Sangeeta B. Joshi

Protein–excipient interactions: Mechanisms and biophysical characterization applied to protein formulation development

The purpose of this review is to demonstrate the critical importance of understanding protein-excipient interactions as a key step in the rational design of formulations to stabilize and deliver protein-based therapeutic drugs and vaccines. Biophysical methods used to examine various molecular interactions between solutes and protein molecules are discussed with an emphasis on applications to pharmaceutical excipients in terms of their effects on protein stability. Key mechanisms of protein-excipient interactions such as electrostatic and cation-pi interactions, preferential hydration, dispersive forces, and hydrogen bonding are presented in the context of different physical states of the formulation such as frozen liquids, solutions, gels, freeze-dried solids and interfacial phenomenon. An overview of the different classes of pharmaceutical excipients used to formulate and stabilize protein therapeutic drugs is also presented along with the rationale for use in different dosage forms including practical pharmaceutical considerations. The utility of high throughput analytical methodologies to examine protein-excipient interactions is presented in terms of expanding formulation design space and accelerating experimental timelines.

Multidimensional Methods for the Formulation of Biopharmaceuticals and Vaccines

Determining and preserving the higher order structural integrity and conformational stability of proteins, plasmid DNA, and macromolecular complexes such as viruses, virus-like particles, and adjuvanted antigens are often a significant barrier to the successful stabilization and formulation of biopharmaceutical drugs and vaccines. These properties typically must be investigated with multiple lower resolution experimental methods because each technique monitors only a narrow aspect of the overall conformational state of a macromolecular system. This review describes the use of empirical phase diagrams (EPDs) to combine large amounts of data from multiple high-throughput instruments and construct a map of a target macromolecules physical state as a function of temperature, solvent conditions, and other stress variables. We present a tutorial on the mathematical methodology, an overview of some of the experimental methods typically used, and examples of some of the previous major formulation applications. We also explore novel applications of EPDs including potential new mathematical approaches as well as possible new biopharmaceutical applications such as analytical comparability, chemical stability, and protein dynamics.

Properties of Synthetic Spider Silk Fibers Based on Argiope aurantia MaSp2

Spiders have evolved a complex system of silk producing glands. Each of the glands produces silk with strength and elasticity tailored to its biological purpose. Sequence analysis of the major ampullate silk reveals four highly conserved concatenated blocks of amino acids: (GA) n , A n , GPGXX, and GGX. While the GPGXX motif, which has been hypothesized to be responsible for the extensibility of the fiber, displays natural variation in its precise sequence arrangement and content, correlating these differences with particular fiber properties has been difficult. Three genetic constructs based on the Argiope aurantia sequence were engineered to progressively increase the number of GPGXX repeats in a head-to-tail assembly prior to interruption by another motif. Circular dichroism and Fourier transform infrared spectroscopy of synthetic spider silk spin dopes show secondary structures that correspond to an increase in the repeat number of GPGXX regions and an increase in the extensibility of synthetically spun recombinant fibers.

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, Nanoparticle Tracking Analysis, 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 transmission electron microscopy and MFI images. Shaking samples without NaCl generated the most fibrillar particles, whereas 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 cross-links, 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.

Formulation design and high-throughput excipient selection based on structural integrity and conformational stability of dilute and highly concentrated IgG1 monoclonal antibody solutions

A systematic approach is presented to characterize and stabilize the higher order structural integrity of an immunoglobulin G (IgG1) monoclonal antibody (mAb) formulated at both low concentrations and as a highly concentrated solution. The conformational and colloidal stabilities of a recombinant humanized IgG1κ mAb at both 1 and 100 mg/mL were investigated as a function of solution temperature (10°C-87.5°C) and pH (3-8). Protein secondary structure was characterized using circular dichroism, whereas intrinsic (tryptophan) and extrinsic (8-anilino-1-naphthalenesulfonic acid) fluorescence spectroscopy measurements were used to evaluate the tertiary structure of the protein. Light scattering analysis was employed to monitor mAb aggregation behavior as a function of temperature and solution pH. These biophysical data sets were analyzed and summarized using a previously described empirical phase diagrams (EPDs) approach. The different phases observed in the EPD were correlated with the individual physical states of the IgG1 in solution (aggregated, native, unfolded, etc.). The temperature-dependent conformational stability profile of the mAb, at both 1 and 100 mg/mL, generally followed the order pH 6 ≥ pH 7 ≥ pH 8 > pH 5 > pH 4 ≥ pH 3. Analysis of the EPD apparent phase boundaries identified solution conditions of pH 4.5 near 60°C for the development of an excipient screening assay. A supplemented generally regarded as safe excipient library was screened using an aggregation assay (optical density at 350 nm) at low mAb concentrations (4 mg/mL) and potential stabilizers were identified. The ability of these excipients to prevent conformational alterations in high concentration mAb solutions (100 mg/mL) was determined by monitoring tertiary structure changes using an intrinsic fluorescence method. The results suggest that substantial increases in the onset temperature of thermal transitions (>5°C) are obtained in the presence of (a) 20% dextrose, (b) 20% sorbitol, and (c) 5% dextrose + 10% sorbitol. Similar stabilization effects were obtained at an intermediate (50 mg/mL) as well as low mAb concentrations (1 mg/mL).

Stabilization of measles virus for vaccine formulation

An attenuated live measles virus (MV) was characterized by several biophysical methods as a function of temperature and pH. Following a method developed previously, the resultant light scattering and spectroscopic data were synthesized into an empirical phase diagram that visually and simultaneously represents the entire data set. Using this empirically-based phase diagram, screening assays were developed to identify potential vaccine stabilizers. Various compounds are shown by these assays to inhibit the temperature-induced aggregation of viral particles, and also to protect the integrity of the viral envelope. Accelerated stability assays show that, upon thermal challenge, MV formulated with these excipients retains its infectivity to a significant extent. Thus, the enhanced physical stability produced by this method is shown to protect the biological activity of this important but labile vaccine.

Compatibility, Physical Stability, and Characterization of an IgG4 Monoclonal Antibody After Dilution into Different Intravenous Administration Bags

The physical stability of an immunoglobulin G4 monoclonal antibody (mAb) upon dilution into intravenous (i.v.) bags containing 0.9% saline was examined. Soluble aggregates and subvisible particles were observed by size-exclusion high-performance liquid chromatography (SE-HPLC) and light obscuration when formulated with suboptimal levels of polysorbate 20. The formation of soluble aggregates and particulates was further characterized by a combination of SE-HPLC, nanoparticle tracking analysis (NTA), microflow-digital imaging (MFI), and turbidity measurements. With sufficient PS20 levels, particle formation was minimized, although quantification of submicron sized particles by NTA was not possible because of the interference from PS20. Intravenous bags composed of polyvinyl chloride caused more protein particle formation than polyolefin bags. Differences between bag types were affected by removing headspace and by transferring the saline solution into glass vials. Characterization studies with Fourier transform infrared microscopy and extrinsic fluorescence spectroscopy demonstrated that isolated particles contained native-like secondary structure with partially altered tertiary structure, compared with heat-denatured and nonstressed controls. Transmission electron microscopy and MFI analysis showed particles had an amorphous morphology of varying sizes. Particles contained some non-native disulfide bond crosslinks, potentially initiated by low levels of free thiol in the native mAb. The critical role of proper formulation design to stabilize proteins against physical instability during i.v. administration is discussed.

Comparison of high-throughput biophysical methods to identify stabilizing excipients for a model IgG2 monoclonal antibody: Conformational stability and kinetic aggregation measurements

The overall conformational stability of a model IgG2 monoclonal antibody (mAb) was examined as a function of temperature and pH using an empirical phase diagram approach. Stabilizing excipients were then identified based on high-throughput methods including (1) kinetic studies measuring aggregation via increases in optical density and (2) thermally induced structural transitions as measured by differential scanning calorimetry (DSC) and fluorescence spectroscopy. The kinetic profiles of antibody aggregation at 65 °C were pH dependent and correlated well with pH effects on secondary and tertiary structural transitions due to heat stress. For the screening of stabilizing excipients, the inhibition of the rate of protein aggregation at pH 4.5 at 65°C, as represented by changes in optical density, was shown to have a clear trend with a modest correlation coefficient compared with the stabilizing effect of the same excipients on the conformational stability of the antibody as measured by DSC and tryptophan fluorescence spectroscopy. These results demonstrate the utility of combining high-throughput data from protein aggregation kinetic experiments and conformational stability studies to identify stabilizing excipients that minimize the physical degradation of an IgG2 mAb.

Improved data visualization techniques for analyzing macromolecule structural changes

The empirical phase diagram (EPD) is a colored representation of overall structural integrity and conformational stability of macromolecules in response to various environmental perturbations. Numerous proteins and macromolecular complexes have been analyzed by EPDs to summarize results from large data sets from multiple biophysical techniques. The current EPD method suffers from a number of deficiencies including lack of a meaningful relationship between color and actual molecular features, difficulties in identifying contributions from individual techniques, and a limited ability to be interpreted by color‐blind individuals. In this work, three improved data visualization approaches are proposed as techniques complementary to the EPD. The secondary, tertiary, and quaternary structural changes of multiple proteins as a function of environmental stress were first measured using circular dichroism, intrinsic fluorescence spectroscopy, and static light scattering, respectively. Data sets were then visualized as (1) RGB colors using three‐index EPDs, (2) equiangular polygons using radar charts, and (3) human facial features using Chernoff face diagrams. Data as a function of temperature and pH for bovine serum albumin, aldolase, and chymotrypsin as well as candidate protein vaccine antigens including a serine threonine kinase protein (SP1732) and surface antigen A (SP1650) from S. pneumoniae and hemagglutinin from an H1N1 influenza virus are used to illustrate the advantages and disadvantages of each type of data visualization technique.

Using empirical phase diagrams to understand the role of intramolecular dynamics in immunoglobulin G stability

Understanding the relationship between protein dynamics and stability is of paramount importance to the fields of biology and pharmaceutics. Clarifying this relationship is complicated by the large amount of experimental data that must be generated and analyzed if motions that exist over the wide range of timescales are to be included. To address this issue, we propose an approach that utilizes a multidimensional vector-based empirical phase diagram (EPD) to analyze a set of dynamic results acquired across a temperature-pH perturbation plane. This approach is applied to a humanized immunoglobulin G1 (IgG1), a protein of major biological and pharmaceutical importance whose dynamic nature is linked to its multiple biological roles. Static and dynamic measurements are used to characterize the IgG and to construct both static and dynamic EPDs. Between pH 5 and 8, a single, pH-dependent transition is observed that corresponds to thermal unfolding of the IgG. Under more acidic conditions, evidence exists for the formation of a more compact, aggregation resistant state of the immunoglobulin, known as A-form. The dynamics-based EPD presents a considerably more detailed pattern of apparent phase transitions over the temperature-pH plane. The utility and potential applications of this approach are discussed.