A High Threshold of Biotherapeutic Aggregate Numbers is Needed to Induce an Immunogenic Response In Vitro, In Vivo, and in the Clinic.

BACKGROUND AND PURPOSE: There is concern that subvisible aggregates in biotherapeutic drug products pose a risk to patient safety. We investigated the threshold of biotherapeutic aggregates needed to induce immunogenic responses. METHODS AND RESULTS: Highly aggregated samples were tested in cell-based assays and induced cellular responses in a manner that depended on the number of particles. The threshold of immune activation varied by disease state (cancer, rheumatoid arthritis, allergy), concomitant therapies, and particle number. Compared to healthy donors, disease state patients showed an equal or lower response at the late phase (7 days), suggesting they may not have a higher risk of responding to aggregates. Xeno-het mice were used to assess the threshold of immune activation in vivo. Although highly aggregated samples (~ 1,600,000 particles/mL) induced a weak and transient immunogenic response in mice, a 100-fold dilution of this sample (~ 16,000 particles/mL) did not induce immunogenicity. To confirm this result, subvisible particles (up to ~ 18,000 particles/mL, containing aggregates and silicone oil droplets) produced under representative administration practices (created upon infusion of a drug product through an IV catheter) did not induce a response in cell-based assays or appear to increase the rate of adverse events or immunogenicity during phase 3 clinical trials. CONCLUSION: The ability of biotherapeutic aggregates to elicit an immune response in vitro, in vivo, and in the clinic depends on high numbers of particles. This suggests that there is a high threshold for aggregates to induce an immunogenic response which is well beyond that seen in standard biotherapeutic drug products.

Contributions of a disulfide bond to the structure, stability, and dimerization of human IgG1 antibody CH3 domain.

Recombinant human monoclonal antibodies have become important protein-based therapeutics for the treatment of various diseases. The antibody structure is complex, consisting of beta-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.

Sequence swapping does not result in conformation swapping for the beta4/beta5 and beta8/beta9 beta-hairpin turns in human acidic fibroblast growth factor.

The beta-turn is the most common type of nonrepetitive structure in globular proteins, comprising ~25% of all residues; however, a detailed understanding of effects of specific residues upon beta-turn stability and conformation is lacking. Human acidic fibroblast growth factor (FGF-1) is a member of the beta-trefoil superfold and contains a total of five beta-hairpin structures (antiparallel beta-sheets connected by a reverse turn). beta-Turns related by the characteristic threefold structural symmetry of this superfold exhibit different primary structures, and in some cases, different secondary structures. As such, they represent a useful system with which to study the role that turn sequences play in determining structure, stability, and folding of the protein. Two turns related by the threefold structural symmetry, the beta4/beta5 and beta8/beta9 turns, were subjected to both sequence-swapping and poly-glycine substitution mutations, and the effects upon stability, folding, and structure were investigated. In the wild-type protein these turns are of identical length, but exhibit different conformations. These conformations were observed to be retained during sequence-swapping and glycine substitution mutagenesis. The results indicate that the beta-turn structure at these positions is not determined by the turn sequence. Structural analysis suggests that residues flanking the turn are a primary structural determinant of the conformation within the turn.

Symmetric primary and tertiary structure mutations within a symmetric superfold: a solution, not a constraint, to achieve a foldable polypeptide.

In previous studies designed to increase the primary structure symmetry within the hydrophobic core of human acidic fibroblast growth factor (FGF-1) a combination of five mutations were accommodated, resulting in structure, stability and folding kinetic properties similar to wild-type (despite the symmetric constraint upon the set of core residues). A sixth mutation in the core, involving a highly conserved Met residue at position 67, appeared intolerant to substitution. Structural analysis suggested that the local packing environment of position 67 involved two regions of apparent insertions that distorted the tertiary structure symmetry inherent in the beta-trefoil architecture. It was postulated that a symmetric constraint upon the primary structure within the core could only be achieved after these insertions had been deleted (concomitantly increasing the tertiary structure symmetry). The deletion of these insertions is now shown to permit mutation of position 67, thereby increasing the primary structure symmetry relationship within the core. Furthermore, despite the imposed symmetric constraint upon both the primary and tertiary structure, the resulting mutant form of FGF-1 is substantially more stable. The apparent inserted regions are shown to be associated with heparin-binding functionality; however, despite a marked reduction in heparin-binding affinity the mutant form of FGF-1 is surprisingly approximately 70 times more potent in 3T3 fibroblast mitogenic assays. The results support the hypothesis that primary structure symmetry within a symmetric protein superfold represents a possible solution, rather than a constraint, to achieving a foldable polypeptide.

Identification of a key structural element for protein folding within beta-hairpin turns.

Specific residues in a polypeptide may be key contributors to the stability and foldability of the unique native structure. Identification and prediction of such residues is, therefore, an important area of investigation in solving the protein folding problem. Atypical main-chain conformations can help identify strains within a folded protein, and by inference, positions where unique amino acids may have a naturally high frequency of occurrence due to favorable contributions to stability and folding. Non-Gly residues located near the left-handed alpha-helical region (L-alpha) of the Ramachandran plot are a potential indicator of structural strain. Although many investigators have studied mutations at such positions, no consistent energetic or kinetic contributions to stability or folding have been elucidated. Here we report a study of the effects of Gly, Ala and Asn substitutions found within the L-alpha region at a characteristic position in defined beta-hairpin turns within human acidic fibroblast growth factor, and demonstrate consistent effects upon stability and folding kinetics. The thermodynamic and kinetic data are compared to available data for similar mutations in other proteins, with excellent agreement. The results have identified that Gly at the i+3 position within a subset of beta-hairpin turns is a key contributor towards increasing the rate of folding to the native state of the polypeptide while leaving the rate of unfolding largely unchanged.

Free fatty acid particles in protein formulations, part 1: microspectroscopic identification.

We report, for the first time, the identification of fatty acid particles in formulations containing the surfactant polysorbate 20. These fatty acid particles were observed in multiple mAb formulations during their expected shelf life under recommended storage conditions. The fatty acid particles were granular or sand-like in morphology and were several microns in size. They could be identified by distinct IR bands, with additional confirmation from energy-dispersive X-ray spectroscopy analysis. The particles were readily distinguishable from protein particles by these methods. In addition, particles containing a mixture of protein and fatty acids were also identified, suggesting that the particulation pathways for the two particle types may not be distinct. The techniques and observations described will be useful for the correct identification of proteinaceous versus nonproteinaceous particles in pharmaceutical products.

Accommodation of a highly symmetric core within a symmetric protein superfold.

An alternative core packing group, involving a set of five positions, has been introduced into human acidic FGF-1. This alternative group was designed so as to constrain the primary structure within the core region to the same threefold symmetry present in the tertiary structure of the protein fold (the beta-trefoil superfold). The alternative core is essentially indistinguishable from the WT core with regard to structure, stability, and folding kinetics. The results show that the beta-trefoil superfold is compatible with a threefold symmetric constraint on the core region, as might be the case if the superfold arose as a result of gene duplication/fusion events. Furthermore, this new core arrangement can form the basis of a structural "building block" that can greatly simplify the de novo design of beta-trefoil proteins by using symmetric structural complementarity. Remaining asymmetry within the core appears to be related to asymmetry in the tertiary structure associated with receptor and heparin binding functionality of the growth factor.

The identification of free cysteine residues within antibodies and a potential role for free cysteine residues in covalent aggregation because of agitation stress.

Human immunoglobulin G1 (IgG1) and immunoglobulin G2 (IgG2) antibodies contain multiple disulfide bonds, which are an integral part of the structure and stability of the protein. Open disulfide bonds have been detected in a number of therapeutic and serum derived antibodies. This report details a method that fluorescently labels free cysteine residues, quantifies, and identifies the proteolytic fragments by liquid chromatography coupled to online mass spectrometry. The majority of the open disulfide bonds in recombinant and serum derived IgG1 and IgG2 antibodies were in the constant domains. This method was applied to the identification of cysteines in an IgG2 antibody that are involved in the formation of covalent intermolecular bonds because of the application of a severe agitation stress. The free cysteine in the CH 1 domain of the IgG2 decreased upon application of the stress and implicates open disulfide bonds in this domain as the likely source of free cysteines involved in the formation of intermolecular disulfide bonds. The presence of comparable levels of open disulfide bonds in recombinant and endogenous antibodies suggests that open disulfide bonds are an inherent feature of antibodies and that the susceptibility of intermolecular disulfide bond formation is similar for recombinant and serum-derived IgG antibodies.

Increased aggregation propensity of IgG2 subclass over IgG1: role of conformational changes and covalent character in isolated aggregates.

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 ß-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.

Characterization of antibody aggregation: role of buried, unpaired cysteines in particle formation.

Proteins are susceptible to degradation upon exposure to a variety of stresses during product manufacturing, transportation and storage. In this study, we investigated the aggregation properties of a monoclonal antibody during agitation stress. Agitation exclusively led to insoluble aggregates, or particle formation. Removal or modification of the air-liquid interface with a surfactant (e.g., polysorbate) abrogated particle formation. The supernatant postagitation was analyzed using SE-HPLC, FTIR, and AUC analyses and revealed no changes in conformation and aggregation profile when compared to the nonagitated antibody sample. The antibody particles were comprised of a combination of nonnative intermolecular disulfide-linked covalent as well as noncovalent interactions. Analysis of the antibody's unpaired cysteines revealed that the nonnative intermolecular disulfide bonds were formed through buried cysteines, which suggested at least partial unfolding of the antibody domains. FTIR analysis indicated that the particulated antibody maintained significant native-like secondary structure suggesting that particle formation led to minimal structure changes, but capable of exposing free cysteines to solvent to form the nonnative intermolecular disulfide bonds. The results presented in this study indicate the importance of the interactions between the antibody and the air-liquid interface during agitation in the formation of particles and suggests that reduced disulfide bonds may play a significant role in the particulation reaction. This phenomenon can be applicable to other proteins with similar free cysteine and structural characteristics.

Effect of ions on agitation- and temperature-induced aggregation reactions of antibodies.

PURPOSE: The impact of ions on protein aggregation remains poorly understood. We explored the role of ionic strength and ion identity on the temperature- and agitation-induced aggregation of antibodies. METHODS: Stability studies were used to determine the influence of monovalent Hofmeister anions and cations on aggregation propensity of three IgG(2) mAbs. The C(H)2 domain melting temperature (T (m1)) and reduced valence (z*) of the mAbs were measured. RESULTS: Agitation led to increased solution turbidity, consistent with the formation of insoluble aggregates, while soluble aggregates were formed during high temperature storage. The degree of aggregation increased with anion size (F(-) < Cl(-) < Br(-) < I(-) < SCN(-) ~ ClO(4) (-)) and correlated with a decrease in T (m1) and z*. The aggregation propensity induced by the anions increased with the chaotropic nature of anion. The cation identity (Li(+), Na(+), K(+), Rb(+), or Cs(+)) had no effect on T (m1), z* or aggregation upon agitation. CONCLUSIONS: 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.

Self-buffering antibody formulations.

Monoclonal antibodies (mAbs) often require the development of high-concentration formulations. In such cases, and when it is desirable to formulate a mAb around pH 5.0, we explored a novel approach of controlling the formulation pH by harnessing the ability of mAbs to "self-buffer." Buffer capacities of four representative IgG(2) molecules (designated mAb1 through mAb4) were measured in the pH 4-6 range. The buffer capacity results indicated that the mAbs possessed a significant amount of buffer capacity, which increased linearly with concentration. By 60-80 mg/mL, the mAb buffer capacities surpassed that of 10 mM acetate, which is commonly employed in formulations for buffering in the pH 4-6 range. Accelerated high temperature stability studies (50 degrees C over 3 weeks) conducted with a representative antibody in a self-buffered formulation (50 mg/mL mAb1 in 5.25% sorbitol, pH 5.0) and with solutions formulated using conventional buffers (50 mg/mL mAb1 in 5.25% sorbitol, 25 or 50 mM acetate, glutamate or succinate, also at pH 5.0) indicated that mAb1 was most resistant to the formation of soluble aggregates in the self-buffered formulation. Increased soluble aggregate levels were observed in all the conventionally buffered (acetate, glutamate, and succinate) formulations, which further increased with increasing buffer strength. The long-term stability of the self-buffered liquid mAb1 formulation (60 mg/mL in 5% sorbitol, 0.01% polysorbate 20, pH 5.2) was comparable to the conventionally buffered (60 mg/mL in 10 mM acetate or glutamate, 5.25% sorbitol, 0.01% polysorbate 20, pH 5.2) formulations. No significant change in pH was observed after 12 months of storage at 37 and 4 degrees C for the self-buffered formulation. The 60 mg/mL self-buffered formulation of mAb1 was also observed to be stable to freeze-thaw cycling (five cycles, -20 degrees C --> room temperature). Self-buffered formulations may be a better alternative for the development of high-concentration antibody and protein dosage forms.