Effect of Peroxide- Versus Alkoxyl-Induced Chemical Oxidation on the Structure, Stability, Aggregation, and Function of a Therapeutic Monoclonal Antibody.

Current guidelines indicate that the effects of oxidation should be included as part of forced degradation studies on protein drugs. We probed the effect of 3 commonly used oxidants, hydrogen peroxide, tert-butyl hydroperoxide, and 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH), on a therapeutic monoclonal IgG1 antibody (mAb8). Upon oxidation, mAb8 did not show noticeable changes in its secondary structure but showed minor changes in tertiary structure. Significant decrease in conformational stability was observed for all the 3 oxidized forms. Both hydrogen peroxide and tert-butyl hydroperoxide destabilized mainly the CH2 domain, whereas AAPH destabilized the variable domain in addition to CH2. Increased aggregation was found for AAPH-oxidized mAb8. In addition, a significant decrease in Fc receptor binding was observed for all 3 oxidized forms. Antibody dependent cell-mediated cytotoxicity, binding to target protein receptor, and cell proliferation activity were significantly reduced in the case of AAPH-oxidized mAb8. The presence of free methionine in the formulation buffer seems to alleviate the effect of oxidation. The results of this study show that the 3 oxidants differ in terms of their effects on the structure and function of mAb8 because of chemical modification of different sets of residues located in Fab versus Fc.

Effect of photo-degradation on the structure, stability, aggregation, and function of an IgG1 monoclonal antibody.

Photostability testing of therapeutic proteins is a critical requirement in the development of biologics. Upon exposure to light, pharmaceutical proteins may undergo a change in structure, stability, and functional properties that could have a potential impact on safety and efficacy. In this work, we studied how exposure to light, according to ICH guidelines, leads to photo-oxidation of a therapeutic IgG1 mAb. We also tested the ability of five different excipients to prevent such oxidation. In samples that were exposed to light, we found that the CH2 domain was considerably destabilized but there were no major changes in the overall structure of the protein. Aggregation of the protein was observed because of light exposure. Mass spectrometry identified that light exposure oxidizes two key methionine residues in the Fc region of the protein. In terms of function, a loss in binding to the neonatal Fc receptor, decreased antibody-dependent cell-mediated cytotoxicity and cell proliferation activities of the protein were seen. Combined analysis of the photo-oxidation effects on the structure, stability, aggregation, and function of the mAb has identified the underlying unifying mechanism. Among the sugars and amino acids tested, methionine was the most effective in protecting mAb against photo-oxidation.

Comparison of predicted extinction coefficients of monoclonal antibodies with experimental values as measured by the Edelhoch method.

Pace et al. (1995) [1] recommended an equation used to predict extinction coefficient of a protein. However, no antibody data was included in the development of this equation. The main objective of this study was to therefore investigate how the predicted value of the extinction coefficient is comparable to the experimentally determined extinction coefficient of antibodies measured by the Edelhoch method. We have measured the extinction coefficients (ɛ) of 13 IgG1 monoclonal antibodies (mAbs) in phosphate buffer at pH 7.2. The maximum variability in the experimentally measured extinction coefficient of a given mAb molecule was found to be about 2%. Experimentally determined extinction coefficients of all mAbs were found to be lower than the predicted value, with the maximum difference found to being 4.7%. The highest and lowest values of experimental extinction coefficient among the thirteen IgG1 monoclonal antibodies obtained were 230525.9M(-1)cm(-1) (i.e. 1.55(mg/ml)(-1)cm(-1)) and 191,411.6M(-1)cm(-1) (i.e. 1.29(mg/ml)(-1)cm(-1)). A difference of <3% (with respect to mean value) was observed between the experimental and predicted values of the extinction coefficient. A comprehensive analysis and interpretation of the comparison of the predicted and experimentally determined extinction coefficient by the Edelhoch method is discussed in terms of structural characterization and accessible surface area (ASA).

Principles and applications of selective biophysical methods for characterization and comparability assessment of a monoclonal antibody.

The strategy for a comparability assessment is developed on a hierarchical risk-based approach. Critical analysis of physicochemical and biological characterization assays is essential for the development of a good comparability protocol. Therefore, selection and sensitivity of these assays is very important. This article discusses a case study to evaluate the sensitivity of various methods in a comparability assessment of three lots of an IgG1 monoclonal antibody (mAb). Analysis with eighteen methods demonstrated that only six of the methods were sensitive enough to show a measurable difference of comparability under accelerated conditions (40°C). Samples stored at 4°C were found to be comparable by all methods. A brief comparison of the results of biochemical and functional assays with biophysical analysis is discussed. Basic principles, applications, strength, and limitations of different biophysical methods are also discussed here.

Effects of arginine on photostability and thermal stability of IgG1 monoclonal antibodies.

This study demonstrates that arginine is a highly effective solvent additive which significantly reduces the light induced aggregation of four IgG1 type monoclonal antibodies (named as IMC-1A, IMC-1B, IMC-1C and IMC-1D) as measured by size exclusion chromatography. All experiments were performed in a phosphate buffer system containing either sodium chloride or arginine hydrochloride. The protein samples were exposed to light in a photo chamber according to ICH (International Conference on Harmonization) guidelines. Thermal unfolding transition temperature (T(m)) of IMC-1A as determined by differential scanning calorimetry (DSC) was significantly decreased ( approximately 3.3 degrees C) in the presence of arginine hydrochloride as compared to in sodium chloride. However, a noticeable increase in thermal stability was observed for IMC-1B, IMC-1C and IMC-1D in the presence of arginine hydrochloride. The photostability of all these molecules was significantly enhanced by arginine hydrochloride and both a direct and inverse correlation was observed between conformational stability and photostability. To our knowledge, this paper for the first time, demonstrates that arginine hydrochloride considerably reduces the light induced aggregation of monoclonal antibodies. Arginine hydrochloride is also known to increase protein solubility and its ability to extensively reduce light induced aggregation makes it a potential solvent additive for the formulation development of therapeutic proteins.

Effects of pH and arginine on the solubility and stability of a therapeutic protein (Fibroblast Growth Factor 20): relationship between solubility and stability.

The purpose of this study was to dramatically enhance the solubility (> 400 fold) and stability of a therapeutic protein (Fibroblast Growth Factor 20) and to perform detailed biophysical characterization for the optimization of its formulation. The solubility of FGF-20 strongly depends on pH, arginine concentration and anions present in a buffer system. In the absence and presence of arginine, solubility was higher at lower pH (5 < or = pH < or = 6) and then decreased steadily with a minimum solubility at around pH 6.3 and plateaus at around pH 7.5 respectively. For a given pH, the protein was most soluble in arginine-sulfate. The solubility of FGF-20 increases with an increase in arginine-sulfate concentration for a given pH. However, a salting out effect was observed at higher arginine-sulfate concentration. Polysorbate-80 did not have any striking effect on solubility and no effect on thermal stability, but it significantly prevented the loss of protein under agitated conditions. Thermal stability of FGF-20 measured by DSC was increased with an increase in arginine-sulfate concentration (at least up to 0.5M). A sturdy dependence of thermal stability on pH was observed with about a 15 degrees C increase in T(m) (melting temperature) at pH 7.0 in comparison to pH 5.0. From the DSC data, approximate stability curves were generated and cold denaturation temperatures were predicted. Denaturant induced unfolding studies provided better insight of FGF-20 in different solution conditions in terms of structure and stability than the DSC data. An inverse relationship of solubility and thermal stability was observed in the pH range of 5.0 to 8.5 at a fixed arginine concentration and is consistent with Linderstrom-Lange's smeared model. A direct correlation between solubility and thermal stability was observed at different arginine concentrations for a fixed pH. The effect of arginine on the solubility and stability of FGF-20 was dominated by the preferential binding interaction.

Mapping of solution components, pH changes, protein stability and the elimination of protein precipitation during freeze-thawing of fibroblast growth factor 20.

This study discusses the effect of key factors like containers, buffers and the freeze (controlled vs. flash freezing) and thawing processes on the stability of a therapeutic protein fibroblast growth factor 20 (FGF-20). The freezing profiles monitored by 15 temperature probes located at different regions in a 2-L bottle during freezing can be grouped into three categories. A rapid drop in temperature was observed at the bottom followed by the top and middle center of the bottle. The freeze-thawing behavior in a 50 ml tube is considerably uniform, as expected. Among phosphate, HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), citrate and histidine (each containing 0.5 M arginine-sulfate) buffer systems, a minimum pH change (0.4 pH unit vs. approximately 1.7 pH unit) was observed for the phosphate buffer system. Thawing in a 50 ml tube at room temperature standing resulted in a significant phase separation in citrate, histidine and HEPES buffers; however, phase separation was least in the phosphate buffer system. These phase separations were found to be temperature dependent. No effect of Polysorbate 80 on freeze-thawing of FGF-20 was observed. Significant concentration gradients in major buffer components and protein concentration were observed during freeze-thawing in a 2-L bottle. The segregation patterns of the various components were similar with the top and bottom layers containing lowest and highest concentrations, respectively. In the formulation buffer no pH gradient was formed, and the precipitation of FGF-20 during thawing at the top layer was related to an insufficient amount of arginine-sulfate and the precipitation at the bottom layer was due to a salting out effect. The precipitate generated during thawing goes into solution easily upon mixing whole solution of the bottle and the various gradient formations do not cause any irreversible change in structure, stability and isoform distribution of FGF-20. Comparison of slow freezing and flash freezing data suggests that the gradients in excipient and protein concentrations are mainly formed during thawing.

Branching in the sequential folding pathway of cytochrome c.

Previous results indicate that the folding pathways of cytochrome c and other proteins progressively build the target native protein in a predetermined stepwise manner by the sequential formation and association of native-like foldon units. The present work used native state hydrogen exchange methods to investigate a structural anomaly in cytochrome c results that suggested the concerted folding of two segments that have little structural relationship in the native protein. The results show that the two segments, an 18-residue omega loop and a 10-residue helix, are able to unfold and refold independently, which allows a branch point in the folding pathway. The pathway that emerges assembles native-like foldon units in a linear sequential manner when prior native-like structure can template a single subsequent foldon, and optional pathway branching is seen when prior structure is able to support the folding of two different foldons.

Order of steps in the cytochrome C folding pathway: evidence for a sequential stabilization mechanism.

Previous work used hydrogen exchange (HX) experiments in kinetic and equilibrium modes to study the reversible unfolding and refolding of cytochrome c (Cyt c) under native conditions. Accumulated results now show that Cyt c is composed of five individually cooperative folding units, called foldons, which unfold and refold as concerted units in a stepwise pathway sequence. The first three steps of the folding pathway are linear and sequential. The ordering of the last two steps has been unclear because the fast HX of the amino acid residues in these foldons has made measurement difficult. New HX experiments done under slower exchange conditions show that the final two foldons do not unfold and refold in an obligatory sequence. They unfold separately and neither unfolding obligately contains the other, as indicated by their similar unfolding surface exposure and the specific effects of destabilizing and stabilizing mutations, pH change, and oxidation state. These results taken together support a sequential stabilization mechanism in which folding occurs in the native context with prior native-like structure serving to template the stepwise formation of subsequent native-like foldon units. Where the native structure of Cyt c requires sequential folding, in the first three steps, this is found. Where structural determination is ambiguous, in the final two steps, alternative parallel folding is found.

Functional role of a protein foldon--an Omega-loop foldon controls the alkaline transition in ferricytochrome c.

Hydrogen exchange results for cytochrome c and several other proteins show that they are composed of a number of foldon units which continually unfold and refold and account for some functional properties. Previous work showed that one Omega-loop foldon controls the rate of the structural switching and ligand exchange behavior of cytochrome c known as the alkaline transition. The present work tests the role of foldons in the alkaline transition equilibrium. We measured the effects of denaturant and 14 destabilizing mutations. The results show that the ligand exchange equilibrium is controlled by the stability of the same foldon unit implicated before. In addition, the results obtained confirm the epsilon-amino group of Lys79 and Lys73 as the alkaline replacement ligands and bear on the search for a triggering group.

Protein folding: the stepwise assembly of foldon units.

Equilibrium and kinetic hydrogen exchange experiments show that cytochrome c is composed of five foldon units that continually unfold and refold even under native conditions. Folding proceeds by the stepwise assembly of the foldon units rather than one amino acid at a time. The folding pathway is determined by a sequential stabilization process; previously formed foldons guide and stabilize subsequent foldons to progressively build the native protein. Four other proteins have been found to show similar behavior. These results support stepwise protein folding pathways through discrete intermediates.

Equilibrium unfolding of dimeric and engineered monomeric forms of lambda Cro (F58W) repressor and the effect of added salts: evidence for the formation of folded monomer induced by sodium perchlorate.

The equilibrium unfolding transitions of Cro repressor variants, dimeric variant Cro F58W and monomer Cro K56[DGEVK]F58W, have been studied by urea and guanidine hydrochloride to probe the folding mechanism. The unfolding transitions of a dimeric variant are well described by a two state process involving native dimer and unfolded monomer with a free energy of unfolding, DeltaG(0,un)(0), of approximately 10-11 kcal/mol. The midpoint of transition curves is dependent on total protein concentration and DeltaG(0,un)(0) is independent of protein concentration, as expected for this model. Unfolding of Cro monomer is well described by the standard two state model. The stability of both forms of protein increases in the presence of salt but decreases with the decrease in pH. Because of the suggested importance of a N2<-->2F dimerization process in DNA binding, we have also studied the effect of sodium perchlorate, containing the chaotropic perchlorate anion, on the conformational transition of Cro dimer by CD, fluorescence and NMR (in addition to urea and guanidine hydrochloride) in an attempt both to characterize the thermodynamics of the process and to identify conditions that lead to an increase in the population of the folded monomers. Data suggest that sodium perchlorate stabilizes the protein at low concentration (<1.5 M) and destabilizes the protein at higher perchlorate concentration with the formation of a "significantly folded" monomer. The tryptophan residue in the "significantly folded" monomer induced by perchlorate is more exposed to the solvent than in native dimer.

Perchlorate-induced conformational transition of Staphylococcal nuclease: evidence for an equilibrium unfolding intermediate.

The sodium perchlorate-induced conformational transition of Staphylococcal nuclease has been monitored by both circular dichroism (CD) and fluorescence spectroscopy. The perchlorate-induced transition is cooperative as observed by both spectroscopic signals. However, the protein loses only about one-third of its native far-UV CD signal at high perchlorate concentrations, indicating that a significant amount of secondary structure remains in the post-transition state. The remaining CD signal can be further diminished in a cooperative manner by the addition of the strong denaturant, urea. Near-UV CD spectra clearly show that the protein loses its tertiary structure in the perchlorate-induced denatured state. The perchlorate-induced transition curves were fit to the standard two-state model and the standard free energy change and m value of the transition are 2.3kcal/mol and 1.8kcal/(molM), respectively. By comparison, the urea-induced unfolding of Staphylococcal nuclease (in the absence of perchlorate) yields an unfolding free energy change, DeltaG(0,un), of 5.6kcal/mol and an m value of 2.3kcal/(molM). Thus, the thermodynamic state obtained in the post-transition region of perchlorate-induced conformation transition has a significantly lower free energy change, a high content of secondary structure, and diminished tertiary structure. These results suggest that the perchlorate-induced denatured state is a partially folded equilibrium state. Whether this intermediate is relevant to the folding/unfolding path under standard conditions is unknown at this time.

How cytochrome c folds, and why: submolecular foldon units and their stepwise sequential stabilization.

Native state hydrogen exchange experiments have shown that the cytochrome c (Cyt c) protein consists of five cooperative folding-unfolding units, called foldons. These are named, in the order of increasing unfolding free energy, the nested-Yellow, Red, Yellow, Green, and Blue foldons. Previous results suggest that these units unfold in a stepwise sequential way so that each higher energy partially unfolded form includes all of the previously unfolded lower free energy units. If this is so, then selectively destabilizing any given foldon should equally destabilize each subsequent unfolding step above it in the unfolding ladder but leave the lower ones before it unaffected. To perform this test, we introduced the mutation Glu62Gly, which deletes a salt link in the Yellow unit and destabilizes the protein by 0.8 kcal/mol. Native state hydrogen exchange and other experiments show that the stability of the Yellow unit and the states above it in the free energy ladder are destabilized by about the same amount while the lower lying states are unaffected. These results help to confirm the sequential stepwise nature of the Cyt c unfolding pathway and therefore a similar refolding pathway. The steps in the pathway are dictated by the concerted folding-unfolding property of the individual unit foldons; the order of steps is determined by the sequential stabilization of progressively added foldons in the native context. Much related information for Cyt c strongly conforms with this mechanism. Its generality is supported by available information for other proteins.

Folding units govern the cytochrome c alkaline transition.

The alkaline transition of cytochrome c is a model for protein structural switching in which the normal heme ligand is replaced by another group. Stopped flow data following a jump to high pH detect two slow kinetic phases, suggesting two rate-limiting structure changes. Results described here indicate that these events are controlled by the same structural unfolding reactions that account for the first two steps in the reversible unfolding pathway of cytochrome c. These and other results show that the cooperative folding-unfolding behavior of protein foldons can account for a variety of functional activities in addition to determining folding pathways.

Protein hydrogen exchange mechanism: local fluctuations.

Experiments were done to study the dynamic structural motions that determine protein hydrogen exchange (HX) behavior. The replacement of a solvent-exposed lysine residue with glycine (Lys8Gly) in a helix of recombinant cytochrome c does not perturb the native structure, but it entropically potentiates main-chain flexibility and thus can promote local distortional motions and large-scale unfolding. The mutation accelerates amide hydrogen exchange of the mutated residue by about 50-fold, neighboring residues in the same helix by less, and residues elsewhere in the protein not at all, except for Leu98, which registers the change in global stability. The pattern of HX changes shows that the coupled structural distortions that dominate exchange can be several residues in extent, but they expose to exchange only one amide NH at a time. This "local fluctuation" mode of hydrogen exchange may be generally recognized by disparate near-neighbor rates and a low dependence on destabilants (denaturant, temperature, pressure). In contrast, concerted unfolding reactions expose multiple neighboring amide NHs with very similar computed protection factors, and they show marked destabilant sensitivity. In both modes, ionic hydrogen exchange catalysts attack from the bulk solvent without diffusing through the protein matrix.

Fluorescence quenching of dimeric and monomeric forms of yeast hexokinase (PII): effect of substrate binding steady-state and time-resolved fluorescence studies.

Fluorescence quenching studies on the PII isoenzyme of yeast hexokinase have been performed using charged as well as polar uncharged quenchers. In both 'open' (i.e. in the absence of glucose) and 'closed' (i.e. in the presence of glucose) forms of the enzyme, bimolecular quenching rate constant (kq) for acrylamide is significantly larger than that of KI, indicating that all the tryptophans are not fully exposed to the solvent. Overall accessibility of tryptophans towards KI was greater in the presence of glucose than in the absence of glucose. At high ionic strength, the value of bimolecular quenching rate constant (kq) for KI did not change suggesting that the average environment of the accessible tryptophan residue(s) is almost neutral. Quenching by KI is dynamic in nature. Accessibility of tryptophans towards acrylamide at concentration > or = 0.2 M was more in the 'open' form of the enzyme than that observed in the 'closed' form whereas at concentration < or = 0.2 M no significant difference in the extent of quenching was observed. It is reasonable to conclude that glucose induced conformational change leads some tryptophan residue(s) to be more exposed and at the same time some tryptophan residue(s) in the hydrophobic region become more buried. Dimeric and monomeric forms of the enzyme behave similarly towards the quenching by acrylamide. In the unfolded state, the accessibility of tryptophans was considerably higher for both the quenchers. Temperature dependent study and the fluorescence lifetime data indicate that the mechanism of quenching by acrylamide is primarily dynamic in nature.

Structure of triphosphoryl nucleotide bound at the active site of yeast hexokinase: 1H-nuclear magnetic resonance study.

Conformation of a nonhydrolyzable adenosine triphosphate (ATP) analogue, adenylyl-(beta,gamma-methylene)-diphosphonate (AMPPCP) bound at the active site of yeast hexokinase-PII was determined by proton two-dimensional transferred nuclear Overhauser effect spectroscopy (TRNOESY) and molecular dynamics simulations. The effect of the glucose-induced domain closure on the conformation of the nucleotide was evaluated by making measurements on two different complexes: PII AMPPCPMg(II) and PII-Glc.AMPPCPMg(Il). TRNOE measurements were made at 500 MHz, 10 degress C, as a function of several mixing times varying in the range of 40 to 200 ms. Interproton distances derived from the analysis of NOE buildup curves were used as restraints in molecular dynamics simulations to determine the conformation of the enzyme bound nucleotide. The adenosine moiety was found to bind in high anti conformation with a glycosidic torsion angle chi = 48 +/- 5 degrees in both complexes. However, significant differences in the conformations of the ribose and triphosphoryl chain of the nucleotide are observed between the two complexes. The phase angles of pseudorotation P in PII.AMPPCPMg(II) and PII.Glc.AMPPCPMg(II) are 87 degrees and 77 degrees, describing a OE and OT4 sugar pucker and the amplitudes of the sugar pucker (tau) are 37 degrees and 61 degrees, respectively.