Structural Characterization of IgG1 mAb Aggregates and Particles Generated under Various Stress Conditions

IgG1 mAb solutions were prepared with and without sodium chloride and subjected to different environmental stresses. Formation of aggregates and particles of varying size was monitored by a combination of size exclusion chromatography (SEC), Nanosight Tracking Analysis (NTA), Micro-flow Imaging (MFI), turbidity, and visual assessments. Stirring and heating induced the highest concentration of particles. In general, the presence of NaCl enhanced this effect. The morphology of the particles formed from mAb samples exposed to different stresses was analyzed from TEM and MFI images. Shaking samples without NaCl generated the most fibrillar particles, while stirring created largely spherical particles. The composition of the particles was evaluated for covalent cross-linking by SDS-PAGE, overall secondary structure by FTIR microscopy, and surface apolarity by extrinsic fluorescence spectroscopy. Freeze-thaw and shaking led to particles containing protein with native-like secondary structure. Heating and stirring produced IgG1 containing aggregates and particles with some non-native disulfide crosslinks, varying levels of intermolecular beta sheet content, and increased surface hydrophobicity. These results highlight the importance of evaluating protein particle morphology and composition, in addition to particle number and size distributions, to better understand the effect of solution conditions and environmental stresses on the formation of protein particles in mAb solutions

Characterization of the Physical Stability of a Lyophilized IgG1 mAb After Accelerated Shipping-like Stress

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

Physical characterixation and in vitro biological impact of highly aggregated antibodies separated into size-enriched populations by fluorescence-activated cell sorting

An IgG2 monoclonal antibody (mAb) solution was subjected to stirring, generating high concentrations of nanometer and subvisible particles, which were then successfully size enriched into different size bins by low speed centrifugation or a combination of gravitational sedimentation and Fluorescence-Activated Cell Sorting (FACS). The size-fractionated mAb particles were assessed for their ability to elicit the release of cytokines from a population of donor-derived human peripheral blood mononuclear cells (PBMC) at two phases of the immune response. Fractions enriched in nanometer-sized particles showed a lower response than those enriched in micron-sized particles in this assay. Particles of 5–10 μm in size displayed elevated cytokine release profiles compared to other size ranges. Stir-stressed mAb particles had amorphous morphology, contained protein with partially altered secondary structure, elevated surface hydrophobicity (compared to controls), and trace levels of elemental fluorine. FACS size-enriched the mAb particle samples, yet did not notably alter the overall morphology or composition of particles as measured by Microflow imaging, Transmission Electron Microscopy, and Scanning Electron Microscopy-Energy Dispersive X-ray Spectroscopy. The utility and limitations of FACS for size separation of mAb particles and potential of in-vitro PBMC studies to rank order the immunogenic potential of various types of mAb particles is discussed

Hybrid Necrosis in Wheat-A Genetic System Showing Reduced Capacity to Detoxify Reactive Oxygen Species Leading to Programmed Cell Death

17-25Hybrid necrosis in wheat is the premature gradual death of leaves and leaf sheath caused by two complementary genes NeJ and Ne2 when brought together in a hybrid combination. Many promising wheat varieties are carriers of these genes, which limit the parental choice for transfer of desirable traits as the necrotic plants die without producing seeds. The leaves of Kalyansona x C306 and WL711 x C306 hybrids showed enhanced generation of superoxide radical and H202 than parents before and during progression of necrosis. Higher lipid peroxidation was an early event in hybrid necrosis and was accompanied by a loss in membrane permeability and cell viability. The anti-oxidant defense in necrotic hybrids was not well coordinated culminating in a persistent oxidative stress in the leaf especialiy in the chloroplast. The hybrid necrosis barrier was overcome in some crosses by culturing ears in medium containing antioxidants and F2 seeds have been obtained. Hybrid necrosis is a unique genetic system for studying the molecular mechanism of programmed cell death in plants. The cloning and characterization of Nel and Ne2 may provide a tool having wide applications in agriculture. The review compares hybrid necrosis with other PCD phenomenon in plants