A comparison of biophysical characterization techniques in predicting monoclonal antibody stability.

With the rapid growth of biopharmaceutical product development, knowledge of therapeutic protein stability has become increasingly important. We evaluated assays that measure solution-mediated interactions and key molecular characteristics of 9 formulated monoclonal antibody (mAb) therapeutics, to predict their stability behavior. Colloidal interactions, self-association propensity and conformational stability were measured using effective surface charge via zeta potential, diffusion interaction parameter (kD) and differential scanning calorimetry (DSC), respectively. The molecular features of all 9 mAbs were compared to their stability at accelerated (25°C and 40°C) and long-term storage conditions (2-8°C) as measured by size exclusion chromatography. At accelerated storage conditions, the majority of the mAbs in this study degraded via fragmentation rather than aggregation. Our results show that colloidal stability, self-association propensity and conformational characteristics (exposed tryptophan) provide reasonable prediction of accelerated stability, with limited predictive value at 2-8°C stability. While no correlations to stability behavior were observed with onset-of-melting temperatures or domain unfolding temperatures, by DSC, melting of the Fab domain with the CH2 domain suggests lower stability at stressed conditions. The relevance of identifying appropriate biophysical assays based on the primary degradation pathways is discussed.

The influence of specific binding of collagen-silk chimeras to silk biomaterials on hMSC behavior.

Collagen-like proteins in the bacteria Streptococcus pyogenes adopt a triple-helix structure with a thermal stability similar to that of animal collagens, can be expressed in high yield in Escherichia coli and can be easily modified through molecular biology techniques. However, potential applications for such recombinant collagens are limited by their lack of higher order structure to achieve the physical properties needed for most biomaterials. To overcome this problem, the S. pyogenes collagen domain was fused to a repetitive Bombyx mori silk consensus sequence, as a strategy to direct specific non-covalent binding onto solid silk materials whose superior stability, mechanical and material properties have been previously established. This approach resulted in the successful binding of these new collagen-silk chimeric proteins to silk films and porous scaffolds, and the binding affinity could be controlled by varying the number of repeats in the silk sequence. To explore the potential of collagen-silk chimera for regulating biological activity, integrin (Int) and fibronectin (Fn) binding sequences from mammalian collagens were introduced into the bacterial collagen domain. The attachment of bioactive collagen-silk chimeras to solid silk biomaterials promoted hMSC spreading and proliferation substantially in comparison to the controls. The ability to combine the biomaterial features of silk with the biological activities of collagen allowed more rapid cell interactions with silk-based biomaterials, improved regulation of stem cell growth and differentiation, as well as the formation of artificial extracellular matrices useful for tissue engineering applications.

Interruptions in the collagen repeating tripeptide pattern can promote supramolecular association.

The standard collagen triple-helix requires a perfect (Gly-Xaa-Yaa)(n) sequence, yet all nonfibrillar collagens contain interruptions in this tripeptide repeating pattern. Defining the structural consequences of disruptions in the sequence pattern may shed light on the biological role of sequence interruptions, which have been suggested to play a role in molecular flexibility, collagen degradation, and ligand binding. Previous studies on model peptides with 1- and 4-residue interruptions showed a localized perturbation within the triple-helix, and this work is extended to introduce natural collagen interruptions up to nine residue in length within a fixed (Gly-Pro-Hyp)(n) peptide context. All peptides in this set show decreases in triple-helix content and stability, with greater conformational perturbations for the interruptions longer than five residue. The most stable and least perturbed structure is seen for the 5-residue interruption peptide, whose sequence corresponds to a Gly to Ala missense mutation, such as those leading to collagen genetic diseases. The triple-helix peptides containing 8- and 9-residue interruptions exhibit a strong propensity for self-association to fibrous structures. In addition, a small peptide modeling only the 9-residue sequence within the interruption aggregates to form amyloid-like fibrils with antiparallel beta-sheet structure. The 8- and 9-residue interruption sequences studied here are predicted to have significant cross-beta aggregation potential, and a similar propensity is reported for approximately 10% of other naturally occurring interruptions. The presence of amyloidogenic sequences within or between triple-helix domains may play a role in molecular association to normal tissue structures and could participate in observed interactions between collagen and amyloid.

Self-association of streptococcus pyogenes collagen-like constructs into higher order structures.

A number of bacterial collagen-like proteins with Gly as every third residue and a high Pro content have been observed to form stable triple-helical structures despite the absence of hydroxyproline (Hyp). Here, the high yield cold-shock expression system is used to obtain purified recombinant collagen-like protein (V-CL) from Streptococcus pyogenes containing an N-terminal globular domain V followed by the collagen triple-helix domain CL and the modified construct with two tandem collagen domains V-CL-CL. Both constructs and their isolated collagenous domains form stable triple-helices characterized by very sharp thermal transitions at 35-37 degrees C and by high values of calorimetric enthalpy. Procedures for the formation of collagen SLS crystallites lead to parallel arrays of in register V-CL-CL molecules, as well as centrosymmetric arrays of dimers joined at their globular domains. At neutral pH and high concentrations, the bacterial constructs all show a tendency towards aggregation. The isolated collagen domains, CL and CL-CL, form units of diameter 4-5 nm which bundle together and twist to make larger fibrillar structures. Thus, although this S. pyogenes collagen-like protein is a cell surface protein with no indication of participation in higher order structure, the triple-helix domain has the potential of forming fibrillar structures even in the absence of hydroxyproline. The formation of fibrils suggests bacterial collagen proteins may be useful for biomaterials and tissue engineering applications.

NMR monitoring of chain-specific stability in heterotrimeric collagen peptides.

NMR spectroscopy is used to investigate the heterotrimeric nature of a collagen model peptide. Two distinct peptide chains (A and B) were synthesized to model a site in heterotrimeric basement membrane type IV collagen. For NMR studies, four amino acids in the B chain were labeled with 15N/13C. Circular dichroism spectroscopy and differential scanning calorimetry thermal stability results on a solution with both A and B peptides (molar ratio 2A:1B) are consistent with the presence of one heterotrimeric triple-helical molecular species. Heteronuclear single quantum coherence experiments on homotrimers of the B peptide show trimer peaks which disappear at temperatures higher than 10 degrees C, while the 2A:1B mixture has trimer peaks with increased stability and altered chemical shifts. The reduction in the number of Leu trimer peaks from three to one and the increased stability of trimer resonances confirm the participation of B chains in an AAB heterotrimer molecule.

Triple-helical peptides: an approach to collagen conformation, stability, and self-association.

Peptides have been an integral part of the collagen triple-helix structure story, and have continued to serve as useful models for biophysical studies and for establishing biologically important sequence-structure-function relationships. High resolution structures of triple-helical peptides have confirmed the basic Ramachandran triple-helix model and provided new insights into the hydration, hydrogen bonding, and sequence dependent helical parameters in collagen. The dependence of collagen triple-helix stability on the residues in its (Gly-X-Y)(n) repeating sequence has been investigated by measuring melting temperatures of host-guest peptides and an on-line collagen stability calculator is now available. Although the presence of Gly as every third residue is essential for an undistorted structure, interruptions in the repeating (Gly-X-Y)(n) amino acid sequence pattern are found in the triple-helical domains of all nonfibrillar collagens, and are likely to play a role in collagen binding and degradation. Peptide models indicate that small interruptions can be incorporated into a rod-like triple-helix with a highly localized effect, which perturbs hydrogen bonds and places the standard triple-helices on both ends out of register. In contrast to natural interruptions, missense mutations which replace one Gly in a triple-helix domain by a larger residue have pathological consequences, and studies on peptides containing such Gly substitutions clarify their effect on conformation, stability, and folding. Recent studies suggest peptides may also be useful in defining the basic principles of collagen self-association to the supramolecular structures found in tissues.

Physicochemical properties and antibacterial activity of the precipitate of vancomycin and ceftazidime: implications in the management of endophthalmitis.

OBJECTIVE: To study the physicochemical properties, bioavailability, and microbicidal activity of vancomycin and ceftazidime in the precipitate formed by the drugs in mixture. METHODS: The precipitation of the drugs prepared in buffers in the pH range of 5.8 to 8.9, in normal saline, in balanced salt solution (BSS), and in Ringer lactate, was studied by light scatter analysis. Bioavailability and antibacterial activity of the precipitate and the supernatant were estimated using high-performance liquid chromatography (HPLC) and microbiologic assay respectively. RESULTS: The scatter analysis showed precipitation of vancomycin alone at pH 7.5 and in BSS. When mixed together precipitation was observed in the mixture of the two drugs within the pH range of 6.8 to 8.0 and in all solutions. HPLC of both supernatant and precipitate showed the presence of active drugs and the microbiologic assay confirmed its inhibitory activity against bacteria. The precipitate had greater activity against Gram-positive bacteria while the supernatant showed greater activity against Gram-negative bacteria. CONCLUSIONS: Vancomycin's pH dependent precipitation occurs regardless of the presence of ceftazidime. The precipitate as well as the supernatant retains significant antibacterial activity thus confirming the efficacy of combination therapy with vancomycin and ceftazidime in the management of bacterial endophthalmitis.

Common interruptions in the repeating tripeptide sequence of non-fibrillar collagens: sequence analysis and structural studies on triple-helix peptide models.

Interruptions in the repeating (Gly-X1-X2)(n) amino acid sequence pattern are found in the triple-helix domains of all non-fibrillar collagens, and perturbations to the triple-helix at such sites are likely to play a role in collagen higher-order structure and function. This study defines the sequence features and structural consequences of the most common interruption, where one residue is missing from the tripeptide pattern, Gly-X1-X2-Gly-AA(1)-Gly-X1-X2, designated G1G interruptions. Residues found within G1G interruptions are predominantly hydrophobic (70%), followed by a significant amount of charged residues (16%), and the Gly-X1-X2 triplets flanking the interruption are atypical. Studies on peptide models indicate the degree of destabilization is much greater when Pro is in the interruption, GP, than when hydrophobic residues (GF, GY) are present, and a rigid Gly-Pro-Hyp tripeptide adjacent to the interruption leads to greater destabilization than a flexible Gly-Ala-Ala sequence. Modeling based on NMR data indicates the Phe residue within a GF interruption is located on the outside of the triple helix. The G1G interruptions resemble a previously studied collagen interruption GPOGAAVMGPO, designated G4G-type, in that both are destabilizing, but allow continuation of rod-like triple helices and maintenance of the single residue stagger throughout the imperfection, with a loss of axial register of the superhelix on both sides. Both kinds of interruptions result in a highly localized perturbation in hydrogen bonding and dihedral angles, but the hydrophobic residue of a G4G interruption packs near the central axis of the superhelix, while the hydrophobic residue of a G1G interruption is located on the triple-helix surface. The different structural consequences of G1G and G4G interruptions in the repeating tripeptide sequence pattern suggest a physical basis for their differential susceptibility to matrix metalloproteinases in type X collagen.

Kinetics and thermodynamics of amyloid assembly using a high-performance liquid chromatography-based sedimentation assay.

Nonnative protein aggregation has been classically treated as an amorphous process occurring by colloidal coagulation kinetics and proceeding to an essentially irreversible endpoint often ascribed to a chaotic tangle of unfolded chains. However, some nonnative aggregates, particularly amyloid fibrils, exhibit ordered structures that appear to assemble according to ordered mechanisms. Some of these fibrils, as illustrated here with the Alzheimer's plaque peptide amyloid beta, assemble to an endpoint that is a dynamic equilibrium between monomers and fibrils exhibiting a characteristic equilibrium constant with an associated free energy of formation. Some fibrils, as illustrated here with the polyglutamine repeat sequences associated with Huntington's disease, assemble via highly regular mechanisms exhibiting nucleated growth polymerization kinetics. Here, we describe a series of linked methods for quantitative analysis of such aggregation kinetics and thermodynamics, focusing on a robust high-performance liquid chromatography (HPLC)-based sedimentation assay. An integrated group of protocols is provided for peptide disaggregation, setting up the HPLC sedimentation assay, the preparation of fibril seed stocks and determination of the average functional molecular weight of the fibrils, elongation and nucleation kinetics analysis, and the determination of the critical concentration describing the thermodynamic endpoint of fibril elongation.

High incidence of later-onset fabry disease revealed by newborn screening.

The classic phenotype of Fabry disease, X-linked alpha -galactosidase A (alpha -Gal A) deficiency, has an estimated incidence of approximately 1 in 50,000 males. The recent recognition of later-onset variants suggested that this treatable lysosomal disease is more frequent. To determine the disease incidence, we undertook newborn screening by assaying the alpha-Gal A activity in blood spots from 37,104 consecutive Italian male neonates. Enzyme-deficient infants were retested, and "doubly screened-positive" infants and their relatives were diagnostically confirmed by enzyme and mutation analyses. Twelve (0.03%) neonates had deficient alpha-Gal A activities and specific mutations, including four novel missense mutations (M51I, E66G, A73V, and R118C), three missense mutations (F113L, A143T, and N215S) identified previously in later-onset patients, and one splicing defect (IVS5(+1G-->T)) reported in a patient with the classic phenotype. Molecular modeling and in vitro overexpression of the missense mutations demonstrated structures and residual activities, which were rescued/enhanced by an alpha-Gal A-specific pharmacologic chaperone, consistent with mutations that cause the later-onset phenotype. Family studies revealed undiagnosed Fabry disease in affected individuals. In this population, the incidence of alpha-Gal A deficiency was 1 in approximately 3,100, with an 11 : 1 ratio of patients with the later-onset : classic phenotypes. If only known disease-causing mutations were included, the incidence would be 1 in approximately 4,600, with a 7 : 1 ratio of patients with the later-onset : classic phenotypes. These results suggest that the later-onset phenotype of Fabry disease is underdiagnosed among males with cardiac, cerebrovascular, and/or renal disease. Recognition of these patients would permit family screening and earlier therapeutic intervention. However, the higher incidence of the later-onset phenotype in patients raises ethical issues related to when screening should be performed--in the neonatal period or at early maturity, perhaps in conjunction with screening for other treatable adult-onset disorders.

Oligoproline effects on polyglutamine conformation and aggregation.

There are nine known expanded CAG repeat neurological diseases, including Huntington's disease (HD), each involving the repeat expansion of polyglutamine (polyGln) in a different protein. Similar conditions can be induced in animal models by expression of the polyGln sequence alone or in other protein contexts. Besides the polyGln sequence, the cellular context of the disease protein, and the sequence context of the polyGln within the disease protein, are both likely to contribute to polyGln physical behavior and to pathology. In HD, the N-terminal, exon-1 segment of the protein huntingtin contains the polyGln sequence immediately followed by an oligoproline region. We show here that introduction of a P10 sequence C-terminal to polyGln in synthetic peptides decreases both the rate of formation and the apparent stability of the amyloid-like aggregates associated with this family of diseases. The sequence can be trimmed to P6 without altering the suppression, but a P3 sequence is ineffective. Spacers up to at least three amino acid residues in length can be inserted between polyGln and P10 without altering this effect. There is no suppression, however, when the P10 sequence is either placed on the N-terminal side of polyGln or attached to polyGln via a side-chain tether. The nucleation mechanism of a Q40 sequence is unchanged upon addition of a P10 C-terminal extension, yielding a critical nucleus of one. The effects of oligoPro length and structural context on polyGln aggregation are correlated strongly with alterations in the circular dichroism spectra of the monomeric peptides. For example, the P10 sequence eliminates the small amount of alpha helical content otherwise exhibited by the Q40 sequence. The P10 sequence may suppress aggregation by stabilizing an aggregation-incompetent conformation of the monomer. The effect is transportable: a P10 sequence fixed to the C terminus of the sequence Abeta similarly modulates amyloid fibril formation.

Peroxynitrite reaction with eye lens proteins: alpha-crystallin retains its activity despite modification.

PURPOSE: . Peroxynitrite is a highly potent reactive oxygen/nitrogen species present in the environment and also endogenously in the eye, that causes a variety of disorders. This study was undertaken to look at the oxidative damage that peroxynitrite causes to the proteins of the lens and the functional consequences thereof. METHODS: . Peroxynitrite was allowed to react with alpha-, beta-, and gamma-crystallins. The formation of nitrotyrosine and nitrotryptophan, dityrosine, protein covalent cross-links, and chain degradation products were monitored by spectroscopy and SDS-PAGE. Conformational changes occurring in the protein were monitored with circular dichroism spectroscopy. The chaperoning ability of alpha-crystallin was assayed by monitoring its ability to inhibit the self-aggregation of two test proteins: beta-crystallin and insulin. RESULTS: . Peroxynitrite reaction produced nitrotyrosine, nitrotryptophan, and dityrosine, nondisulfide covalent cross-linked aggregates, and peptide chain degradation. The hydroxyl radicals produced by peroxynitrite caused more chain degradation than did the carbonate radicals. The oxidative reaction caused increased conformational disorder. The yield was highest in gamma-crystallin and least in alpha-crystallin. The chaperoning ability of alpha-crystallin was not affected. CONCLUSIONS: . Peroxynitrite reacts with lens proteins, causing extensive covalent chemical changes. However, alpha-crystallin retains its chaperoning ability, despite the oxidative changes caused by the peroxynitrite reaction, indicating its functional robustness.

Modified high-performance liquid chromatography technique for detection of vancomycin in human vitreous.

PURPOSE: To standardize the technique and methodology for estimating the level of vancomycin in human vitreous using a modified high-performance liquid chromatographic method. METHODS: Sample preparation involved spiking the vitreous with known quantities of the drug followed by a brief treatment with 30% trichloroacetic acid. Samples were analyzed on a C18 reverse-phase column using a mobile phase of 50 mM phosphate buffer (pH 4.0) and 10% acetonitrile. RESULTS: Vancomycin was detected at 198 nm. It showed a retention time of 2.2 min in the vitreous. A linear increase in the area under the peak corresponding to the drug was observed with increase in concentration of vancomycin in the vitreous. The method was applied to detect the vancomycin levels in vitreous of 5 patients with exogenous endophthalmitis, 24-48 h after intravitreal injection of vancomycin 1 mg/0.1 ml. The mean concentration of vancomycin in these samples was 120.022 +/- 59.87 microg/ml (43.859-179.09 microg/ml). The present technique allowed a quantification limit of 1 microg/ml. CONCLUSION: This technique is suitable to estimate vancomycin levels in human vitreous in a variety of clinical and experimental studies. It has the added advantages of being less expensive, simple and rapid.

Molecular and cellular assessment of ginkgo biloba extract as a possible ophthalmic drug.

We have investigated the biochemical and cell biological basis of the reported beneficiary effects of the leaf extracts of the plant Ginkgo biloba, which has been used as a possible ophthalmic drug. The antioxidant, antimicrobial, anti-apoptotic and cytoprotective properties of the standardized extract called EGb761 were assayed. Chemical stresses were induced in cells using alloxan or dexamethasone, and the effect of EGb761 on them was studied using the MTT and TUNEL assays. Its ability to modulate the activities of some antioxidant enzymes was tested in vitro. In addition, cataract was induced in rats through selenite injection, and the effect of EGb761 administration on the progression of cataract was studied using slit lamp examination. Ginkgo biloba was found to be an excellent antioxidant. It readily scavenges reactive oxygen and nitrogen radicals and inhibits oxidative modifications that occur to proteins in vitro. It enters intact cells and protects them from alloxan-mediated and light-mediated stress, and the nuclear DNA from single strand breaks. It also effectively inhibits chemically induced apoptosis. It does not modulate the activities of endogenous antioxidant enzymes, nor does it have any significant antimicrobial activity. Unlike some other plant extracts, it is not phototoxic. In experiments wherein selenite cataract was induced in laboratory rats, treatment with the extract significantly retards the progression of lens opacification in vivo. Ginkgo biloba's inherent antioxidant, antiapoptotic and cytoprotective action and potential anticataract ability appear to be some of the factors responsible for its beneficial effects.

Role of xanthurenic acid 8-O-beta-D-glucoside, a novel fluorophore that accumulates in the brunescent human eye lens.

We have been able to identify a blue fluorophore from the low-molecular weight soluble fraction of human adult nondiabetic brunescent cataract lenses as xanthurenic acid 8-O-beta-D-glucoside (XA8OG) (excitation = 338 nm and emission = 440 nm). To determine the role of this fluorophore in the lens, we have examined its photophysical and photodynamic properties. We found XA8OG to have a fluorescence quantum yield (phi) of 0.22 and a major emission lifetime of 12 ns. We found it to be a UVA-region sensitizer, capable of efficiently generating singlet oxygen species but little of superoxide. We also demonstrated that XA8OG oxidizes proteins when irradiated with UVA light, causing photodynamic covalent chemical damage to proteins. Its accumulation in the aging human lens (and the attendant decrease of its precursor O-beta-D-glucoside of 3-hydroxykynurenine) can, thus, add to the oxidative burden on the system. XA8OG, thus, appears to be an endogenous chromophore in the lens, which can act as a cataractogenic agent.