Several growth factors are expressed in distinct temporal and spatial patterns during fracture repair. Of these, vascular endothelial growth factor, VEGF, is of particular interest because of its ability to induce neovascularization (angiogenesis). To determine whether VEGF is required for bone repair, we inhibited VEGF activity during secondary bone healing via a cartilage intermediate (endochondral ossification) and during direct bone repair (intramembranous ossification) in a novel mouse model. Treatment of mice with a soluble, neutralizing VEGF receptor decreased angiogenesis, bone formation, and callus mineralization in femoral fractures. Inhibition of VEGF also dramatically inhibited healing of a tibial cortical bone defect, consistent with our discovery of a direct autocrine role for VEGF in osteoblast differentiation. In separate experiments, exogenous VEGF enhanced blood vessel formation, ossification, and new bone (callus) maturation in mouse femur fractures, and promoted bony bridging of a rabbit radius segmental gap defect. Our results at specific time points during the course of healing underscore the role of VEGF in endochondral vs. intramembranous ossification, as well as skeletal development vs. bone repair. The responses to exogenous VEGF observed in two distinct model systems and species indicate that a slow-release formulation of VEGF, applied locally at the site of bone damage, may prove to be an effective therapy to promote human bone repair.
Increasing the in vivo residence times of protein therapeutics could decrease their dosing frequencies. We show that genetic fusion of an unstructured recombinant polypeptide of 864 amino acids, called XTEN, to a peptide or protein provides an apparently generic approach to extend plasma half-life. Allometric scaling suggests that a fusion of XTEN to the exenatide peptide should increase exenatide half-life in humans from 2.4 h to a projected time of 139 h. We confirmed the biological activity of the exenatide-XTEN fusion in mice. As extended stability might exacerbate undesirable side effects in some cases, we show that truncating the XTEN sequence can regulate plasma half-life. XTEN lacks hydrophobic amino acid residues that often contribute to immunogenicity and complicate manufacture. Based on data on XTEN fusions to exenatide, glucagon, GFP and human growth hormone, we expect that XTEN will enable dosing of otherwise rapidly cleared protein drugs at up to monthly intervals in humans.
Recombinant humanized monoclonal antibody HER2, rhuMAb HER2, in liquid formulations undergoes oxidation when exposed to intense light and elevated temperatures (30 & 40 degrees C). Met-255 in the heavy chain of the Fc region of the antibody is the primary site of oxidation. Met-431 of the Fc fragment can also be oxidized under extreme conditions. The amount of oxidation was determined by cleaving the Fab and Fc fragments by papain digestion, and the oxidized Fc fragment was detected by hydrophobic interaction chromatography. Oxidation of rhuMAb HER2 was also formulation dependent. The presence of NaCl in the rhuMAb HER2 formulation caused an increase in oxidation at higher temperatures after contact with stainless steel containers or stainless steel components in the filling process. The corrosion of stainless steel by chloride ions at the low pH of the formulation buffer generated iron ions that catalyzed methionine oxidation in rhuMAb HER2. Temperature-induced oxidation of rhuMAb HER2 occurred by the formation of free radicals, and light-induced oxidation of rhuMAb HER2 occurred via single oxygen pathway. Antioxidants, such as methionine, sodium thiosulfate, catalase, or platinum, prevented Met oxidation in rhuMAb HER2, presumably as free radicals or oxygen scavengers. The minimum effective levels (molar ratios of protein to antioxidant) required to inhibit temperature-induced oxidation were 1:5 and 1:25 for methionine and thiosulfate, respectively. A thiosulfate adduct of rhuMAb HER2 was observed by cation-exchange chromatography. These studies demonstrate that stoichiometric amounts of methionine and thiosulfate are sufficient to eliminate temperature-induced oxidation of rhuMAb HER2 caused by free radicals that were generated by the presence of metal ion and peroxide impurities in the formulation.
Aggregation of proteins, even under conditions favoring the native state, is a ubiquitous problem in biotechnology and biomedical engineering. Providing a mechanistic basis for the pathways that lead to aggregation should allow development of rational approaches for its prevention. We have chosen recombinant human interferon-␥ (rhIFN-␥) as a model protein for a mechanistic study of aggregation. In the presence of 0.9 M guanidinium hydrochloride, rhIFN-␥ aggregates with first order kinetics, a process that is inhibited by addition of sucrose. We describe a pathway that accounts for both the observed first-order aggregation of rhIFN-␥ and the effect of sucrose. In this pathway, aggregation proceeds through a transient expansion of the native state. Sucrose shifts the equilibrium within the ensemble of rhIFN-␥ native conformations to favor the most compact native species over more expanded ones, thus stabilizing rhIFN-␥ against aggregation. This phenomenon is attributed to the preferential exclusion of sucrose from the protein surface. In addition, kinetic analysis combined with solution thermodynamics shows that only a small (9%) expansion surface area is needed to form the transient native state that precedes aggregation. The approaches used here link thermodynamics and aggregation kinetics to provide a powerful tool for understanding both the pathway of protein aggregation and the rational use of excipients to inhibit the process.Formation of biologically inactive proteins by aggregation is a problem of considerable importance in many disciplines (1-3). For example, protein aggregates can be formed in vivo and in vitro during folding of nascent polypeptide chains, eliminating or reducing the protein's biological function (4, 5). Misfolded protein aggregates often can be observed as inclusion bodies (6, 7) and are implicated in amyloid deposition in vivo (8). In the biotechnology industry, protein aggregation is encountered routinely during purification, refolding, sterilization, shipping, and storage processes because of the presence of chemical, physical, and thermal stresses (9).In addition to significant losses in protein activity, clinical dangers result from parenteral administration of aggregated material (10). Even aggregation levels as low as 1% over a 2-year shelf life can render a product clinically unacceptable. Thus, proteins must be protected against even relatively mild stresses by the addition of proper excipients. Rational choice of these excipients requires insight into the mechanism of aggregation.The detailed mechanism of protein aggregation is still unclear. Usually, the aggregation pathway is modeled as shown in Scheme 1 by using the Lumry-Erying framework (11,12). The model involves a first-order reversible unfolding of the protein and subsequent aggregation of nonnative species in a higher order process (2, 11-13):In this scheme, N refers to native protein, and A refers to an intermediate conformational state preceding aggregation. A m refers to an aggregated form composed of m pro...
In the absence of surfactants, recombinant human growth hormone (rhGH) rapidly forms insoluble aggregates during agitation. The nonionic surfactant Tween 20, when present at Tween:protein molar ratios >4, effectively inhibits this aggregation. Differential scanning calorimetry (DSC) of rhGH solutions showed melting transitions that decreased by ca. 2 degrees C in the presence of Tween. Circular dichroism (CD) studies of the same thermal transition showed that the decrease is specific to the relatively high protein concentrations required for DSC. CD studies showed melting transitions that decreased with lower protein concentrations. Tween has an insignificant effect on the melting transition of rhGH at lower protein concentrations (0.18 mg/mL). Injection titration microcalorimetry showed that the interaction of Tween with rhGH is characterized by a weak enthalpy of binding. For comparison, interferon-g, another protein which has been shown to bind Tween, also shows weak enthalpy of binding. Fluorescent probe binding studies and infrared spectroscopic investigations of rhGH secondary structure support suggestions in the literature (Bam, N. B.; Cleland, J. L., Randolph, T. W. Molten globule intermediate of recombinant human growth hormone: stabilization with surfactants. Biotechnol. Prog. 1996. 12, 801-809) that Tween binding is driven by hydrophobic interactions, with little perturbation of protein secondary structure.
Protein aggregation is a complex phenomenon that can occur in vitro and in vivo, usually resulting in the loss of the protein's biological activity. While many aggregation studies focus on a mechanism due to a specific stress, this study focuses on the general nature of aggregation. Recombinant human interferon-gamma (rhIFN-gamma) provides an ideal model for studying protein aggregation, as it has a tendency to aggregate under mild denaturing stresses (low denaturant concentration, temperature below the Tm, and below pH 5). All of the aggregates induced by these stresses have a similar structure (high in intermolecular beta-sheet content and a large loss of alpha-helix) as determined by infrared and circular dichroism spectroscopy. Thermally induced and denaturant-induced aggregation processes follow first-order kinetics under the conditions of this study. Spectroscopic and kinetic data suggest that rhIFN-gamma aggregates through an intermediate form possessing a large amount of residual secondary structure. In contrast to the aggregates formed under denaturing stresses, the salted-out protein has a remarkably nativelike secondary structure.
Bovine carbonic anhydrase B (CAB) is chosen as the model protein to study the phenomenon of protein aggregation, which often occurs during the refolding process. Refolding of CAB from 5 M GuHCl has been observed by quasi-elastic light scattering (QLS), which confirms the formation of a molten globular protein structure as reported previously [Semisotnov, G. V., Rodionova, N. A., Kutyshenko, V. P., Ebert, B., Blanck, J., & Ptitsyn, O. B. (1987) FEBS Lett. 224, 9-13]. QLS analysis reveals the formation of multimeric species prior to precipitation. Activity and cross-linking studies have confirmed the presence of inactive multimeric protein species. The dimer formation has been determined to be the initiating step in the aggregation of CAB during refolding. Activity studies have indicated that the first intermediate observed in the refolding pathway of CAB aggregates to form the inactive dimer. The rate of formation of the dimer has a stoichiometric dependence on the final protein concentration. The dimer formation rate is a function of the final guanidine hydrochloride (GuHCl) concentration to the inverse 6.7 power, which correlates well with the binding of GuHCl to the native protein in 0.60-0.80 M GuHCl. These rate dependencies require the refolding of CAB to be performed at high GuHCl concentrations (1 M GuHCl) and low protein concentrations (less than 1 mg/mL) to avoid the formation of aggregates. Alternatively, refolding can be performed by allowing the first intermediate to form the second intermediate prior to further dilution or dialysis. The aggregation of a hydrophobic first intermediate species is likely to be common to the refolding of other molten globular proteins.
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