A high-resolution multi-attribute method for product characterization, process characterization, and quality control of therapeutic proteins.

During the manufacturing of therapeutic proteins, Critical Quality Attributes (CQAs) have been monitored by conventional methods, such as cation exchange chromatography (CEX), reduced capillary electrophoresis-sodium dodecyl sulfate (rCE-SDS), and 1,2-diamino-4,5-methylenedioxybenzene (DMB) labelling method. The conventional methods often generate individual peaks that contain multiple components, which may obscure the detection and the quantification of individual critical quality attributes (CQAs). Alternatively, Multi-Attribute Method (MAM) enables detection and quantification of specific CQAs. A high resolution MAM has been developed and qualified to replace several conventional methods in monitoring product quality attributes, such as oxidation, deamidation, clipping, and glycosylation. The qualified MAM was implemented in process characterization, as well as release and stability assays in quality control (QC). In combination with a design-of-experiments (DoE), the MAM method identified multivariate process parameter ranges that yield acceptable CQA level, which provides operational flexibility for manufacturing.

Identification of critical chemical modifications and paratope mapping by size exclusion chromatography of stressed antibody-target complexes.

Therapeutic proteins including antibodies and Fc-fusion proteins undergo a large number of chemical modifications during cell culture, purification, storage and in human circulation. They are also exposed to harsh conditions during stress studies, including elevated temperature, extremes of pH, forced oxidation, physiological pH, UV light to assess the possible degradation pathways and suitability of methods for detecting them. Some of these modifications are located on residues in binding regions, leading to loss of binding and potency and classified as critical quality attributes. Currently, criticality of modifications is assessed by a laborious process of collecting antibody fractions from the soft chromatography techniques ion exchange and hydrophobic interaction chromatography and characterizing the fractions one-by-one for potency and chemical modifications. Here, we describe a method for large-scale, parallel identification of all critical chemical modifications in one experiment. In the first step, the antibody is stressed by one or several stress methods. It is then mixed with target protein and separated by size-exclusion chromatography (SEC) on bound antibody-target complex and unbound antibody. Peptide mapping of fractions and statistical analysis are performed to identify modifications on amino acid residues that affect binding. To identify the modifications leading to slight decreases in binding, competitive SEC of antibody and antigen mixtures was developed and described in a companion study by Shi et al, where target protein is provided at lower level, below the stoichiometry. The newly described method was successfully correlated to crystallography for assessing criticality of chemical modifications and paratope mapping. It is more sensitive to low-level modifications, better streamlined and platform ready.

Identification of critical chemical modifications by size exclusion chromatography of stressed antibody-target complexes with competitive binding.

Chemical modifications (attributes) in the binding regions of stressed therapeutic proteins may affect binding to target and efficacy of therapeutic proteins. The method presented here describes the criticality assessment of therapeutic antibody modifications by size-exclusion chromatography (SEC) of competitive binding between a stressed antibody and its target, human epidermal growth factor receptor-2 (HER2), followed by SEC fractionation and peptide mapping characterization of bound and unbound antibodies. When stressed antibody and its target were mixed at a stoichiometric molar ratio of 1:2, only antibody-receptor complex eluted from SEC, indicating that binding was not decreased to break the complex. When a smaller amount of the receptor was provided (1:1), the antibody species with modifications reducing binding eluted as unbound from SEC, while the antibody-receptor complex eluted as the bound fraction. Peptide mapping revealed ratios of modifications between unbound and bound fractions. Statistical analysis after triplicate measurements (n = 3) indicated that heavy chain (HC) D102 isomerization and light chain (LC) N30 deamidation were four-fold higher in unbound fraction with high statistical significance. Although HC N55 deamidation and M107 oxidation were also abundant, they were not statistically different between unbound and bound. Our findings agree with previously published potency measurements of collected CEX fractions and the crystal structure of antibody and HER2. Overall, competitive SEC of stressed antibody-receptor mixture followed by peptide mapping is a useful tool in revealing critical residues and modifications involved in the antibody-target binding, even if they elute as a complex from SEC when mixed at 1:2 stoichiometric ratio.

Characterization of therapeutic proteins by cation exchange chromatography-mass spectrometry and top-down analysis.

Recently, cation exchange chromatography (CEX) using aqueous volatile buffers was directly coupled with mass spectrometry (MS) and applied for intact analysis of therapeutic proteins and antibodies. In our study, chemical modifications responsible for charge variants were identified by CEX-UV-MS for a monoclonal antibody (mAb), a bispecific antibody, and an Fc-fusion protein. We also report post-CEX column addition of organic solvent and acid followed by mixing at elevated temperatures, which unfolded proteins, increased ion intensity (sensitivity) and facilitated top-down analysis. mAb stressed by hydrogen peroxide oxidation was used as a model system, which produced additional CEX peaks. The on-line CEX-UV-MS top-down analysis produced gas-phase fragments containing one or two methionine residues. Oxidation of some methionine residues contributed to earlier (acidic), some to later (basic) eluting peaks, while oxidation of other residues did not change CEX elution. The abundance of the oxidized and non-oxidized fragment ions also allowed estimation of the oxidation percentage of different methionine residues in stressed mAb. CEX-UV-MS measurement revealed a new intact antibody proteoform at 5% that eluted as a basic peak and included paired modifications: high-mannose glycosylation and remaining C-terminal lysine residue (M5/M5 + K). This finding was confirmed by peptide mapping and on-column disulfide reduction coupled with reversed-phase liquid chromatography - top-down MS analysis of the collected basic peak. Overall, our results demonstrate the utility of the on-line method in providing site-specific structural information of charge modifications without fraction collection and laborious peptide mapping.

Comparison of Acid Titration, Conductivity, Flame Photometry, ICP-MS, and Accelerated Lamellae Formation Techniques in Determining Glass Vial Quality.

Certain types of glass vials used as primary containers for liquid formulations of biopharmaceutical drug products have been observed with delamination that produced small glass like flakes termed lamellae under certain conditions during storage. The cause of this delamination is in part related to the glass surface defects, which renders the vials susceptible to flaking, and lamellae are formed during the high-temperature melting and annealing used for vial fabrication and shaping. The current European Pharmacopoeia method to assess glass vial quality utilizes acid titration of vial extract pools to determine hydrolytic resistance or alkalinity. Four alternative techniques with improved throughput, convenience, and/or comprehension were examined by subjecting seven lots of vials to analysis by all techniques. The first three new techniques of conductivity, flame photometry, and inductively coupled plasma mass spectrometry measured the same sample pools as acid titration. All three showed good correlation with alkalinity: conductivity (R(2) = 0.9951), flame photometry sodium (R(2) = 0.9895), and several elements by inductively coupled plasma mass spectrometry [(sodium (R(2) = 0.9869), boron (R(2) = 0.9796), silicon (R(2) = 0.9426), total (R(2) = 0.9639)]. The fourth technique processed the vials under conditions that promote delamination, termed accelerated lamellae formation, and then inspected those vials visually for lamellae. The visual inspection results without the lot with different processing condition correlated well with alkalinity (R(2) = 0.9474). Due to vial processing differences affecting alkalinity measurements and delamination propensity differently, the ratio of silicon and sodium measurements from inductively coupled plasma mass spectrometry was the most informative technique to assess overall vial quality and vial propensity for lamellae formation. The other techniques of conductivity, flame photometry, and accelerated lamellae formation condition may still be suitable for routine screening of vial lots produced under consistent processes. LAY ABSTRACT: Recently, delamination that produced small glass like flakes termed lamellae has been observed in glass vials that are commonly used as primary containers for pharmaceutical drug products under certain conditions during storage. The main cause of these lamellae was the quality of the glass itself related to the manufacturing process. Current European Pharmacopoeia method to assess glass vial quality utilizes acid titration of vial extract pools to determine hydrolytic resistance or alkalinity. As alternative to the European Pharmacopoeia method, four other techniques were assessed. Three new techniques of conductivity, flame photometry, and inductively coupled plasma mass spectrometry measured the vial extract pool as acid titration to quantify quality, and they demonstrated good correlation with original alkalinity. The fourth technique processed the vials under conditions that promote delamination, termed accelerated lamellae formation, and the vials were then inspected visually for lamellae. The accelerated lamellae formation technique also showed good correlation with alkalinity. Of the new four techniques, inductively coupled plasma mass spectrometry was the most informative technique to assess overall vial quality even with differences in processing between vial lots. Other three techniques were still suitable for routine screening of vial lots produced under consistent processes.

Do not drop: mechanical shock in vials causes cavitation, protein aggregation, and particle formation.

Industry experience suggests that g-forces sustained when vials containing protein formulations are accidentally dropped can cause aggregation and particle formation. To study this phenomenon, a shock tower was used to apply controlled g-forces to glass vials containing formulations of two monoclonal antibodies and recombinant human growth hormone (rhGH). High-speed video analysis showed cavitation bubbles forming within 30 µs and subsequently collapsing in the formulations. As a result of echoing shock waves, bubbles collapsed and reappeared periodically over a millisecond time course. Fluid mechanics simulations showed low-pressure regions within the fluid where cavitation would be favored. A hydroxyphenylfluorescein assay determined that cavitation produced hydroxyl radicals. When mechanical shock was applied to vials containing protein formulations, gelatinous particles appeared on the vial walls. Size-exclusion chromatographic analysis of the formulations after shock did not detect changes in monomer or soluble aggregate concentrations. However, subvisible particle counts determined by microflow image analysis increased. The mass of protein attached to the vial walls increased with increasing drop height. Both protein in bulk solution and protein that became attached to the vial walls after shock were analyzed by mass spectrometry. rhGH recovered from the vial walls in some samples revealed oxidation of Met and/or Trp residues.

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.

Novel Mechanism of Glass Delamination in Type 1A Borosilicate Vials Containing Frozen Protein Formulations.

Storing protein formulations in the frozen state typically improves stability during long-term storage as a drug substance or as a drug product. The frozen state minimizes chemical degradation and physical instability. However, the frozen state is not an optimal storage condition for the glass vial itself. A significant issue was observed when small, flake-like pieces of glass particles (lamellae) appeared in vials containing thawed protein product. The occurrence of glass particles during freeze-thaw results in product rejection and potentially, adverse events. In recent years, glass flakes due to chemical delamination have been observed in parenteral liquid formulations after long-term storage, resulting in a number of product recalls. In this study, for the first time, glass delamination is reported in pharmaceutical glass vials containing frozen protein formulation, caused by a novel mechanism involving thermally-induced mechanical stress. In this article, a monoclonal antibody drug product in glass vials and the corresponding placebo vials were studied to identify the contributing factors from the freeze-thaw process, such as freezing temperature, the presence or absence of protein, and other handling conditions. Freezing temperature was found to be the most critical factor. Glass lamellae were only observed when the products were frozen to -70 °C, while freezing only to -30 °C did not cause any lamellae formation even after multiple freeze-thaw cycles. Protein concentration and the handling of the vials were also identified as contributing factors. A concentration gradient which formed after freeze-thaw induced a higher rate of lamellae occurrence in a subsequent freeze-thaw cycle compared to vials without the concentration gradient. Analyses by Fourier transform infrared spectroscopy and scanning electron microscopy/energy dispersive spectroscopy confirmed that the flake-like lamellae were thin, flat glass particles. Defects corresponding to the glass flakes were observed by scanning electron microscopy on the inner surface of the vials that contained lamellae. In addition, inductively coupled plasma mass spectrometry testing did not show elevated levels of silicon in the drug product solution, suggesting that the glass lamellae formed in the frozen vials was a local, event-based phenomenon rather than silica dissolution from the product contact surface or glass degradation caused by corrosive attack. These findings can be explained by the same thermally-induced mechanical stress which caused vial breakage. Frozen protein formulations contracted below -30 °C, causing an inward glass deformation and a subsequent rapid movement of the glass when the frozen plug of drug product solution separated from the vial inner surface at approximately -50 to -60 °C. The mechanical stress released during this separation caused vial breakage. The incidence of vial breakage increased with more concentrated product and higher fill volume-to-vial volume ratios. The same mechanism applies to lamellae formation. As the rapid surface separation occurred, small, thin pieces of glass were pulled from the glass surface by the frozen plug, and, as a result, glass lamellae particles appeared in the drug product solution after thawing. LAY ABSTRACT: In recent years, glass flakes have been observed in parenteral liquid formulations due to chemical delamination during long-term storage, resulting in a number of product recalls. In our study, we discovered a novel mechanism of glass delamination in vials containing frozen protein formulations. This glass delamination mechanism has never been reported before, and we believe this work will benefit the pharmaceutical scientific community, especially the biotechnology and parenteral drug industries. Storing protein formulations in the frozen state typically improves stability during long-term storage as a drug substance or as a drug product. The frozen state minimizes chemical degradation and physical instability. However, the frozen state is not an optimal storage condition for the glass vial itself. In this study, we observed that after thawing, small, flake-like pieces of glass particles (i.e., lamellae) appeared in vials containing frozen protein formulation. To investigate the root cause, we performed a series of freeze-thaw experiments and characterized the lamellae particles, the vial inner surface, and the elemental composition of the solution. The root cause was determined to be mechanical stress caused by thermal contraction of frozen protein formulations below -30 °C. This contraction caused an inward glass deformation on the vial sidewall and, subsequently, the glass vial surface abruptly separated from frozen protein formulation. Under this mechanical stress, small, thin glass pieces were peeled from the vial inner surface by the frozen formulation, causing lamellae formation. The experimental design and results leading to the discovery of the novel glass delamination mechanism are presented in detail in this article.

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.

Influence of process conditions on the crystallization and transition of metastable mannitol forms in protein formulations during lyophilization.

PURPOSE: To study the impact of different process conditions and formulation compositions on metastable mannitol forms in protein formulations during lyophilization. METHODS: Mannitol 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. RESULTS: Amorphous 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. CONCLUSION: Mannitol 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.

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.

A molecular mechanism of hydrolysis of peptide bonds at neutral pH using a model compound.

The covalent stability of peptide bonds is a critical aspect of biological chemistry and therapeutic protein applications. In this computational study, the hydrolytic reaction of peptide bonds at neutral pH was studied using a model compound, N-MAA. The most probable reaction pathway and intermediate(s) involved are controversial in previous studies. In addition, most previous computational studies focus on the energetics of chemical species involved, rather than providing a dynamic picture of the reaction process in aqueous conditions. However, fluctuations at finite temperatures are quite important, as we show. Thus, a path sampling method was used to generate an ensemble of trajectories according to their statistical weights in trajectory space. An ab initio molecular dynamics technique was applied to advance the time of the reaction in order to collect trajectories. The likelihood maximization procedure and its modification were used in extracting dynamically relevant degrees of freedom in the system, and approximations of the reaction coordinate were compared. It was found that this hydrolytic reaction is very complex because it involves many degrees of freedom. The reaction coordinate C-O distance previously assumed was found to be inadequate in describing the dynamic progress of the reaction. In addition to affecting atoms directly involved in bond-making and -breaking processes, the water network also has determining effects on the hydrolytic reaction, a fact which is manifest in the expression of the best one-dimensional reaction coordinate that we found, which includes five geometric quantities. p(B) histograms were computed to verify the results of the likelihood maximization and to evaluate the accuracy of our best reaction coordinate to the "true" reaction coordinate. The relation with previous suggested reaction pathways and intermediate(s) is discussed in terms of computational system, method, and accuracy.

Molecular mechanism of acid-catalyzed hydrolysis of peptide bonds using a model compound.

The stability of peptide bonds is a critical aspect of biological chemistry and therapeutic protein applications. Recent studies found elevated nonenzymatic hydrolysis in the hinge region of antibody molecules, but no mechanism was identified. As a first step in providing a mechanistic interpretation, this computational study examines the rate-determining step of the hydrolytic reaction of a peptide bond under acidic pH by a path sampling technique using a model compound N-MAA. Most previous computational studies did not include explicit water molecules, whose effects are significant in solution chemistry, nor did they provide a dynamic picture for the reaction process in aqueous conditions. Because no single trajectory can be used to describe the reaction dynamics due to fluctuations at finite temperatures, a variant version of the transition path sampling technique, the aimless shooting algorithm, was used to sample dynamic trajectories and to generate an ensemble of transition trajectories according to their statistical weights in the trajectory space. Each trajectory was computed as the time evolution of the molecular system using the Car-Parrinello molecular dynamics technique. The likelihood maximization procedure and its modification were used in extracting dynamically relevant degrees of freedom in the system, and approximations of the reaction coordinate were compared. Its low log-likelihood score and poor p(B) histogram indicate that the C-O distance previously assumed as the reaction coordinate for the rate-determining step is inadequate in describing the dynamics of the reaction. More than one order parameter in a candidate set including millions of geometric quantities was required to produce a convergent reaction coordinate model; its involvement of many degrees of freedom suggests that this hydrolytic reaction step is very complex. In addition to affecting atoms directly involved in bond-making and -breaking processes, the water network also has determining effects on the hydrolytic reaction, a fact that is manifest in the expression of the one-dimensional best-ranked reaction coordinate, which includes three geometric quantities. The p(B) histograms were computed to verify the results of the likelihood maximization and to verify the accuracy of approximation to the "true" reaction coordinate.

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.

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.

The LC/MS analysis of glycation of IgG molecules in sucrose containing formulations.

Glycation of a recombinant monoclonal IgG2 molecule, in sucrose containing liquid formulations, was studied using reversed-phase LC/MS analysis of the intact IgG, the F(ab')2 fragments and after complete tryptic digestion. The extent of glycation in sucrose containing formulations was monitored at different temperatures over a period of 21 months using the Hexose index (Hex(I)). Hex(I) represents the average number of hexose molecules per molecule of IgG and was calculated by using the intensity values of peaks corresponding to hexose isoforms in the deconvoluted mass spectra. The rate of glycation in mildly acidic sucrose containing formulations was proportional to the incubation temperature. No glycation was observed in sucrose containing formulations incubated at 4 degrees C even after 18 months. However, when the same formulations were incubated at 37 degrees C glycation was observed after just 1 month. The glycation sites were mapped to 10 lysine residues distributed throughout the molecule. The amino terminal end of the light chain was also shown to contain glycation. The surface accessibility of the lysine side chain could influence its susceptibility to glycation.

Oxidation of methionine residues in recombinant human interleukin-1 receptor antagonist: implications of conformational stability on protein oxidation kinetics.

Oxidation of methionine residues is involved in several biochemical processes and in degradation of therapeutic proteins. The relationship between conformational stability and methionine oxidation in recombinant human interleukin-1 receptor antagonist (rhIL-1ra) was investigated to document how thermodynamics of unfolding affect methionine oxidation in proteins. Conformational stability of rhIL-1ra was monitored by equilibrium urea denaturation, and thermodynamic parameters of unfolding (DeltaGH2O, m, and Cm) were estimated at different temperatures. Methionine oxidation induced by hydrogen peroxide at varying temperatures was monitored during "coincubation" of rhIL-1ra with peptides mimicking specific regions of the reactive methionine residues in the protein. The coincubation study allowed estimation of oxidation rates in protein and peptide at each temperature from which normalized oxidation rate constants and activation energies were calculated. The rate constants for buried Met-11 in the protein were lower than for methionine in the peptide with an associated increase in activation energy. The rate constants and activation energy of solvent exposed methionines in protein and peptide were similar. The results showed that conformational stability, monitored using the Cm value, has an effect on oxidation rates of buried methionines. The rate constant of buried Met-11 correlated well with the Cm value but not DeltaGH2O. No correlation was observed for the oxidation rates of solvent-exposed methionines with any thermodynamic parameters of unfolding. The findings presented have implications in protein engineering, in design of accelerated stability studies for protein formulation development, and in understanding disease conditions involving protein oxidation.