Interactions between Therapeutic Proteins and Acrylic Acid Leachable.

Leachables are chemical compounds that migrate from manufacturing equipment, primary containers and closure systems, and packaging components into biopharmaceutical and pharmaceutical products. Acrylic acid (at concentration around 5 µg/mL) was detected as leachable in syringes from one of the potential vendors (X syringes). In order to evaluate the potential impact of acrylic acid on therapeutic proteins, an IgG 2 molecule was filled into a sterilized X syringe and then incubated at 45 °C for 45 days in a pH 5 acetate buffer. We discovered that acrylic acid can interact with proteins at three different sites: (1) the lysine side chain, (2) the N-terminus, and (3) the histidine side chain, by the Michael reaction. In this report, the direct interactions between acrylic acid leachable and a biopharmaceutical product were demonstrated and the reaction mechanism was proposed. Even thought a small amount (from 0.02% to 0.3%) of protein was found to be modified by acrylic acid, the modified protein can potentially be harmful due to the toxicity of acrylic acid. After being modified by acrylic acid, the properties of the therapeutic protein may change due to charge and hydrophobicity variations. LAY ABSTRACT: Acrylic acid was detected to migrate from syringes (Vendor X) into a therapeutic protein solution (at a concentration around 5 µg/mL). In this study, we discovered that acrylic acid can modify proteins at three different sites: (1) the lysine side chain, 2) the N-terminus, and 3) the histidine side chain, by the Michael reaction. In this report, the direct interactions between acrylic acid leachable and a biopharmaceutical product were demonstrated and the reaction mechanism was proposed.

Comparison of high-throughput biophysical methods to identify stabilizing excipients for a model IgG2 monoclonal antibody: conformational stability and kinetic aggregation measurements.

The overall conformational stability of a model IgG2 monoclonal antibody (mAb) was examined as a function of temperature and pH using an empirical phase diagram approach. Stabilizing excipients were then identified based on high-throughput methods including (1) kinetic studies measuring aggregation via increases in optical density and (2) thermally induced structural transitions as measured by differential scanning calorimetry (DSC) and fluorescence spectroscopy. The kinetic profiles of antibody aggregation at 65 °C were pH dependent and correlated well with pH effects on secondary and tertiary structural transitions due to heat stress. For the screening of stabilizing excipients, the inhibition of the rate of protein aggregation at pH 4.5 at 65°C, as represented by changes in optical density, was shown to have a clear trend with a modest correlation coefficient compared with the stabilizing effect of the same excipients on the conformational stability of the antibody as measured by DSC and tryptophan fluorescence spectroscopy. These results demonstrate the utility of combining high-throughput data from protein aggregation kinetic experiments and conformational stability studies to identify stabilizing excipients that minimize the physical degradation of an IgG2 mAb.

Determination of the dipole moments of RNAse SA wild type and a basic mutant.

In this study, we report the effects of acidic to basic residue point mutations (5K) on the dipole moment of RNAse SA at different pHs. Dipole moments were determined by measuring solution capacitance of the wild type (WT) and the 5K mutant with an impedance analyzer. The dipole moments were then (1) compared with theoretically calculated dipole moments, (2) analyzed to determine the effect of the point mutations, and (3) analyzed for their contribution to overall protein-protein interactions (PPI) in solution as quantitated by experimentally derived second virial coefficients. We determined that experimental and calculated dipoles were in reasonable agreement. Differences are likely due to local motions of residue side chains, which are not accounted for by the calculated dipole. We observed that the proteins' dipole moments increase as the pH is shifted further from their isoelectric points and that the wild-type dipole moments were greater than those of the 5K. This is likely due to an increase in the proportion of one charge (either negative or positive) relative to the other. A greater charge disparity corresponded to a larger dipole moment. Finally, the larger dipole moments of the WT resulted in greater attractive overall PPI for that protein as compared to the 5K.

Effective charge measurements reveal selective and preferential accumulation of anions, but not cations, at the protein surface in dilute salt solutions.

Specific-ion effects are ubiquitous in nature; however, their underlying mechanisms remain elusive. Although Hofmeister-ion effects on proteins are observed at higher (>0.3 M) salt concentrations, in dilute (<0.1 M) salt solutions nonspecific electrostatic screening is considered to be dominant. Here, using effective charge (Q*) measurements of hen-egg white lysozyme (HEWL) as a direct and differential measure of ion-association, we experimentally show that anions selectively and preferentially accumulate at the protein surface even at low (<100 mM) salt concentrations. At a given ion normality (50 mN), the HEWL Q* was dependent on anion, but not cation (Li(+), Na(+), K(+), Rb(+), Cs(+), GdnH(+), and Ca(2+)), identity. The Q* decreased in the order F(-) > Cl(-) > Br(-) > NO(3)(-) ∼ I(-) > SCN(-) > ClO(4)(-) ≫ SO(4)(2-), demonstrating progressively greater binding of the monovalent anions to HEWL and also show that the SO(4)(2-) anion, despite being strongly hydrated, interacts directly with the HEWL surface. Under our experimental conditions, we observe a remarkable asymmetry between anions and cations in their interactions with the HEWL surface.

Diffusion and sedimentation interaction parameters for measuring the second virial coefficient and their utility as predictors of protein aggregation.

The concentration-dependence of the diffusion and sedimentation coefficients (k(D) and k(s), respectively) of a protein can be used to determine the second virial coefficient (B2), a parameter valuable in predicting protein-protein interactions. Accurate measurement of B2 under physiologically and pharmaceutically relevant conditions, however, requires independent measurement of k(D) and k(s) via orthogonal techniques. We demonstrate this by utilizing sedimentation velocity (SV) and dynamic light scattering (DLS) to analyze solutions of hen-egg white lysozyme (HEWL) and a monoclonal antibody (mAb1) in different salt solutions. The accuracy of the SV-DLS method was established by comparing measured and literature B2 values for HEWL. In contrast to the assumptions necessary for determining k(D) and k(s) via SV alone, k(D) and ks were of comparable magnitudes, and solution conditions were noted for both HEWL and mAb1 under which 1), k(D) and k(s) assumed opposite signs; and 2), k(D) ≥k(s). Further, we demonstrate the utility of k(D) and k(s) as qualitative predictors of protein aggregation through agitation and accelerated stability studies. Aggregation of mAb1 correlated well with B2, k(D), and k(s), thus establishing the potential for k(D) to serve as a high-throughput predictor of protein aggregation.

High-throughput dynamic light scattering method for measuring viscosity of concentrated protein solutions.

We propose a new method to measure the viscosity of concentrated protein solutions in a high-throughput format. This method measures the apparent hydrodynamic radius of polystyrene beads with known sizes using a dynamic light scattering (DLS) system with a microplate reader. Glycerol solution viscosities obtained by the DLS method were in good agreement with those reported in the literature. Viscosity of the solutions of two monoclonal antibody molecules was acquired using both DLS and cone-and-plate techniques, and the results were comparable. The DLS method described here has the potential to be used in many aspects of protein characterization.

Denaturant-dependent conformational changes in a beta-trefoil protein: global and residue-specific aspects of an equilibrium denaturation process.

Conformational properties of the folded and unfolded ensembles of human interleukin-1 receptor antagonist (IL-1ra) are strongly denaturant-dependent as evidenced by high-resolution two-dimensional nuclear magnetic resonance (NMR), limited proteolysis, and small-angle X-ray scattering (SAXS). The folded ensemble was characterized in detail in the presence of different urea concentrations by (1)H-(15)N HSQC NMR. The beta-trefoil fold characteristic of native IL-1ra was preserved until the unfolding transition region beginning at 4 M urea. At the same time, a subset of native resonances disappeared gradually starting at low denaturant concentrations, indicating noncooperative changes in the folded state. Additional evidence of structural perturbations came from the chemical shift analysis, nonuniform and bell-shaped peak intensity profiles, and limited proteolysis. In particular, the following nearby regions of the tertiary structure became progressively destabilized with increasing urea concentrations: the beta-hairpin interface of trefoils 1 and 2 and the H2a-H2 helical region. These regions underwent small-scale perturbations within the native baseline region in the absence of populated molten globule-like states. Similar regions were affected by elevated temperatures known to induce irreversible aggregation of IL-1ra. Further evidence of structural transitions invoking near-native conformations came from an optical spectroscopy analysis of its single-tryptophan variant W17A. The increase in the radius of gyration was associated with a single equilibrium unfolding transition in the case of two different denaturants, urea and guanidine hydrochloride (GuHCl). However, the compactness of urea- and GuHCl-unfolded molecules was comparable only at high denaturant concentrations and deviated under less denaturing conditions. Our results identified the role of conformational flexibility in IL-1ra aggregation and shed light on the nature of structural transitions within the folded ensembles of other beta-trefoil proteins, such as IL-1beta and hFGF-1.

An improved trypsin digestion method minimizes digestion-induced modifications on proteins.

Trypsin digestion can induce artificial modifications such as asparagine deamidation and N-terminal glutamine cyclization on proteins due to the temperature and the alkaline pH buffers used during digestion. The amount of these artificial modifications is directly proportional to the incubation time of protein samples in the reduction/alkylation buffer and, more important, in the digestion buffer where the peptides are completely solvent exposed. To minimize these artificial modifications, we focused on minimizing the trypsin digestion time by maximizing trypsin activity. Trypsin activity was optimized by the complete removal of guanidine, which is a known trypsin inhibitor, from the digestion buffer. As a result, near complete trypsin digestion was achieved on reduced and alkylated immunoglobulin gamma molecules in 30min. The protein tryptic fragments and their modification products were analyzed and quantified by reversed-phase liquid chromatography/tandem mass spectrometry using an in-line LTQ Orbitrap mass spectrometer. The reduction and alkylation reaction time was also minimized by monitoring the completeness of the reaction using a high-resolution time-of-flight mass spectrometer. Using this 30-min in-solution trypsin digestion method, little protocol-induced deamidation or N-terminal glutamine cyclization product was observed and cleaner tryptic maps were obtained due to less trypsin self-digestion and fewer nonspecific cleavages. The throughput of trypsin digestion was also improved significantly compared with conventional trypsin digestion methods.

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.

Ion-specific modulation of protein interactions: anion-induced, reversible oligomerization of a fusion protein.

Ions can significantly modulate the solution interactions of proteins. We aim to demonstrate that the salt-dependent reversible heptamerization of a fusion protein called peptibody A or PbA is governed by anion-specific interactions with key arginyl and lysyl residues on its peptide arms. Peptibody A, an E. coli expressed, basic (pI = 8.8), homodimer (65.2 kDa), consisted of an IgG1-Fc with two, C-terminal peptide arms linked via penta-glycine linkers. Each peptide arm was composed of two, tandem, active sequences (SEYQGLPPQGWK) separated by a spacer (GSGSATGGSGGGASSGSGSATG). PbA was monomeric in 10 mM acetate, pH 5.0 but exhibited reversible self-association upon salt addition. The sedimentation coefficient (s(w)) and hydrodynamic diameter (D(H)) versus PbA concentration isotherms in the presence of 140 mM NaCl (A5N) displayed sharp increases in s(w) and D(H), reaching plateau values of 9 s and 16 nm by 10 mg/mL PbA. The D(H) and sedimentation equilibrium data in the plateau region (>12 mg/mL) indicated the oligomeric ensemble to be monodisperse (PdI = 0.05) with a z-average molecular weight (M(z)) of 433 kDa (stoichiometry = 7). There was no evidence of reversible self-association for an IgG1-Fc molecule in A5N by itself or in a mixture containing fluorescently labeled IgG1-Fc and PbA, indicative of PbA self-assembly being mediated through its peptide arms. Self-association increased with pH, NaCl concentration, and anion size (I(-) > Br(-) > Cl(-) > F(-)) but could be inhibited using soluble Trp-, Phe-, and Leu-amide salts (Trp > Phe > Leu). We propose that in the presence of salt (i) anion binding renders PbA self-association competent by neutralizing the peptidyl arginyl and lysyl amines, (ii) self-association occurs via aromatic and hydrophobic interactions between the ..xxCTRWPWMC..xxxCTRWPWMCxx.. motifs, and (iii) at >10 mg/mL, PbA predominantly exists as heptameric clusters.

Effect of ions on agitation- and temperature-induced aggregation reactions of antibodies.

PURPOSE: The impact of ions on protein aggregation remains poorly understood. We explored the role of ionic strength and ion identity on the temperature- and agitation-induced aggregation of antibodies. METHODS: Stability studies were used to determine the influence of monovalent Hofmeister anions and cations on aggregation propensity of three IgG(2) mAbs. The C(H)2 domain melting temperature (T (m1)) and reduced valence (z*) of the mAbs were measured. RESULTS: Agitation led to increased solution turbidity, consistent with the formation of insoluble aggregates, while soluble aggregates were formed during high temperature storage. The degree of aggregation increased with anion size (F(-) < Cl(-) < Br(-) < I(-) < SCN(-) ~ ClO(4) (-)) and correlated with a decrease in T (m1) and z*. The aggregation propensity induced by the anions increased with the chaotropic nature of anion. The cation identity (Li(+), Na(+), K(+), Rb(+), or Cs(+)) had no effect on T (m1), z* or aggregation upon agitation. CONCLUSIONS: The results indicate that anion binding mediates aggregation by lowering mAb conformational stability and reduced valence. Our observations support an agitation-induced particulation model in which anions enhance the partitioning and unfolding of mAbs at the air/water interface. Aggregation predominantly occurs at this interface; refreshing of the surface during agitation releases the insoluble aggregates into bulk solution.

Degradation products analysis of an Fc fusion protein using LC/MS methods.

The following analytical methods have been used to identify and quantify degradation products in an E. coli expressed human immunoglobulin G Fc fusion protein in both liquid and lyophilized forms: two-dimensional AEX/RP/MS, limited proteolysis followed by LC/MS, and tryptic digestion followed by LC/MS/MS. After aging in a potassium phosphate pH 7.0 buffer for 3 months at 29 degrees C, peptide map analysis revealed that asparagine N78 (N297 according to Edelman sequencing) of the CH2 domain was the most rapidly deamidated site in the molecule probably due to the lack of the N-linked glycan on this asparagine, but this deamidation can be prevented under properly formulated conditions. This is the first report on the rate of deamidation on N297 of an IgG molecule without glycosylation. The active protein portion of the Fc fusion protein contains two methionine residues that are potentially susceptible to oxidation. Limited proteolysis was employed to cleave the active protein portion and measure the amount of oxidation. LC/MS analysis identified that the liquid sample aged at 29 degrees C for 3 months produced 40% oxidation, while the control sample contained only 4% oxidation on the active protein. In contrast to the aged liquid sample, the aged lyophilized sample showed no increase of deamidation or oxidation after storage at 37 degrees C for 8 months.

Anion binding mediated precipitation of a peptibody.

PURPOSE: Understand the underlying mechanism governing the salt-induced precipitation of a basic (pI = 8.8) protein, Peptibody A (PbA), in acidic solutions. METHODS: The rate, extent, and reversibility of PbA precipitation was monitored over 4-weeks as a function of pH (3.7-5.0), salt concentration (0-400 mM), and ion identity using a series of monovalent, Hofmeister anions (F(-), Cl(-), Br(-), I(-), ClO(4) (-), SCN(-)) and cations (Li+, Na+, K+, Rb+, Cs+). The effects of salt on conformational stability and reduced valence were determined using Fourier-transform infrared spectroscopy, circular dichroism, and capillary electrophoresis/analytical ultracentrifugation. RESULTS: PbA precipitation occurred upon salt addition and could be modulated with solution pH, salt identity & concentration. The precipitation was sensitive to anions, but not cations, and increased with anion size. A reverse Hofmeister effect (SCN(-) approximately ClO(4) (-)>I(-)>Cl(-)>Br(-)>F(-)) was observed with "salting-in" anions being the more effective precipitants. An increase in the precipitation rate below pH 4.3 indicated that protonation of aspartyl and glutamyl side-chains was also important for precipitation. The reversibility of precipitation was excellent (100%) at 4 degrees C but decreased upon storage at 25 degrees C and 37 degrees C; the loss in reversibility correlated with an increase in intermolecular beta-sheet content of the precipitate. CONCLUSION: Salts, employed as buffering, tonicifying, and viscosity modifying agents, may adversely affect the solubility of basic proteins formulated under acidic conditions.

Structure and stability changes of human IgG1 Fc as a consequence of methionine oxidation.

The Fc region has two highly conserved methionine residues, Met 33 (C(H)3 domain) and Met 209 (C(H)3 domain), which are important for the Fc's structure and biological function. To understand the effect of methionine oxidation on the structure and stability of the human IgG1 Fc expressed in Escherichia coli, we have characterized the fully oxidized Fc using biophysical (DSC, CD, and NMR) and bioanalytical (SEC and RP-HPLC-MS) methods. Methionine oxidation resulted in a detectable secondary and tertiary structural alteration measured by circular dichroism. This is further supported by the NMR data. The HSQC spectral changes indicate the structures of both C(H)2 and C(H)3 domains are affected by methionine oxidation. The melting temperature (Tm) of the C(H)2 domain of the human IgG1 Fc was significantly reduced upon methionine oxidation, while the melting temperature of the C(H)3 domain was only affected slightly. The change in the C(H)2 domain T m depended on the extent of oxidation of both Met 33 and Met 209. This was confirmed by DSC analysis of methionine-oxidized samples of two site specific methionine mutants. When incubated at 45 degrees C, the oxidized Fc exhibited an increased aggregation rate. In addition, the oxidized Fc displayed an increased deamidation (at pH 7.4) rate at the Asn 67 and Asn 96 sites, both located on the C(H)2 domain, while the deamidation rates of the other residues were not affected. The methionine oxidation resulted in changes in the structure and stability of the Fc, which are primarily localized to the C(H)2 domain. These changes can impact the Fc's physical and covalent stability and potentially its biological functions; therefore, it is critical to monitor and control methionine oxidation during manufacturing and storage of protein therapeutics.

Isomerization of a single aspartyl residue of anti-epidermal growth factor receptor immunoglobulin gamma2 antibody highlights the role avidity plays in antibody activity.

A new isoform of the light chain of a fully human monoclonal immunoglobulin gamma2 (IgG2) antibody panitumumab against human epidermal growth factor receptor (EGFR) was generated by in vitro aging. The isoform was attributed to the isomerization of aspartate 92 located between phenylalanine 91 and histidine 93 residues in the antigen-binding region. The isomerization rate increased with increased temperature and decreased pH. A size-exclusion chromatography binding assay was used to show that one antibody molecule was able to bind two soluble extracellular EGFR molecules in solution, and isomerization of one or both Asp-92 residues deactivated one or both antigen-binding regions, respectively. In addition, isomerization of Asp-92 showed a decrease in in vitro potency as measured by a cell proliferation assay with a 32D cell line that expressed the full-length human EGFR. The data indicate that antibodies containing either one or two isomerized residues were not effective in inhibiting EGFR-mediated cell proliferation, and that two unmodified antigen binding regions were needed to achieve full efficacy. For comparison, the potency of an intact IgG1 antibody cetuximab against the same receptor was correlated with the bioactivity of its individual antigen-binding fragments. The intact IgG1 antibody with two antigen-binding fragments was also much more active in suppressing cell proliferation than the individual fragments, similar to the IgG2 results. These results indicated that avidity played a key role in the inhibition of cell proliferation by these antibodies against the human EGFR, suggesting that their mechanisms of action are similar.

Contributions of a disulfide bond to the structure, stability, and dimerization of human IgG1 antibody CH3 domain.

Recombinant human monoclonal antibodies have become important protein-based therapeutics for the treatment of various diseases. The antibody structure is complex, consisting of beta-sheet rich domains stabilized by multiple disulfide bridges. The dimerization of the C(H)3 domain in the constant region of the heavy chain plays a pivotal role in the assembly of an antibody. This domain contains a single buried, highly conserved disulfide bond. This disulfide bond was not required for dimerization, since a recombinant human C(H)3 domain, even in the reduced state, existed as a dimer. Spectroscopic analyses showed that the secondary and tertiary structures of reduced and oxidized C(H)3 dimer were similar, but differences were observed. The reduced C(H)3 dimer was less stable than the oxidized form to denaturation by guanidinium chloride (GdmCl), pH, or heat. Equilibrium sedimentation revealed that the reduced dimer dissociated at lower GdmCl concentration than the oxidized form. This implies that the disulfide bond shifts the monomer-dimer equilibrium. Interestingly, the dimer-monomer dissociation transition occurred at lower GdmCl concentration than the unfolding transition. Thus, disulfide bond formation in the human C(H)3 domain is important for stability and dimerization. Here we show the importance of the role played by the disulfide bond and how it affects the stability and monomer-dimer equilibrium of the human C(H)3 domain. Hence, these results may have implications for the stability of the intact antibody.

Self-buffering antibody formulations.

Monoclonal antibodies (mAbs) often require the development of high-concentration formulations. In such cases, and when it is desirable to formulate a mAb around pH 5.0, we explored a novel approach of controlling the formulation pH by harnessing the ability of mAbs to "self-buffer." Buffer capacities of four representative IgG(2) molecules (designated mAb1 through mAb4) were measured in the pH 4-6 range. The buffer capacity results indicated that the mAbs possessed a significant amount of buffer capacity, which increased linearly with concentration. By 60-80 mg/mL, the mAb buffer capacities surpassed that of 10 mM acetate, which is commonly employed in formulations for buffering in the pH 4-6 range. Accelerated high temperature stability studies (50 degrees C over 3 weeks) conducted with a representative antibody in a self-buffered formulation (50 mg/mL mAb1 in 5.25% sorbitol, pH 5.0) and with solutions formulated using conventional buffers (50 mg/mL mAb1 in 5.25% sorbitol, 25 or 50 mM acetate, glutamate or succinate, also at pH 5.0) indicated that mAb1 was most resistant to the formation of soluble aggregates in the self-buffered formulation. Increased soluble aggregate levels were observed in all the conventionally buffered (acetate, glutamate, and succinate) formulations, which further increased with increasing buffer strength. The long-term stability of the self-buffered liquid mAb1 formulation (60 mg/mL in 5% sorbitol, 0.01% polysorbate 20, pH 5.2) was comparable to the conventionally buffered (60 mg/mL in 10 mM acetate or glutamate, 5.25% sorbitol, 0.01% polysorbate 20, pH 5.2) formulations. No significant change in pH was observed after 12 months of storage at 37 and 4 degrees C for the self-buffered formulation. The 60 mg/mL self-buffered formulation of mAb1 was also observed to be stable to freeze-thaw cycling (five cycles, -20 degrees C --> room temperature). Self-buffered formulations may be a better alternative for the development of high-concentration antibody and protein dosage forms.

Reversed-phase liquid chromatography of immunoglobulin G molecules and their fragments with the diphenyl column.

A diphenyl column was able to resolve two closely related monoclonal IgG2 molecules, while a C8 column failed to separate these IgGs under identical chromatographic conditions. The diphenyl column also showed a better separation of a mixture of two light and two heavy chains than the C8 column. The influence of amino acid side chains from protein sequences in binding to the diphenyl and C8 stationary phases was studied by using a set of synthetic peptides with the sequence GXXLLLKK, where X represents substitution with all of the 20 amino acids. Peptides containing aromatic amino acids showed a greater binding on the diphenyl column than on the C8 column. This increase in retention was attributed to pi-pi interactions between the aromatic amino acid side chains and the diphenyl ligand. Based on the retention of peptides on the diphenyl column, new retention coefficients were assigned for the separation of proteins. A good correlation was observed between the sum of retention coefficients (SigmaRc) for IgGs and their retention time on the diphenyl column. On-column hydrogen-deuterium exchange showed that the diphenyl column had a larger surface of interaction with protein than the C8 column. pi-pi interactions and the large contact surface resulted in improved resolution of IgGs and their fragments on the diphenyl column.

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.

Biophysical characterization of structural properties and folding of interleukin-1 receptor antagonist.

Structural properties and folding of interleukin-1 receptor antagonist (IL-1ra), a therapeutically important cytokine with a symmetric beta-trefoil topology, are characterized using optical spectroscopy, high-resolution NMR, and size-exclusion chromatography. Spectral contributions of two tryptophan residues, Trp17 and Trp120, present in the wild-type protein, have been determined from mutational analysis. Trp17 dominates the emission spectrum of IL-1ra, while Trp120 is quenched presumably by the nearby cysteine residues in both folded and unfolded states. The same Trp17 gives rise to two characteristic negative peaks in the aromatic CD. Urea denaturation of the wild-type protein is probed by measuring intrinsic and extrinsic (binding of 1-anilinonaphthalene-8-sulfonic acid) fluorescence, near- and far-UV CD, and 1D and 2D ((1)H-(15)N heteronuclear single quantum coherence (HSQC)) NMR. Overall, the data suggest an essentially two-state equilibrium denaturation mechanism with small, but detectable structural changes within the pretransition region. The majority of the (1)H-(15)N HSQC cross-peaks of the folded state show only a limited chemical shift change as a function of the denaturant concentration. However, the amide cross-peak of Leu31 demonstrates a significant urea dependence that can be fitted to a two-state binding model with a dissociation constant of 0.95+/-0.04 M. This interaction has at least a five times higher affinity than reported values for nonspecific urea binding to denatured proteins and peptides, suggesting that the structural context around Leu31 stabilizes the protein-urea interaction. A possible role of denaturant binding in inducing the pretransition changes in IL-1ra is discussed. Urea unfolding of wild-type IL-1ra is sufficiently slow to enable HPLC separation of folded and unfolded states. Quantitative size-exclusion chromatography has provided a hydrodynamic view of the kinetic denaturation process. Thermodynamic stability and unfolding kinetics of IL-1ra resemble those of structurally and evolutionary close IL-1beta, suggesting similarity of their free energy landscapes.