RESUMO
Screening for pharmaceutically viable stability from measurements of thermally induced protein unfolding and short-term accelerated stress underpins much molecule design, selection, and formulation in the pharmaceutical biotechnology industry. However, the interrelationships among intrinsic protein conformational stability, thermal denaturation, and pharmaceutical stability are complex. There are few publications in which predictions from thermal unfolding-based screening methods are examined together with pharmaceutically relevant long-term storage stability performance. We have studied eight developable therapeutic IgG molecules under solution conditions optimized for large-scale commercial production and delivery. Thermal unfolding profiles were characterized by differential scanning calorimetry (DSC) and intrinsic fluorescence recorded simultaneously with static light scattering (SLS). These molecules exhibit a variety of thermal unfolding profiles under common reference buffer conditions and under individually optimized formulation conditions. Aggregation profiles by SE-HPLC and bioactivity upon long-term storage at 5, 25, and 40 °C establish that IgG molecules possessing a relatively wide range of conformational stabilities and thermal unfolding profiles can be formulated to achieve pharmaceutically stable drug products. Our data suggest that a formulation design strategy that increases the thermal unfolding temperature of the Fab transition may be a better general approach to improving pharmaceutical storage stability than one focused on increasing Tonset or Tm of the first unfolding transition.
Assuntos
Anticorpos Monoclonais/química , Varredura Diferencial de Calorimetria , Química Farmacêutica , Cromatografia Líquida de Alta Pressão , Estabilidade de Medicamentos , Humanos , Imunoglobulina G/química , Luz , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Estabilidade Proteica , Espalhamento de Radiação , Espectrometria de Fluorescência , TemperaturaRESUMO
A simultaneous multiple sample light scattering (SMSLS) prototype instrument was built to simultaneously measure light scattering from many independent monoclonal antibody (mAb) solutions in order to monitor their time-dependent aggregation behavior and to characterize, via absolute Rayleigh scattering ratios, their molecular masses and second, third, and fourth virial coefficients under non-aggregating conditions at concentrations up to 190mg/ml. One stable mAb and another prone to aggregation were studied. Early phase aggregation rates spanned six orders of magnitude over temperatures 30 to 83°C for both mAbs and divided into "Arrhenius" and "Stochastic" regimes. The Arrhenius regimes comprise two thermal regimes whose breakpoint occurs near the first thermal unfolding temperature of the mAb domain structure. The Stochastic regime occurs for T⩽40°C. Rates yielded activation energies and temperature and concentration crossovers among rate-limiting regimes. Virial coefficients were closely related to aggregation kinetics. Hydrodynamic diameter relationship to virial coefficients provided further insight into stability. SMSLS detected as few as three dimerization events among 1000 monomeric proteins. Although early phase aggregation is linear in time and reproducible, aggregation becomes chaotic in later phases. SMSLS dramatically increases protein monitoring throughput, providing continuous monitoring for hours, weeks, and longer. New samples can be changed in and out without affecting other sample measurements in progress.
Assuntos
Proteínas/química , Análise Espectral/instrumentação , Análise Espectral/métodos , Anticorpos Monoclonais , Cinética , Proteínas/metabolismoRESUMO
Characterizing the denatured state ensemble is crucial to understanding protein stability and the mechanism of protein folding. The aim of this research was to see if fluorescence could be used to gain new information on the denatured state ensemble. Ribonuclease Sa (RNase Sa) contains no Trp residues. We made five variants of RNase Sa by adding Trp residues at locations where they are found in other members of the microbial ribonuclease family. To better understand the protein denatured state, we also studied the fluorescence properties of the following peptides: N-acetyl-Trp-amide (NATA), N-acetyl-Ala-Trp-Ala-amide (AWA), N-acetyl-Ala-Ala-Trp-Ala-Ala-amide (AAWAA), and the five pentapeptides with the same sequence as the Trp substitution sites in RNase Sa. The major conclusions are: 1), the wavelength of maximum fluorescence intensity, lambda(max), does not differ significantly for the peptides and the denatured proteins; 2), the fluorescence intensity at lambda(max), I(F), differs significantly for the five Trp containing variants of RNase Sa; 3), the I(F) differences for the denatured proteins are mirrored in the peptides, showing that the short-range effects giving rise to the I(F) differences in the peptides are also present in the proteins; 4) the I(F) values for the denatured proteins are more than 30% greater than for the peptides, showing the presence of long-range effects in the proteins; 5), fluorescence quenching of Trp by acrylamide and iodide is more than 50% greater in the peptides than in the denatured proteins, showing that long-range effects limit the accessibility of the quenchers to the Trp side chains in the proteins; and 6), these results show that nonlocal effects in the denatured states of proteins influence Trp fluorescence and accessibility significantly.
Assuntos
Ribonucleases/química , Espectrometria de Fluorescência/métodos , Ureia/química , Acrilamida/química , Acrilamidas/química , Sequência de Aminoácidos , Dissulfetos/química , Fluorescência , Iodetos/química , Conformação Molecular , Dados de Sequência Molecular , Peptídeos/química , Proteínas/química , Fatores de Tempo , TriptofanoRESUMO
This article probes the denatured state ensemble of ribonuclease Sa (RNase Sa) using fluorescence. To interpret the results obtained with RNase Sa, it is essential that we gain a better understanding of the fluorescence properties of tryptophan (Trp) in peptides. We describe studies of N-acetyl-L-tryptophanamide (NATA), a tripeptide: AWA, and six pentapeptides: AAWAA, WVSGT, GYWHE, HEWTV, EAWQE, and DYWTG. The latter five peptides have the same sequence as those surrounding the Trp residues studied in RNase Sa. The fluorescence emission spectra, the fluorescence lifetimes, and the fluorescence quenching by acrylamide and iodide were measured in concentrated solutions of urea and guanidine hydrochloride. Excited-state electron transfer from the indole ring of Trp to the carbonyl groups of peptide bonds is thought to be the most important mechanism for intramolecular quenching of Trp fluorescence. We find the maximum fluorescence intensities vary from 49,000 for NATA with two carbonyls, to 24,400 for AWA with four carbonyls, to 28,500 for AAWAA with six carbonyls. This suggests that the four carbonyls of AWA are better able to quench Trp fluorescence than the six carbonyls of AAWAA, and this must reflect a difference in the conformations of the peptides. For the pentapeptides, EAWQE has a fluorescence intensity that is more than 50% greater than DYWTG, showing that the amino acid sequence influences the fluorescence intensity either directly through side-chain quenching and/or indirectly through an influence on the conformational ensemble of the peptides. Our results show that peptides are generally better models for the Trp residues in proteins than NATA. Finally, our results emphasize that we have much to learn about Trp fluorescence even in simple compounds.
Assuntos
Biofísica/métodos , Peptídeos/química , Espectrometria de Fluorescência/métodos , Triptofano/análogos & derivados , Triptofano/química , Acrilamidas/química , Biologia Computacional/métodos , Guanidina/química , Iodetos/química , Conformação Molecular , Distribuição Normal , Temperatura , Fatores de Tempo , Tirosina/química , Ureia/químicaRESUMO
Ribonuclease Sa (RNase Sa) contains no tryptophan (Trp) residues. We have added single Trp residues to RNase Sa at sites where Trp is found in four other microbial ribonucleases, yielding the following variants of RNase Sa: Y52W, Y55W, T76W, and Y81W. We have determined crystal structures of T76W and Y81W at 1.1 and 1.0 A resolution, respectively. We have studied the fluorescence properties and stabilities of the four variants and compared them to wild-type RNase Sa and the other ribonucleases on which they were based. Our results should help others in selecting sites for adding Trp residues to proteins. The most interesting findings are: 1), Y52W is 2.9 kcal/mol less stable than RNase Sa and the fluorescence intensity emission maximum is blue-shifted to 309 nm. Only a Trp in azurin is blue-shifted to a greater extent (308 nm). This blue shift is considerably greater than observed for Trp71 in barnase, the Trp on which Y52W is based. 2), Y55W is 2.1 kcal/mol less stable than RNase Sa and the tryptophan fluorescence is almost completely quenched. In contrast, Trp59 in RNase T1, on which Y55W is based, has a 10-fold greater fluorescence emission intensity. 3), T76W is 0.7 kcal/mol more stable than RNase Sa, indicating that the Trp side chain has more favorable interactions with the protein than the threonine side chain. The fluorescence properties of folded Y76W are similar to those of the unfolded protein, showing that the tryptophan side chain in the folded protein is largely exposed to solvent. This is confirmed by the crystal structure of the T76W which shows that the side chain of the Trp is only approximately 7% buried. 4), Y81W is 0.4 kcal/mol less stable than RNase Sa. Based on the crystal structure of Y81W, the side chain of the Trp is 87% buried. Although all of the Trp side chains in the variants contribute to the unusual positive circular dichroism band observed near 235 nm for RNase Sa, the contribution is greatest for Y81W.