Mitigation of Oxidation in Therapeutic Antibody Formulations: a Biochemical Efficacy and Safety Evaluation of N-Acetyl-Tryptophan and L-Methionine.

PURPOSE: Biotherapeutics can be susceptible to oxidation during manufacturing and storage. Free L-methionine is known to protect methionine residues in proteins from oxidation. Similarly, free tryptophan and other indole derivatives have been shown to protect tryptophan residues from oxidation. N-acetyl-DL-tryptophan was previously identified as a potentially superior antioxidant to tryptophan as it has a lower oxidation potential and produces less peroxide upon light exposure. This study sought to confirm the antioxidant efficacy and safety of N-acetyl-DL-tryptophan and L-methionine as formulation components for biotherapeutic drugs. METHODS: Antibodies were subjected to AAPH and light exposure in the presence of N-acetyl-DL-tryptophan and L-methionine. Oxidation in relevant CDR and Fc residues was quantified by peptide map. In silico, in vitro, and in vivo studies were performed to evaluate the safety of N-acetyl-DL-tryptophan and L-methionine. RESULTS: Peptide mapping demonstrated that N-acetyl-DL-tryptophan was effective at protecting tryptophans from AAPH stress, and that the combination of N-acetyl-DL-tryptophan and L-methionine protected both tryptophan and methionine from AAPH stress. The safety assessment suggested an acceptable safety profile for both excipients. CONCLUSIONS: N-acetyl-tryptophan and L-methionine effectively reduce the oxidation of susceptible tryptophan and methionine residues in antibodies and are safe for use in parenteral biotherapeutic formulations.

In silico selection of therapeutic antibodies for development: viscosity, clearance, and chemical stability.

For mAbs to be viable therapeutics, they must be formulated to have low viscosity, be chemically stable, and have normal in vivo clearance rates. We explored these properties by observing correlations of up to 60 different antibodies of the IgG1 isotype. Unexpectedly, we observe significant correlations with simple physical properties obtainable from antibody sequences and by molecular dynamics simulations of individual antibody molecules. mAbs viscosities increase strongly with hydrophobicity and charge dipole distribution and decrease with net charge. Fast clearance correlates with high hydrophobicities of certain complementarity determining regions and with high positive or high negative net charge. Chemical degradation from tryptophan oxidation correlates with the average solvent exposure time of tryptophan residues. Aspartic acid isomerization rates can be predicted from solvent exposure and flexibility as determined by molecular dynamics simulations. These studies should aid in more rapid screening and selection of mAb candidates during early discovery.

Evaluating the Use of Antibody Variable Region (Fv) Charge as a Risk Assessment Tool for Predicting Typical Cynomolgus Monkey Pharmacokinetics.

The pharmacokinetic (PK) behavior of monoclonal antibodies in cynomolgus monkeys (cynos) is generally translatable to that in humans. Unfortunately, about 39% of the antibodies evaluated for PKs in cynos have fast nonspecific (or non-target-mediated) clearance (in-house data). An empirical model relating variable region (Fv) charge and hydrophobicity to cyno nonspecific clearance was developed to gauge the risk an antibody would have for fast nonspecific clearance in the monkey. The purpose of this study was to evaluate the predictability of this empirical model on cyno nonspecific clearance with antibodies specifically engineered to have either high or low Fv charge. These amino acid changes were made in the Fv region of two test antibodies, humAb4D5-8 and anti-lymphotoxin α. The humAb4D5-8 has a typical nonspecific clearance in cynos, and by making it more positively charged, the antibody acquires fast nonspecific clearance, and making it less positively charged did not impact its clearance. Anti-lymphotoxin α has fast nonspecific clearance in cynos, and making it more positively charged caused it to clear even faster, whereas making it less positively charged caused it to clear slower and within the typical range. These trends in clearance were also observed in two other preclinical species, mice and rats. The effect of modifying Fv charge on subcutaneous bioavailability was also examined, and in general bioavailability was inversely related to the direction of the Fv charge change. Thus, modifying Fv charge appears to impact antibody PKs, and the changes tended to correlate with those predicted by the empirical model.

Specific catalysis of asparaginyl deamidation by carboxylic acids: kinetic, thermodynamic, and quantitative structure-property relationship analyses.

Asparaginyl (Asn) deamidation could lead to altered potency, safety, and/or pharmacokinetics of therapeutic protein drugs. In this study, we investigated the effects of several different carboxylic acids on Asn deamidation rates using an IgG1 monoclonal antibody (mAb1*) and a model hexapeptide (peptide1) with the sequence YGKNGG. Thermodynamic analyses of the kinetics data revealed that higher deamidation rates are associated with predominantly more negative ΔS and, to a lesser extent, more positive ΔH. The observed differences in deamidation rates were attributed to the unique ability of each type of carboxylic acid to stabilize the energetically unfavorable transition-state conformations required for imide formation. Quantitative structure property relationship (QSPR) analysis using kinetic data demonstrated that molecular descriptors encoding for the geometric spatial distribution of atomic properties on various carboxylic acids are effective determinants for the deamidation reaction. Specifically, the number of O-O and O-H atom pairs on carboxyl and hydroxyl groups with interatomic distances of 4-5 Å on a carboxylic acid buffer appears to determine the rate of deamidation. Collectively, the results from structural and thermodynamic analyses indicate that carboxylic acids presumably form multiple hydrogen bonds and charge-charge interactions with the relevant deamidation site and provide alignment between the reactive atoms on the side chain and backbone. We propose that carboxylic acids catalyze deamidation by stabilizing a specific, energetically unfavorable transition-state conformation of l-asparaginyl intermediate II that readily facilitates bond formation between the γ-carbonyl carbon and the deprotonated backbone nitrogen for cyclic imide formation.

Investigation into temperature-induced aggregation of an antibody drug conjugate.

Conjugation of an antibody to a drug can produce heterogeneous species that may have different physical stabilities and safety profiles. We explored the effect of thermal stress on the physical stability, specifically aggregation, of an antibody drug conjugate (ADC), ADC 1, wherein the antibody was linked to the val-cit-Monomethyl Auristatin E (vc-MMAE) linker drug through the reduction of interchain disulfides. We also explored the effects of conjugation on the secondary and tertiary structures of ADC 1. Circular dichroism, intrinsic tryptophan fluorescence, and differential scanning calorimetry showed that for species with high drug loading, conjugation does not measurably alter the secondary structure, but it does render the CH2 domain less stable to thermal stress such that ADC 1 rapidly forms high molecular weight species (HMWS) at 40 °C. Characterization of the HMWS using chromatographic and electrophoretic methods showed that it is an irreversible, noncovalent, and structurally altered form of ADC 1 primarily composed of molecules with six or eight drugs. Furthermore, the variable domain of the antibody may contribute to the extent of aggregation, since eight ADCs with over 90% sequence homology exhibited monthly rates of HMWS formation that differ by up to a factor of 2.

Culture temperature modulates aggregation of recombinant antibody in cho cells.

During production of therapeutic monoclonal antibodies (mAb), it is highly desirable to remove and control antibody aggregates in the manufacturing process to minimize the potential risk of immunogenicity to patients. During process development for the production of a recombinant IgG in a CHO cell line, we observed atypical high variability from 1 to 20% mAb aggregates formed during cell culture that negatively impacted antibody purification. Analytical characterization revealed the IgG aggregates were mediated by hydrophobic interactions likely caused by misfolded antibody during intracellular processing. Strikingly, data analysis showed an inverse correlation of lower cell culture temperature producing higher aggregate levels. All cultures at 37°C exhibited ≤ 5% aggregates at harvest. Aggregate levels increased 4-12-fold in 33°C cultures when compared to 37°C, with a corresponding 2-4-fold increase in heavy chain (HC) and light chain (LC) mRNA. Additionally, 37°C cases showed a greater excess of LC to HC mRNA levels. Endoplasmic reticulum (ER) chaperone expression and ER size also increased 25-75% at 33°C versus 37°C but to a lesser extent than LC and HC mRNA, consistent with a potential limiting ER folding capacity at 33°C for this cell line. Finally, we identified a 2-5-fold increase in mAb aggregate formation at 33°C compared to 37°C cultures for three additional CHO cell lines. Taken together, our observations indicate that low culture temperature can increase antibody aggregate formation in CHO cells by increasing LC and HC transcripts coupled with limited ER machinery.

Opposite effects of polyols on antibody aggregation: thermal versus mechanical stresses.

PURPOSE: To investigate the physical stability of antibody-polyol formulations under thermal and mechanical stresses. METHODS: mAb-U was analyzed in buffer, trehalose, sucrose, glycerol and ethylene glycol solutions at pH 7.0. T(m1) of mAb-U was determined using DSC. Thermal stress studies were performed by incubating mAb-U-polyol solutions at 40°C (2 months), 50°C (3 weeks) and 65°C (5 days). Mechanical stress studies were conducted by shaking mAb-U-polyol solutions at 200 rpm for 5 days at 25°C. RESULTS: Trehalose and glycerol increased the T(m1) of mAb-U, whereas ethylene glycol decreased it. The trend observed in the order of increasing aggregation of mAb-U after thermal stress (40°C and 50°C) was buffer = trehalose = sucrose

Physicochemical stability of the antibody-drug conjugate Trastuzumab-DM1: changes due to modification and conjugation processes.

In the manufacture of the antibody-drug conjugate Trastuzumab-DM1 (T-DM1), the lysine residues on the antibody trastuzumab (Tmab) are modified to form the intermediate Tmab-MCC (T-MCC) and then conjugated with the drug DM1. Our goal is to understand the effects of modification and conjugation steps on the physicochemical stability of the antibody. The structural stability of Tmab relative to its modified and conjugated forms was assessed, employing thermally induced stress conditions to formulations containing Tmab, T-MCC, and T-DM1. DSC, SEC, CE-SDS, and LC-MS were used to study the stability of Tmab, T-MCC, and T-DM1 to thermal stress. The DSC thermograms show a decrease in melting temperature for the CH2 transition, in the order Tmab > T-MCC > T-DM1. As per SEC analysis, a significant increase in level of aggregation was detected in T-MCC (∼32%) and T-DM1 (∼5%) after 14 days at 40 °C. Tmab did not show significant aggregate formation. CE-SDS and LC-MS data demonstrate that the aggregation in the case of T-MCC is largely covalent and involves mechanisms other than formation of intermolecular disulfide cross-links. The aggregation observed for T-MCC was significantly inhibited upon addition of amino acids with nucleophilic side chains containing thiol (Cys) and hydroxyl moieties (Ser, Tyr). The covalent aggregation observed for T-MCC and the ability of nucleophilic amino acids, particularly Cys, to inhibit it indicate that the maleimide moiety in the MCC linker may react to form intermolecular covalent cross-links between T-MCC molecules, possibly through a Michael addition mechanism. In addition, DSC results demonstrate that the conjugation of the drug moiety DM1 to Tmab results in destabilization of the CH2 domain of the antibody.

Triple light chain antibodies: factors that influence its formation in cell culture.

THIOMABs are recombinant antibodies engineered with reactive cysteines, which can be covalently conjugated to drugs of interest to generate targeted therapeutics. During the analysis of THIOMABs secreted by stably transfected Chinese Hamster Ovary (CHO) cells, we discovered the existence of a new species--Triple Light Chain Antibody (3LC). This 3LC species is the product of a disulfide bond formed between an extra light chain and one of the engineered cysteines on the THIOMAB. We characterized the 3LC by size exclusion chromatography, mass spectrometry, and microchip electrophoresis. We also investigated the potential causes of 3LC formation during cell culture, focusing on the effects of free light chain (LC) polypeptide concentration, THIOMAB amino acid sequence, and glutathione (GSH) production. In studies covering 12 THIOMABs produced by 66 stable cell lines, increased free LC polypeptide expression--evaluated as the ratio of mRNA encoding for LC to the mRNA encoding for heavy chain (HC)--correlated with increased 3LC levels. The amino acid sequence of the THIOMAB molecule also impacted its susceptibility to 3LC formation: hydrophilic LC polypeptides showed elevated 3LC levels. Finally, increased GSH production--evaluated as the ratio of the cell-specific production rate of GSH (q(GSH)) to the cell-specific production rate of THIOMAB (q(p))--corresponded to decreased 3LC levels. In time-lapse studies, changes in extracellular 3LC levels during cell culture corresponded to changes in mRNA LC/HC ratio and q(GSH)/q(p) ratio. In summary, we found that cell lines with low mRNA LC/HC ratio and high q(GSH)/q(p) ratio yielded the lowest levels of 3LC. These findings provide us with factors to consider in selecting a cell line to produce THIOMABs with minimal levels of the 3LC impurity.

In situ precipitation and vacuum drying of interferon alpha-2a: development of a single-step process for obtaining dry, stable protein formulation.

Feasibility studies were performed to develop a process for obtaining stable dry protein formulations based on in situ polyethylene glycol (PEG)-induced precipitation and vacuum drying of interferon alpha-2a (IFNalpha2a) solution in a vial. Using a laboratory scale freeze dryer, the process was carried out in two phases: first, protein solution containing PEG was concentrated to achieve protein precipitation, and second, remaining water was removed by further reducing the chamber pressure. Drying conditions, i.e. temperature and pressure, and solution composition were selected to ensure maximal precipitation (solubility of IFNalpha2a), to achieve precipitation without boiling, and to ensure stability. Dried formulations were subjected to stability studies (40 degrees C). Concentration and precipitation could be achieved at a fast rate by utilizing pressures slightly above the vapor pressure of water. Fluorescence and circular dichroism (CD) studies showed that precipitated IFNalpha2a maintained its native structure. Fourier transform infrared spectroscopy (FTIR) studies showed that IFNalpha2a when dried in the presence of trehalose, maintained its secondary structure. Trehalose also prevented formation of aggregates during drying. Moisture contents of 1% (w/w) were achieved within 48 h of drying. Dry formulation containing 1:20:100 (w/w) IFNalpha2a:trehalose:mannitol was stable against aggregation and oxidation (6% oxidized at 40 degrees C, 6 months). Stability profile was comparable to a similar lyophilized formulation.

Effect of polyols on polyethylene glycol (PEG)-induced precipitation of proteins: Impact on solubility, stability and conformation.

Effect of polyols on the solubility of bovine serum albumin (BSA) in the presence of polyethylene glycols (PEGs) was investigated in order to strengthen the understanding of the observed effects of polyols and PEGs on protein properties in solution. Effect of polyols and/or PEGs on the thermodynamic (conformational) stability of BSA was measured using DSC and circular dichroism (CD). Glucose, sucrose, raffinose, glycerol and sorbitol, all reduced the extent of protein precipitation. Solubility of BSA in the presence of ethylene glycol increased in the case of PEG 1450 and PEG 8000, but was unaffected in the case of PEG 400. DSC studies indicated that smaller PEGs have destabilizing influence on protein structure. CD studies showed that smaller PEGs (ethylene glycol) induce subtle unfolding while stabilizing polyols induce subtle compaction. Results show that, effect of polyols on the apparent solubility of the protein correlates with their effect on the thermodynamic stability of the protein, smaller PEGs are not appropriate for estimating the activity of proteins in saturated solutions, and subtle changes in protein conformation can significantly affect protein precipitation. Though smaller PEGs have weak attractive interactions with protein molecules, perturbation of protein structure by PEGs can be balanced by utilizing appropriate stabilizing solutes.

Second derivative tryptophan fluorescence spectroscopy as a tool to characterize partially unfolded intermediates of proteins.

The application of second derivative tryptophan (Trp) fluorescence spectroscopy to characterize partially unfolded intermediates of proteins relevant to protein formulation was investigated. The second derivatives of the normalized emission scans of N-acetyl tryptophanamide (NATA), single-Trp containing proteins, somatostatin and human serum albumin (HSA), and two-Trp containing proteins previously shown to form partially unfolded intermediates, beta-lactoglobulin (beta Lg) and interferon alpha-2a (IFN alpha 2a), were studied in solution. The second derivative of NATA in water showed three bands at 340, 348 and 367 nm. The 340 nm band showed a blue shift, whereas the intensity of all three bands was affected by a decrease in solution polarity. Second derivative of single-Trp containing proteins, somatostatin and HSA, showed three negative bands, whereas, the second derivative of the two-Trp containing proteins, beta Lg and IFN alpha 2a, showed four bands, two of which lie in the 320-340 nm range. These two bands were attributed to the presence of the Trps in different microenvironments. The characteristic changes in the intensities of these two bands on addition of guanidine hydrochloride (beta Lg) and with a decrease in solution pH (IFN alpha 2a) were related to the presence of partially unfolded intermediates of these proteins. Thus, second derivative Trp fluorescence spectroscopy can be used as an important tool to identify partially unfolded states of proteins during formulation utilizing order of magnitude lower concentrations compared to such other technique as near UV CD.

Steady-state tryptophan fluorescence spectroscopy study to probe tertiary structure of proteins in solid powders.

The purpose of this work was to obtain information about protein tertiary structure in solid state by using steady state tryptophan (Trp) fluorescence emission spectroscopy on protein powders. Beta-lactoglobulin (betaLg) and interferon alpha-2a (IFN) powder samples were studied by fluorescence spectroscopy using a front surface sample holder. Two different sets of dried betaLg samples were prepared by vacuum drying of solutions: one containing betaLg, and the other containing a mixture of betaLg and guanidine hydrochloride. Dried IFN samples were prepared by vacuum drying of IFN solutions and by vacuum drying of polyethylene glycol precipitated IFN. The results obtained from solid samples were compared with the emission scans of these proteins in solutions. The emission scans obtained from protein powders were slightly blue-shifted compared to the solution spectra due to the absence of water. The emission scans were red-shifted for betaLg samples dried from solutions containing GuHCl. The magnitude of the shifts in lambda(max) depended on the extent of drying of the samples, which was attributed to the crystallization of GuHCl during the drying process. The shifts in the lambda(max) of the Trp emission spectrum are associated with the changes in the tertiary structure of betaLg. In the case of IFN, the emission scans obtained from PEG-precipitated and dried sample were different compared to the emission scans obtained from IFN in solution and from vacuum dried IFN. The double peaks observed in this sample were attributed to the unfolding of the protein. In the presence of trehalose, the two peaks converged to form a single peak, which was similar to solution emission spectra, whereas no change was observed in the presence of mannitol. We conclude that Trp fluorescence spectroscopy provides a simple and reliable means to characterize Trp microenvironment in protein powders that is related to the tertiary conformation of proteins in the solid state. This study shows that the use of fluorescence spectroscopy of proteins can be extended from simple protein aqueous solutions to protein powders, precipitates, and semidried protein samples to gain understanding of protein tertiary structure in these physical states.

Polyethylene glycol-induced precipitation of interferon alpha-2a followed by vacuum drying: development of a novel process for obtaining a dry, stable powder.

Feasibility studies were performed on the development of a novel process based on polyethylene glycol (PEG)-induced precipitation of proteins followed by vacuum drying in the presence of sugars to obtain dry protein powders. Apparent solubility of interferon alpha-2a (IFNalpha2a) was determined in the presence of various PEGs and the effect of solution pH, ionic strength, and temperature was investigated. IFNalpha2a precipitate was dried at a shelf temperature of 25 degrees C at 100 mTorr either as it is or in the presence of mannitol and/or trehalose. The dried IFNalpha2a formulations were subjected to accelerated stability studies at 40 degrees C (3 months), and the stability was compared with that of a similar lyophilized formulation. The results indicated that more than 90% of the protein could be precipitated using 10% wt/vol PEG 1450 at pH 6.5 at a solution ionic strength of 71 mM. Vacuum drying of the precipitate only resulted in the formation of insoluble aggregates of IFNalpha2a; however, this was prevented by the addition of either mannitol or trehalose. The addition of excess mannitol resulted in low residual moisture content and better handling of the final dried product. Accelerated storage stability did not show any aggregation and showed less than 5% formation of oxidized IFNalpha2a in the dried formulation containing IFNalpha2a:trehalose:mannitol in a 1:10:100 wt/wt ratio upon storage at 40 degrees C for 3 months. The stability of this vacuum dried formulation was comparable with that of a similar lyophilized formulation.

Effect of vacuum drying on protein-mannitol interactions: the physical state of mannitol and protein structure in the dried state.

The purpose of the present studies was to systematically investigate protein-mannitol interactions using vacuum drying, to obtain a better understanding of the effect of protein/mannitol wt/wt ratios on the physical state of mannitol and protein secondary structure in the dried state. Solutions containing beta-lactoglobulin (betaLg):mannitol (1:1-1:15 wt/wt) were vacuum dried at 5 degrees C under 3000 mTorr of pressure. The physical state of mannitol was studied using x-ray powder diffractometry (XRPD), polarized light microscopy (PLM), Fourier-transform infrared (FTIR) spectroscopy, and modulated differential scanning calorimetry (MDSC). XRPD studies indicated that mannitol remained amorphous up to 1:5 wt/wt betaLg:mannitol ratio, whereas PLM showed the presence of crystals of mannitol in all dried samples except for the 1:1 wt/wt betaLg:mannitol dried sample. FTIR studies indicated that a small proportion of crystalline mannitol was present along with the amorphous mannitol in dried samples at lower (less than 1:5 wt/wt) betaLg:mannitol ratios. The T(g) of the dried 1:1 wt/wt betaLg:mannitol sample was observed at 33.4 degrees C in MDSC studies, which indicated that at least a part of mannitol co-existed with protein in a single amorphous phase. Evaluation of the crystallization exotherms indicated that irrespective of the betaLg:protein wt/wt ratio in the initial sample, the protein to amorphous mannitol ratio was below 1:1 wt/wt in all dried samples. Second-derivative FTIR studies on dried betaLg and recombinant human interferon alpha-2a samples showed that mannitol affected protein secondary structure to a varying degree depending on the overall mannitol content in the dried sample and the type of protein.

Characterization of antibody-polyol interactions by static light scattering: implications for physical stability of protein formulations.

In this study, the nature of interactions between monoclonal antibodies and polyols was studied using static light scattering. Solutions of mAb-U and mAb-P (4-12 mg/mL) were analyzed using static light scattering in buffer, 10% w/v trehalose and ethylene glycol solutions at pH 5.0, 7.0 and 9.0. Mechanical stress studies were conducted by shaking the mAb-U solutions (5mg/mL, pH 5.0, 7.0 and 9.0) and mAb-P solutions (5mg/mL, pH 7.0) at 200 rpm for 5 days at 25°C. Addition of trehalose and ethylene glycol resulted in a decrease in the attractive interactions between mAb-U molecules at pH 7.0 and 9.0, and at pH 9.0 between mAb-P molecules. At a higher ionic strength (300 mM, pH 5.0) trehalose and ethylene glycol decreased attractive interactions for both mAbs. Mechanical stress studies showed higher aggregation of mAb-U in trehalose solutions than ethylene glycol and buffer solutions at pH 7.0 and 9.0. A converse trend was seen for mAb-P at pH 7.0. This study showed that polyols, conformational stabilizers or destabilizers, decrease attractive interactions between protein molecules. The decrease is a result of masking of the hydrophobic sites on a protein as polyols can have favorable hydrophobic interactions with the surface exposed hydrophobic groups.

Application of second-derivative fluorescence spectroscopy to monitor subtle changes in a monoclonal antibody structure.

In this study, the tertiary structure of a monoclonal antibody was analyzed under thermal and chemical stresses using second-derivative fluorescence spectroscopy. The effect of polyols, sucrose, and ethylene glycol on the tertiary structure of monoclonal antibody-U (mAb-U) (pH 7.0) was studied under thermal stress (25°C-75°C). The tertiary structure of mAb-U was also analyzed upon chemical denaturation using urea (2.0-8.0 M). The second derivative of mAb-U showed three bands corresponding to the three spectral classes of tryptophan, class I (330 nm), class II (340 nm), and class III (350 nm). Class II was higher in intensity in the presence of polyols compared with the solution without any polyol. Thermally denatured structure of mAb-U in sucrose and ethylene glycol was distinctly different than that in buffer. Addition of urea resulted in a decrease in intensity of class I and II, and an increase in intensity of class III implying unfolding. This study showed that second-derivative fluorescence spectroscopy is an effective tool to monitor subtle alterations in the tertiary structure of proteins. The unfolding of a protein is reflected as an increase in the intensity of the polar class III accompanied with a decrease in the intensity of class I.

Isomerization of Asp-Asp motif in model peptides and a monoclonal antibody Fab fragment.

Isomerization of aspartyl (Asp or D) residues is a critical degradation route to consider for stable monoclonal antibody formulations. Among the known hotspot sequences, the DD motif is relatively understudied. To gain mechanistic insights, we used model hexapeptides, YADXFK, YADDXK, and DIDDDM, as surrogates for the hotspots in a Fab protein (YADDFK and DIDDDM), to characterize the rate-pH profile of Asp isomerization. Compared with the YADGFK peptide, isomerization of D3 (the first D in the DD pair) in YADDFK was highly pH dependent. Comparison of rate-pH profiles of YADDFK, YADNFK, and YADHFK revealed a charge effect of the n + 1 residue-isomerization rate is accelerated by the positive side chain and reduced by negative side chain at n + 1 residue. Studies on YADDFK, YADDAK, and YADDGK indicated a mutual impact of D3 and D4 on their respective isomerization rates through charge effect. Comparison of rate-pH profile of DIDDDM sequence in peptide models with that in the complementary determining region of the Fab showed a faster rate in the Fab than in peptides, presumably because of contribution from structural factors in the former.

Solubilities and transfer free energies of hydrophobic amino acids in polyol solutions: importance of the hydrophobicity of polyols.

The purpose of this work was to investigate the difference in the hydrophobicities of various polyols and the nature of interactions between hydrophobic amino acid side chains and polyols. The interactions were explored by conducting solubility studies of three amino acid derivatives, N-acetyl tryptophanamide (NATA), N-acetyl leucinamide (NALA), and N-acetyl glycinamide (NAGA), in the solutions of sorbitol, sucrose, trehalose, glycerol, ethylene glycol, ribose, and deoxyribose. Hydrophobicity index of polyols was calculated using molecular modeling. An increase in the solubility of the hydrophobic side chains of tryptophan and leucine was observed with an increase in the hydrophobicity index of polyols. Transfer free energies of NATA from water to polyols solutions were negative, whereas those for NALA were positive for all polyols except glycol. This study shows that the hydrophobic nature of polyols plays an important role in polyol-side chain interactions. Solubility behavior observed for NATA and NALA in different polyols indicates that polyols can interact differently with the same side chain depending on the nature of the polyol and the side chain.

Modulation of the thermodynamic stability of proteins by polyols: significance of polyol hydrophobicity and impact on the chemical potential of water.

The exact mechanism of the modulation of chemical potential of proteins by polyols is not yet well understood. Present study investigates the role of hydrophobicity of polyols, and their impact on water activity and/or surface tension, in determining their stabilization/destabilization potential. Results with ribose and methyl-glucose show that the enhanced stability of proteins is not mediated via the effect on interfacial tension, a hypothesis that has so far been restricted to glycerol. An exemplary correlation between thermodynamic stabilization (ΔG(f-uf)), and polyol osmolality, confirms/generalizes the prominent role of water activity in the observed stabilization effects. Results show that even seemingly hydrophilic sugars such as deoxy-ribose can interact favorably with proteins, suggesting that properties other than the presence of hydroxyl groups also contribute to the net effect of polyols. We demonstrate that the hydrophobicity index of polyols and the net stabilization effect afforded to proteins have an excellent inverse correlation. These studies show that the weak hydrophobicity of polyols is critical for promoting their interactions with proteins, weakening of the hydrophobic forces within the protein interior and counteracting the polyol induced-solvent mediated stabilization effect.