobulin G1 (IgG1) and IgG2 monoclonal antibodies. Using this method, cleavage products of a degraded IgG1 antibody were clearly separated and identified by in-line electrospray ionization time-of-flight (ESI-TOF) mass spectrometry generating exact masses and unique terminal ladder sequences. The glycosylation profile, including mapping of the terminal galactose and fucose heterogeneity of the N-linked sugars, was determined by mass spectrometry of intact MAbs. In addition, we discovered that several IgG2 MAbs exhibited greater structural heterogeneity compared to IgG1s. Mass spectral characterization data and reduction data suggested that the heterogeneity is disulfide related. This reversed-phase LC/MS method represents a key advancement in monitoring intact MAb production and stability.

Quantitative methods for developing Fc mutants with extended half-lives.

Fc mutants with increased binding affinity for the neonatal receptor, FcRn, exhibit increased half-lives in vivo, and represent an attractive means for extending the half-lives of therapeutic antibodies. The half-lives of other therapeutic molecules (e.g., proteins) may also be extended by conjugating them to Fc fragments, thus decreasing the frequency of patient injections and allowing the administration of low and potentially nontoxic concentrations of the therapeutics. To investigate the possibility for further increasing the half-life of Fc, a pair of quantitative methods is presented to complement combinatorial screening and in vivo testing. Specifically, a simple molecular modeling procedure was developed to predict relative Gibbs free energies of binding values (DeltaDeltaGbind) between Fc and FcRn across different mutants and species. This procedure was found to reasonably reproduce experimental DeltaDeltaGbind values from our experiments and the literature, and may be used as an initial screen to explore Fc sequence space more fully prior to experimental testing. In addition, a mathematical model of Fc trafficking was formulated and combined with a cell-level pulse-chase assay to obtain a quantitative recycling parameter in human T84 cells. This Fc recycling parameter was found to be correlated with binding affinity, but captures the pH dependent nature of the interaction between Fc and FcRn and may serve as an additional screen following combinatorial experiments.

Effects of excipients on the hydrogen peroxide-induced oxidation of methionine residues in granulocyte colony-stimulating factor.

PURPOSE: The objective of this study was to elucidate the different mechanisms of action of different excipients on the oxidation of Met1, Met122, Met127, and Met138 in granulocyte colony-stimulating factor (G-CSF) by using hydrogen peroxide as the oxidant. METHODS: The oxidation of Met1, Met127, and Met138 was quantified by peptide mapping analysis. The oxidation of Met122 has biphasic oxidation kinetics with a faster second phase. Therefore, the oxidation of Met122 was quantified by two different methods: peptide mapping analysis for the first phase of oxidation and direct reverse-phase HPLC for the second phase of oxidation. RESULTS: The current work reveals that the preferential excluding excipients sorbitol, sucrose, and trehalose, in the concentration range 0-30% (w/v), and the preferential binding excipients urea and guanidine hydrochloride, in the concentration range 0-0.8 M, do not affect the oxidation of methionine residues in G-CSF at pH 4.5. The chelating agents citrate and EDTA have different effects on the rates of oxidation of methionine residues in G-CSF. At low concentrations, citrate decreases the rates, while at high concentrations, citrate increases the rates. EDTA decreases the rates of oxidation of methionine residues in G-CSF, such that its effect becomes more and more as its concentration is increased from 0 to 200 mM. The efficacy of EDTA on the rates of oxidation of the four methionine residues in G-CSF follows the order Met122 > Met127 > Met138 > Met1. CONCLUSIONS: Our results indicate that EDTA can protect the methionine residues in G-CSF against oxidation induced by hydrogen peroxide. The more exposed the methionine residue is, the more difficult it is to be protected by EDTA. The mechanism may be due to the specific ion binding of EDTA to proteins.

Common structural stability properties of 4-helical bundle cytokines: possible physiological and pharmaceutical consequences.

Biological activity and clinical efficacy of a therapeutic protein are contingent upon the structural stability, bioavailability, and clearance rates of the protein. In this review, we examine the class of 4-helical bundle cytokines for common stability properties that may affect biological structure and efficacy. Three critical stability features that are hallmarks of this class of cytokines are the pH dependence of structural stability, the presence of folding intermediates, and the population of aggregation intermediates. We hypothesize that certain cytokines have increased stability in acid to enable receptor-mediated clearance, and that reengineering local endocytic trafficking can result in dramatic improvements in global serum half-life and therapeutic efficacy. The common feature of folding and aggregation intermediates has implications on kinetic folding pathways, membrane permeability, solubility, and precipitation properties that are critical for commercial production, formulation, and delivery. Understanding the structural stability properties of this class of cytokines may help elucidate new approaches to improving therapeutic efficacy.

A comprehensive picture of non-site specific oxidation of methionine residues by peroxides in protein pharmaceuticals.

In this article, a comprehensive picture of the oxidation of protein pharmaceuticals by peroxides is developed based on our earlier computational and experimental studies. We propose a new mechanism, the water-mediated mechanism, for the oxidation of methionine residues, and it has been shown to satisfy all available experimental data including new data reported here. Based on the water-mediated mechanism, we found a structural property, average 2-shell water coordination number, that correlates well to the relative rates of oxidation of methionine groups. We used this to study the oxidation of granulocyte colony-stimulating factor (G-CSF) and 1-34 human parathyroid hormone hPTH(1-34). We believe that this comprehensive picture should aid researchers in the pharmaceutical sciences to develop solvent formulations for therapeutic proteins in a more rational way.

Effects of antioxidants on the hydrogen peroxide-mediated oxidation of methionine residues in granulocyte colony-stimulating factor and human parathyroid hormone fragment 13-34.

PURPOSE: The effects and mechanisms of different antioxidants, methionine, glutathione, acetylcysteine, and ascorbic acid (AscH2), on the oxidation of methionine residues in granulocyte colony-stimulating factor (G-CSF) and human parathyroid hormone fragment 13-34 (hPTH 13-34) by hydrogen peroxide (H2O2) were quantified and analyzed. METHODS: The rates of oxidation of methionine residues in G-CSF were determined by peptide mapping analyses, and the oxidation of methionine residue in hPTH 13-34 was quantified by reverse-phase HPLC. RESULTS: At pH 4.5, free methionine reduces, glutathione and acetylcysteine have no obvious effect on, and AscH2 promotes the rates of oxidation of methionine residues in G-CSF. The H2O2-induced oxidation rate constants for free methionine, acetylcysteine, and glutathione at pH 4.5 were measured to be 32.07, 1.00, and 1.63 M(-1)h(-1), respectively, while the oxidation rate constant for Met1, the most readily oxidizable methionine residue in G-CSF, is 13.95 M(-1)h(-1). Therefore, the different effects of free methionine, acetylcysteine, and glutathione on the rates of oxidation of methionine residues in G-CSF are consistent with their different reactivity toward oxidation by H2O2. By using hPTH 13-34, the effect of AscH2 on the H2O2-induced oxidation of methionine residue was quantified, and the mechanisms involved were proposed. Because of the presence of trace transition metal ions in solution, at low concentrations, AscH2 is prone to be a prooxidant, increasing the hydroxyl radical (.OH) production rate via Fenton-type reactions. In addition to peroxide oxidation, these radicals lead to the degradation of hPTH 13-34 to smaller peptide fragments. At high concentrations, AscH2 tends to act as an OH scavenger. EDTA inhibits OH production and thus eliminates the degradation of hPTH 13-34 by forming complexes with transition metal ions. However, the rate of oxidation of the methionine residue in hPTH 13-34 increases as the concentration of AscH2 is increased from 0 to 200 mM, and the reason for this is still not clear. CONCLUSIONS: Our results demonstrate that free methionine is an effective antioxidant to protect G-CSF against methionine oxidation at pH 4.5. Acetylcysteine and glutathione are not effective antioxidants at pH 4.5. Their oxidation rates at different pH values imply that they would be much more effective antioxidants than free methionine at alkaline conditions. AscH2 is a powerful electron donor. It acts as a prooxidant in the conditions in this study and is unlikely to prevent oxidation by H2O2 in protein formulation, whether or not EDTA is present.

pH Dependence of structural stability of interleukin-2 and granulocyte colony-stimulating factor.

After a cytokine binds to its receptor on the cell surface (pH approximately 7), the complex is internalized into acidic endosomal compartments (pH approximately 5-6), where partially unfolded intermediates can form. The nature of these structural transitions was studied for wild-type interleukin-2 (IL-2) and wild-type granulocyte colony-stimulating factor (G-CSF). A noncoincidence of denaturation transitions in the secondary and tertiary structure of IL-2 and tertiary structural perturbations in G-CSF suggest the presence of an intermediate state for each, a common feature of this structural family of four-helical bundle proteins. Unexpectedly, both IL-2 and G-CSF display monotonic increases in stability as the pH is decreased from 7 to 4. We hypothesize that such cytokines with cell-based clearance mechanisms in vivo may have evolved to help stabilize endosomal complexes for sorting to lysosomal degradation. We show that mutants of both IL-2 and G-CSF have differential stabilities to their wild-type counterparts as a function of pH, and that these differences may explain the differences in ligand trafficking and depletion. Further understanding of the structural changes accompanying unfolding may help guide cytokine design with respect to ligand binding, endocytic trafficking, and, consequently, therapeutic efficacy.