The results indicate that anion binding mediates aggregation by lowering mAb conformational stability and reduced valence. Our observations support an agitation-induced particulation model in which anions enhance the partitioning and unfolding of mAbs at the air/water interface. Aggregation predominantly occurs at this interface; refreshing of the surface during agitation releases the insoluble aggregates into bulk solution.

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Recombinant human monoclonal antibodies have become important protein-based therapeutics for the treatment of various diseases. The antibody structure is complex, consisting of b-sheet rich domains stabilized by multiple disulfide bridges. The dimerization of the C H 3 domain in the constant region of the heavy chain plays a pivotal role in the assembly of an antibody. This domain contains a single buried, highly conserved disulfide bond. This disulfide bond was not required for dimerization, since a recombinant human C H 3 domain, even in the reduced state, existed as a dimer. Spectroscopic analyses showed that the secondary and tertiary structures of reduced and oxidized C H 3 dimer were similar, but differences were observed. The reduced C H 3 dimer was less stable than the oxidized form to denaturation by guanidinium chloride (GdmCl), pH, or heat. Equilibrium sedimentation revealed that the reduced dimer dissociated at lower GdmCl concentration than the oxidized form. This implies that the disulfide bond shifts the monomer-dimer equilibrium. Interestingly, the dimer-monomer dissociation transition occurred at lower GdmCl concentration than the unfolding transition. Thus, disulfide bond formation in the human C H 3 domain is important for stability and dimerization. Here we show the importance of the role played by the disulfide bond and how it affects the stability and monomer-dimer equilibrium of the human C H 3 domain. Hence, these results may have implications for the stability of the intact antibody.Keywords: human antibody; C H 3 domain; disulfide bond; stability; dimerization With the recent advances in heterologous expression of antibodies, more than a dozen monoclonal antibodybased drugs have reached the market. Hundreds more are at various stages of research and development for the treatment of a wide range of diseases, from cancer to inflammation. A number of key scientific and technological advancements, such as the generation of fully human antibody by transgenic mice (Green 1999), have contributed to the successful development of antibody-based therapeutics. Stability and homogeneity of therapeutic antibodies are of prime importance for safety and efficacy. Undesired biochemical, structural, and conformational forms can lead to loss of efficacy and risk of adverse side effects.IgG1 (hereafter referred to as antibody) is a complex molecule, composed of two identical light (L) and heavy Reprint requests to: Masazumi Matsumura,

Human acidic fibroblast growth factor (FGF-1) is a member of the ␤-trefoil hyperfamily and exhibits a characteristic threefold symmetry of the tertiary structure. However, evidence of this symmetry is not readily apparent at the level of the primary sequence. This suggests that while selective pressures may exist to retain (or converge upon) a symmetric tertiary structure, other selective pressures have resulted in divergence of the primary sequence during evolution. Using intra-chain and homologue sequence comparisons for 19 members of this family of proteins, we have designed mutants of FGF-1 that constrain a subset of corepacking residues to threefold symmetry at the level of the primary sequence. The consequences of these mutations regarding structure and stability were evaluated using a combination of X-ray crystallography and differential scanning calorimetry. The mutational effects on structure and stability can be rationalized through the characterization of "microcavities" within the core detected using a 1.0Å probe radius. The results show that the symmetric constraint within the primary sequence is compatible with a well-packed core and near wild-type stability. However, despite the general maintenance of overall thermal stability, a noticeable increase in non-two-state denaturation follows the increase in primary sequence symmetry. Therefore, properties of folding, rather than stability, may contribute to the selective pressure for asymmetric primary core sequences within symmetric protein architectures.

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Aggregation of human therapeutic antibodies represents a significant hurdle to product development. In a test across multiple antibodies, it was observed that IgG1 antibodies aggregated less, on average, than IgG2 antibodies under physiological pH and mildly elevated temperature. This phenomenon was also observed for IgG1 and IgG2 subclasses of anti-streptavidin, which shared 95% sequence identity but varied in interchain disulfide connectivity. To investigate the structural and covalent changes associated with greater aggregation in IgG2 subclasses, soluble aggregates from the two forms of anti-streptavidin were isolated and characterized. Sedimentation velocity analytical ultracentrifugation (SV-AUC) measurements confirmed that the aggregates were present in solution, and revealed that the IgG1 aggregate was composed of a predominant species, whereas the IgG2 aggregate was heterogeneous. Tertiary structural changes accompanied antibody aggregation as evidenced by greater ANS (8-Anilino-1-naphthalene sulfonic acid) binding to the aggregates over monomer, and differences in disulfide character and tryptophan environments between monomer, oligomer and aggregate species, as observed by near-UV circular dichroism (CD). Differences between subclasses were observed in the secondary structural changes that accompanied aggregation, particularly in the intermolecular b-sheet and turn structures between the monomer and aggregate species. Free thiol determination showed 2.4-fold lower quantity of free cysteines in the IgG1 subclass, consistent with the 2.4-fold reduction in aggregation of the IgG1 form when compared with IgG2 under these conditions. These observations suggested an important role for disulfide bond formation, as well as secondary and tertiary structural transitions, during antibody aggregation. Such degradations may be minimized using appropriate formulation conditions.