Aggregation of IgG mAb Biotherapeutics: Sources, Methods of Characterization, and Biological Implications

One of the predominant concerns with protein therapeutics is their tendency to aggregate at various stages of protein production, purification, filling, transportation, and administration. This occurrence has biological significance; while there is no definite, general cause and effect relationship for all protein drugs, many studies suggest that protein aggregates in certain biotherapeutics can decrease efficacy or cause untoward immune responses in human patients. Current research suggests that certain types of protein aggregates may be more immunogenic than others. In this Ph.D. thesis research work, three different IgG monoclonal antibodies (2 IgG1 mAbs, one in solution and one in lyophilized form and one IgG2 mAb in solution) were stressed by a variety of different conditions and the resulting aggregates and particles were characterized using a broad array of methods. Some of the characteristics examined included aggregate/ particle size, count, and morphology, as well as the covalent cross-linking and structural integrity of the protein within the aggregates. In all cases, accelerated stability studies, similar to those performed in the biopharmaceutical industry, were utilized to generate aggregates. In the first study, an IgG1 mAb in solution was subjected to freeze-thaw, shaking, stirring, and heat stress in the presence and absence of NaCl. Depending on the solution and stress conditions, very different types of aggregates and particles formed. In the second study, an IgG1 mAb in lyophilized form was shaken to mimic worst-case shipping condition, which led to extensive cake breakage and upon reconstitution, displayed increased turbidity and subvisible particles compared to the unstressed sample. This study highlights potential stability concerns regarding lyophilized protein undergoing various shipping processes. In the third study, the impact of protein particle size on inducing an early and late phase immune response in an in-vitro assay using human peripheral blood mononuclear cells (PBMC) was investigated. Stir-induced IgG2 mAb aggregates were size-enriched using fluorescence activated cell sorting (FACS) and tested for their ability to induce PBMC cytokine responses, at two phases of the immune response. The size-enriched particles were simultaneously characterized to determine traits, other than size, that may be responsible for the in-vitro assay responses. Amorphous subvisible particles 5-10 μm in size, containing protein with partially altered secondary structure and elevated surface hydrophobicity (compared to controls), and containing elemental fluorine, displayed relatively elevated cytokine release profiles compared to other size ranges. Studies carried out as part of this Ph.D. thesis highlight the importance of 1) comprehensively characterizing protein aggregates and particles to better understand their formation, 2) the need for closer evaluation of the effects of shaking stress on lyophilized protein formulations during shipping, and 3) studying the potential biological implications of a subset of protein particles in an in vitro system, along with developing a better understanding these aggregate's physicochemical properties, should provide improved insights into why some protein aggregates elicit higher immune responses than others in vivo

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

Morphologically-Directed Raman Spectroscopy as an Analytical Method for Subvisible Particle Characterization in Therapeutic Protein Product Quality

Abstract Subvisible particles (SVPs) are a critical quality attribute of injectable therapeutic proteins (TPs) that needs to be controlled due to potential risks associated with drug product quality. The current compendial methods routinely used to analyze SVPs for lot release provide information on particle size and count. However, chemical identification of individual particles is also important to address root-cause analysis. Herein, we introduce Morphologically-Directed Raman Spectroscopy (MDRS) for SVP characterization of TPs. The following particles were used for method development: (1) polystyrene microspheres, a traditional standard used in industry; (2) photolithographic (SU-8); and (3) ethylene tetrafluoroethylene (ETFE) particles, candidate reference materials developed by NIST. In our study, MDRS rendered high-resolution images for the ETFE particles (> 90%) ranging from 19 to 100 μm in size, covering most of SVP range, and generated comparable morphology data to flow imaging microscopy. Our method was applied to characterize particles formed in stressed TPs and was able to chemically identify individual particles using Raman spectroscopy. MDRS was able to compare morphology and transparency properties of proteinaceous particles with reference materials. The data suggests MDRS may complement the current TPs SVP analysis system and product quality characterization workflow throughout development and commercial lifecycle