Characterization, Stabilization and Formulation Design of IgG and Secretory IgA Monoclonal Antibody Candidates during Storage and Administration
ABSTRACT Monoclonal antibodies (mAbs) have become a class of therapeutic protein-based drugs of high importance for treating numerous human diseases. As a complex, delicate three dimensional molecule, a mAb can be sensitive to ambient environments and thus often display colloidal/conformational instability during manufacturing, storage, and administration. The use of formulation strategies, such as employing specific excipients and/or particular solution conditions (e.g., pH and buffers), can significantly improve a mAb’s pharmaceutical stability properties. In this Ph.D. thesis research work, both formulation/storage stability as well as stability during in vitro models of mAb administration in vivo are evaluated with two different of classes of immunoglobulin molecules (IgG and IgA). In addition, the effect of specific classes of excipients and solution conditions are examined by using a wide variety of physicochemical and immunological binding analytical techniques. Specifically, the second and third chapters of the Ph.D. thesis work focus on the formulation development of high-concentration mAb dosage forms for subcutaneous (SC) injections. Reversible self-association (RSA) of mAbs, which is primarily due to intermolecular protein-protein interactions (PPIs) between mAb molecules, has emerged as an important formulation challenge in terms of significantly increasing solution viscosity and turbidity as well as initiating phase separation. In these two chapters, two different human IgG1 molecules (mAb-J and mAb-C), which showed strong RSA propensity at relatively high protein concentrations, were comprehensively studied to better understand their solution properties and molecular behaviors by both biophysical techniques as well as hydrogen-deuterium exchange mass spectrometry (HX-MS). The aim is to not only characterize mAb molecular properties and solution behavior at relatively high protein concentrations, but also to develop a better mechanistic understanding by identifying peptide segments within the mAb involved PPIs in solution. In these two studies, both elevated solution viscosity and turbidity as well as reduced relative solubility and increased protein-protein interaction propensity (as measured by light scattering profiles and observations of phase separation) were determined for two different mAbs (mAb-J and mAb-C) at comparatively high protein concentrations. Concomitantly, based on the amino acid sequence of each mAb’s RSA sites (as determined by lyophilization-reconstitution-based HX-MS methodology), two different dominant non-covalent forces (electrostatic and hydrophobic) are proposed to be the major driving force for PPIs of the two different mAbs (consistent with previous results). More importantly for this work, varying effects of different excipients were investigated particularly for their ability of promote or disrupt PPIs of each mAb. For mAb-J (electrostatic driven RSA), selected ionic excipients showed the ability to disrupt liquid-liquid phase separation and reduce intermolecular interactions to varying extents, with arginine hydrochloride possessing the highest efficiency. For mAb-C (hydrophobic driven RSA), opposing effects were observed for sodium sulfate versus selected hydrophobic additives (e.g., specific salts, amino acids, solvents), showing both enhanced and reduced PPI propensity, respectively. In both studies, not only was the RSA of mAbs shown to be mAb concentration dependent, but the excipient’s ability to mitigate the RSA of mAbs RSA also displayed an excipient concentration dependent pattern. The fourth chapter focuses on the possibility of using various classes of mAbs, including secretory IgA (sIgA) and IgG1, as potential drug candidates for oral delivery to prevent enteric diseases in infants. Specifically, the use of mAbs against enterotoxigenic Escherichia coli (ETEC) is examined with the idea that passive immunization by pathogen-specific immunoglobulins, by oral delivery to infants, is promising approach to provide “instant” protection against ETEC. Secretory IgA (sIgA) is of particular interest because it is naturally found in the mucosal surfaces within the GI tract, is relatively more resistant to proteolysis by digestive enzymes (vs. IgG), and can protect against enteric bacteria by directly neutralizing virulence factors. In this study, three different mAbs, (sIgA1, sIgA2 and IgG1) against heat labile toxin (LT, one of the major virulence factors of ETEC), were used as a model for developing analytical techniques to characterize the structural integrity of the mAbs and to assess their stability profiles under various solution conditions (using physicochemical and immunochemical binding assays). In this work, very different total carbohydrate levels and N-linked glycosylation oligosaccharide composition profiles were observed between sIgAs and IgG1 made from CHO cell lines. According to SDS-PAGE, SE-HPLC, and SV-AUC results, heterogeneous mixtures of higher molecular weight species were observed for sIgAs, while IgG1 samples showed less heterogeneity with more than 90% monomer in solution. The overall physical stability results at both pH 7.2 and pH 3.0 demonstrated that both sIgA1 and sIgA2 were more stable than IgG1, with sIgA1 displaying the best stability profile. The relative solubility profile of each molecule was pH dependent with higher solubility noted at the lower pH. Furthermore, an in vitro digestion model was adapted in the laboratory to mimic in vivo oral gastric degradation conditions using minimal material, and was utilized to monitor the oral delivery stability of the three mAbs. It was shown that F(ab’)2 was the major digestion product by pepsin digestion. Both sIgAs displayed better resistance to degradation by proteases at low pH compared to IgG1. Moreover, the sIgAs showed greater retention of LT-antigen binding activity than that of IgG1, confirming the superior pharmaceutical properties of sIgAs for oral delivery. In summary, we hope to use the information gained by these preformulation characterization studies for the long-term goal to design stable, low-cost liquid formulations for oral delivery of sIgAs to protect against enteric diseases currently affecting infants in the developing world.
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