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C-terminal lysine microheterogeneity and deamidation of Asn141 in the heavy chain and Asn161 in the light chain are the major causes of MMA383 charge heterogeneity. Identification of the two deamidation sites will allow replacement of these amino acids in order to create a product less susceptible to degradation.

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This review focuses on selected applications of the separation and analysis of peptides and proteins published during the period of 1997-1998. Specific topic areas covered include high-performance liquid chromatography (HPLC), ultrafiltration, capillary electrophoresis (CE), affinity-based methods for protein isolation and separation, mass spectrometry (MS), detection of nonenzymatic posttranslational modifications, nuclear magnetic resonance spectroscopy (NMR), infrared (IR) and Raman spectroscopy, circular dichroism (CD), UV-visible absorption spectroscopy, dynamic light scattering, and calorimetry. The quantification and identification of peptides and proteins by chromatographic methods and MS have become fairly routine, as has the conformational analysis of peptides and small proteins in solution by CD, IR, and NMR. Therefore, these topics are not reviewed in detail here. In this review, we have attempted to highlight new technological developments or unique applications of analytical methods that impact the analysis of peptides and proteins.

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Capillary electrophoresis (CE) is a separation technique particularly suited to the analysis of pharmaceutical compounds. This review offers a detailed discussion of the four common modes of detection coupled to CE-UV absorption, fluorescence, electrochemical, and mass spectrometry-and gives examples of the use of these methods in pharmaceutical analyses. Sample preparation and pretreatment techniques used for CE separations are described, as well as methods of preconcentration including hydrophobic retention, affinity concentration, sample stacking, and isotachophoresis. The use of affinity CE, chiral CE, and capillary gel electrophoresis for analysis of pharmaceuticals is covered in detail, and recent advances in capillary electrochromatography and CE on a chip are also discussed.

Upon exposure to shaking stress, an IgG1 mAb formulation in both liquid and lyophilized state formed subvisible particles. Since freeze-drying is expected to minimize protein physical instability under these conditions, the extent and nature of aggregate formation in the lyophilized preparation was examined using a variety of particle characterization techniques. The effect of formulation variables such as residual moisture content, reconstitution rate, and reconstitution medium were examined. Upon reconstitution of shake-stressed lyophilized mAb, differences in protein particle size and number were observed by Microflow Digital Imaging (MFI), with the reconstitution medium having the largest impact. Shake-stress had minor effects on the structure of protein within the particles as shown by SDS-PAGE and FTIR analysis. The lyophilized mAb was shake-stressed to different extents and stored for 3 months at different temperatures. Both extent of cake collapse and storage temperature affected the physical stability of the shake-stressed lyophilized mAb upon subsequent storage. These findings demonstrate that physical degradation upon shaking of a lyophilized IgG1 mAb formulation includes not only cake breakage, but also results in an increase in subvisible particles and turbidity upon reconstitution. The shaking-induced cake breakage of the lyophilized IgG1 mAb formulation also resulted in decreased physical stability upon storage.

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