The objective of this study was to characterize the thermal properties of systems containing various ratios of amorphous and crystalline components using both differential scanning calorimetry (DSC) and freeze-drying microscopy. The glycine/sucrose system was used as a model system, since it is routinely used in protein formulations. DSC analysis revealed that the addition of glycine to sucrose solutions resulted in a decrease in the glass transition (T'g) of the system. The T'g of a pure sucrose solution (7% w/v) decreased from -32.3 to -51.5 degrees C for a mixture containing a sucrose/glycine ratio of 2:5. The glass transition of the sucrose/glycine mixture decreased linearly as more glycine was added to the system. This decrease in glass transition resulted in severe collapse during freeze-drying of these mixtures above T'g. However, collapse was not observed during freeze-drying if the DSC thermogram of the sucrose/glycine mixture exhibited a transition resulting from recrystallization of the amorphous glycine. Mixtures having a sucrose/glycine ratio of 3:4 and 2:5 had a glass transition of -48 degrees C and -51.5 degrees C, respectively. Despite their low glass transition temperatures, these samples freeze-dried readily at a product temperature > T'g using a fast freeze-drying cycle (primary drying at a shelf temperature of +20 degrees C and chamber pressure of 100 mTorr) without any sign of collapse. The crystallization of the amorphous glycine from the frozen mixture of sucrose and glycine provided support during freeze-drying which prevented the macroscopic collapse of the final product. Freeze-drying microscopy visually revealed the crystallization and allowed for prediction of cake quality upon lyophilization. Although the freeze-drying microscope is not as sensitive as the DSC in detecting all transitions (it cannot detect a glass transition), it clarifies the interpretation of DSC, and together they provide valuable information regarding the relevance of each of the transitions to the final freeze-dried product elegance.

High-performance cation-exchange chromatography of recombinant soluble CD4 (rCD4) allowed the resolution of four charge variants. This charge heterogeneity could be eliminated by neuraminidase treatment of rCD4 and therefore can be attributed to different degrees of sialylation of the carbohydrate portion of this glycoprotein. A single acidic variant was observed upon cation-exchange chromatography of neuraminidase-treated rCD4 that had been stored in liquid solution, pH 7.2, at 25 degrees C for 6 months. This acidic variant was isolated by semipreparative cation-exchange chromatography and subjected to tryptic mapping analysis. Tryptic peptides were characterized by fast atom bombardment mass spectrometry (FABMS). The results of this analysis demonstrated that the acidic variant of neuraminidase-treated rCD4 is generated from deamidation at Asn-52. Digestion of the deamidated rCD4 with endoproteinase Asp-N confirmed Asn-52 as the primary site of deamidation. The ability of the deamidated rCD4 variant to bind gp120 was assessed by use of an ELISA-based binding assay. The binding capacity of the deamidated variant was 24% of the binding capacity of unmodified rCD4. The overall structure of the V1 domain in the deamidated variant was not markedly different from that of the native protein as probed with eight conformationally dependent anti-V1 monoclonal antibodies. Therefore, it appears that Asn-52 is directly involved in binding to gp120.

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Trauma, disease, developmental deformities, and tumor resection frequently cause bone defects that seriously challenge the skills of orthopedic and maxillofacial surgeons. Currently, repairing osseous deficiencies involves various medical surgical techniques, including autogenous grafts, allografts, internal and external fixation devices, electrical stimulation, and alloplastic implants. The existing technology, though effective in many cases, still is beset with numerous difficulties and disadvantages. A critical need for improved treatment methods exists today. Biotechnology now provides access to new bone repair concepts via administration of protein growth and morphogenic factors. Implantable device and drug delivery system technologies also have advanced. The converging biopharmaceutical, device, and delivery technologies represent an opportunity to improve the quality of health care for individuals with orthopedic and maxillofacial deficiencies. This report reviews current concepts in fracture healing and bone repair and examines existing treatment modalities. It also addresses novel protein drugs that stimulate osseous regeneration and delivery systems for these drugs.

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