Margaret Speed Ricci

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 (CL) and the heavy chain CH1 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 (CH1-CL-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 κ or λ 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-1β 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 IgG2λ 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.

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.

Conformational implications of an inversed pH-dependent antibody aggregation

Antibody formulation development relies on accelerated stability data at elevated temperatures to optimize formulation parameters. However, the pH- and temperature-dependence of aggregation is complicated for antibody formulations. In this study, a human monoclonal IgG2 antibody exhibited typical pH-dependent dimer formation under normal storage conditions (4 and/or 29 degrees C). However, an inversed pH-dependence was discovered for high molecular weight aggregate formation at elevated temperatures (37 degrees C). The different stability profiles exhibited at the various storage conditions resulted in nonlinearity of the Arrhenius kinetics. Thermal unfolding at or below 37 degrees C was not evident by differential scanning calorimetry. Enriched populations of the structural isoforms of the IgG2 subclass were tested for their unique temperature and pH-dependence of aggregation. The Arrhenius kinetics of aggregation for each of the individual IgG2 isoforms was also nonlinear. However, the temperature-dependence of clipping suggested that clip-mediated aggregation was responsible for the increased higher order aggregates at low pH and elevated temperatures. Unique clip species resulting from the conformational differences between the IgG2 isoforms lead to increased aggregation. These results have implications on the mechanisms of antibody aggregation and on the validity of accelerated data to predict shelf-life accurately.

Effects of Antioxidants on the Hydrogen Peroxide-Mediated Oxidation of Methionine Residues in Granulocyte Colony-Stimulating Factor and Human Parathyroid Hormone Fragment 13-34

No HeadingPurpose.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-1h-1, respectively, while the oxidation rate constant for Met1, the most readily oxidizable methionine residue in G-CSF, is 13.95 M−1h−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.

Elucidation of Degradants in Acidic Peak of Cation Exchange Chromatography in an IgG1 Monoclonal Antibody Formed on Long-Term Storage in a Liquid Formulation

ABSTRACTPurposeAn 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.MethodsPurified 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.ResultsNo 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.ConclusionsThe 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.

Effects of Excipients on the Hydrogen Peroxide-Induced Oxidation of Methionine Residues in Granulocyte Colony-Stimulating Factor

No HeadingPurpose.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.

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 ∼7), the complex is internalized into acidic endosomal compartments (pH ∼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.

Influence of Process Conditions on the Crystallization and Transition of Metastable Mannitol Forms in Protein Formulations During Lyophilization

ABSTRACTPurposeTo study the impact of different process conditions and formulation compositions on metastable mannitol forms in protein formulations during lyophilization.MethodsMannitol was studied with and without other formulation components. A cryostage was used to mimic the different processing steps during lyophilization. The different mannitol forms were monitored and quantified with an in situ Raman spectroscopic method. In addition, a Raman imaging method was developed to characterize the spatial distribution of mannitol forms in final lyophilization samples from the freeze-drying stage.ResultsAmorphous mannitol was observed during fast cooling (10°C/min) and with the addition of other formulation component. Amorphous mannitol crystallized into mainly δ and hemihydrate forms during annealing at −20°C. Under vacuum without moisture, dried amorphous mannitol could transform to mainly α form at 45°C and greater. The transformation mechanism of the hemihydrate mannitol was similar to that of amorphous form.ConclusionMannitol tends to crystallize into stable crystalline forms by itself, but the addition of lyoprotectant (e.g. sucrose) and protein helps stabilize the metastable forms (hemihydrate and amorphous). The metastable forms are capable of transforming into mixtures of different forms, with heat and moisture being the critical processing factors.

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.