A Biopharmaceutical Perspective on Higher-Order Structure and Thermal Stability of mRNA Vaccines.

Preservation of the integrity of macromolecular higher-order structure is a tenet central to achieving biologic drug and vaccine product stability toward manufacturing, distribution, storage, handling, and administration. Given that mRNA lipid nanoparticles (mRNA-LNPs) are held together by an intricate ensemble of weak forces, there are some intriguing parallels to biologic drugs, at least at first glance. However, mRNA vaccines are not without unique formulation and stabilization challenges derived from the instability of unmodified mRNA and its limited history as a drug or vaccine. Since certain learning gained from biologic drug development may be applicable for the improvement of mRNA vaccines, we present a perspective on parallels and contrasts between the emerging role of higher-order structure pertaining to mRNA-LNPs compared to pharmaceutical proteins. In a recent publication, the location of mRNA encapsulated within lipid nanoparticles was identified, revealing new insights into the LNP structure, nanoheterogeneity, and microenvironment of the encapsulated mRNA molecules [Brader et al. Biophys. J. 2021, 120, 2766]. We extend those findings by considering the effect of encapsulation on mRNA thermal unfolding with the observation that encapsulation in LNPs increases mRNA unfolding temperatures.

Encapsulation state of messenger RNA inside lipid nanoparticles.

Understanding the structure of messenger RNA (mRNA) lipid nanoparticles, and specifically the microenvironment of the mRNA molecules within these entities, is fundamental to advancing their biomedical potential. Here, we show that a permeating cationic dye, thionine, can serve as a cryogenic electron microscopy contrasting agent by binding selectively to encapsulated mRNA without disturbing lipid nanoparticle morphology. Cryo-electron microscopy images identify the mRNA location, revealing that mRNA may exist within solvent-filled cavities or may be substantially lipid associated.

Using X-Ray Crystallography to Simplify and Accelerate Biologics Drug Development.

Every major biopharmaceutical company incorporates a protein crystallography unit that is central to its structure-based drug discovery efforts. Yet these capabilities are rarely leveraged toward the formal higher order structural characterization that is so challenging but integral to large-scale biologics manufacturing. Although the biotech industry laments the shortcomings of its favored biophysical techniques, x-ray crystallography is not even considered for drug development. Why not? We suggest that this is due, at least in part, to outdated thinking (for a recent industry-wide survey, see Gabrielson JP, Weiss IV WF. Technical decision-making with higher order structure data: starting a new dialogue. J Pharm Sci. 2015;104(4):1240-1245). We examine some myths surrounding protein crystallography and highlight the inherent properties of protein crystals (molecular identity, biochemical purity, conformational uniformity, and macromolecular crowding) as having practicable commonalities with today's patient-focused liquid drug products. In the new millennium, protein crystallography has become essentially a routine analytical test. Its application may aid the identification of better candidate molecules that are more amenable to high-concentration processing, formulation, and analysis thereby helping to make biologics drug development quicker, simpler, and cheaper.

Evaluation of Incremental Siliconization Levels on Soluble Aggregates, Submicron and Subvisible Particles in a Prefilled Syringe Product.

The evaluation of stability with respect to particles in prefilled syringes is complicated by the presence of silicone oil. The mobility, colloidal characteristics, and kinetic instability of silicone oil in contact with a protein formulation may be influenced in unpredictable ways by pharmaceutical variables, storage, and handling conditions. To provide insight into the impact of these variables on silicone oil originating specifically from the siliconized prefillable syringe (PFS), a series of studies were conducted at incremental syringe barrel siliconization levels. Size-exclusion chromatography and particle counting methods were used to quantitate soluble aggregates and submicron and subvisible particles in peginterferon beta-1a in a PFS siliconized with a fixed nozzle spray-on siliconization process. The effect of silicone oil on the peginterferon beta-1a molecule was examined under pharmaceutically relevant conditions, accelerated degradation, and under denaturing conditions. Resonant mass measurement was used to discriminate silicone oil from protein particles establishing that silicone oil does not mask adverse trends in non-silicone oil particles. The peginterferon beta-1a molecule was shown to be stable in the presence of silicone oil and robust with respect to the formation of soluble aggregates and submicron and subvisible particles in its PFS siliconized over the range of 0-1.2 mg silicone oil per syringe barrel.

Orthogonal Methods for Characterizing the Unfolding of Therapeutic Monoclonal Antibodies: Differential Scanning Calorimetry, Isothermal Chemical Denaturation, and Intrinsic Fluorescence with Concomitant Static Light Scattering.

Evaluating prospective protein pharmaceutical stability from accelerated screening is a critical challenge in biotherapeutic discovery and development. Measurements of protein unfolding transitions are widely employed for comparing candidate molecules and formulations; however, the interrelationships between intrinsic protein conformational stability and pharmaceutical robustness are complex and thermal unfolding measurements can be misleading. Beyond the discovery phase of drug development, astute formulation design is one of the most crucial factors enabling the protein to resist damage to its higher order structure-initially from bioprocessing stresses, then from stresses encountered during its journey from the product manufacturing site to the bloodstream of the patient. Therapeutic monoclonal antibodies are multidomain proteins that represent a large and growing segment of the biotechnology pipeline. In this chapter, we describe how differential scanning calorimetry may be leveraged synergistically with isothermal chemical denaturation and intrinsic fluorescence with concomitant static light scattering to elucidate characteristics of mAb unfolding and aggregation that are helpful toward understanding and designing optimal pharmaceutical compositions for these molecules.

Examination of thermal unfolding and aggregation profiles of a series of developable therapeutic monoclonal antibodies.

Screening for pharmaceutically viable stability from measurements of thermally induced protein unfolding and short-term accelerated stress underpins much molecule design, selection, and formulation in the pharmaceutical biotechnology industry. However, the interrelationships among intrinsic protein conformational stability, thermal denaturation, and pharmaceutical stability are complex. There are few publications in which predictions from thermal unfolding-based screening methods are examined together with pharmaceutically relevant long-term storage stability performance. We have studied eight developable therapeutic IgG molecules under solution conditions optimized for large-scale commercial production and delivery. Thermal unfolding profiles were characterized by differential scanning calorimetry (DSC) and intrinsic fluorescence recorded simultaneously with static light scattering (SLS). These molecules exhibit a variety of thermal unfolding profiles under common reference buffer conditions and under individually optimized formulation conditions. Aggregation profiles by SE-HPLC and bioactivity upon long-term storage at 5, 25, and 40 °C establish that IgG molecules possessing a relatively wide range of conformational stabilities and thermal unfolding profiles can be formulated to achieve pharmaceutically stable drug products. Our data suggest that a formulation design strategy that increases the thermal unfolding temperature of the Fab transition may be a better general approach to improving pharmaceutical storage stability than one focused on increasing Tonset or Tm of the first unfolding transition.

Monitoring protein aggregation kinetics with simultaneous multiple sample light scattering.

A simultaneous multiple sample light scattering (SMSLS) prototype instrument was built to simultaneously measure light scattering from many independent monoclonal antibody (mAb) solutions in order to monitor their time-dependent aggregation behavior and to characterize, via absolute Rayleigh scattering ratios, their molecular masses and second, third, and fourth virial coefficients under non-aggregating conditions at concentrations up to 190mg/ml. One stable mAb and another prone to aggregation were studied. Early phase aggregation rates spanned six orders of magnitude over temperatures 30 to 83°C for both mAbs and divided into "Arrhenius" and "Stochastic" regimes. The Arrhenius regimes comprise two thermal regimes whose breakpoint occurs near the first thermal unfolding temperature of the mAb domain structure. The Stochastic regime occurs for T⩽40°C. Rates yielded activation energies and temperature and concentration crossovers among rate-limiting regimes. Virial coefficients were closely related to aggregation kinetics. Hydrodynamic diameter relationship to virial coefficients provided further insight into stability. SMSLS detected as few as three dimerization events among 1000 monomeric proteins. Although early phase aggregation is linear in time and reproducible, aggregation becomes chaotic in later phases. SMSLS dramatically increases protein monitoring throughput, providing continuous monitoring for hours, weeks, and longer. New samples can be changed in and out without affecting other sample measurements in progress.

Effects of submicron particles on formation of micron-sized particles during long-term storage of an interferon-ß-1a solution.

We present evidence that homogeneous submicron particles can influence the growth rate of larger particles upon long-term storage in a temperature-dependent manner. Interferon-beta-1a was thermally stressed at 50°C for 6 h and characterized using nanoparticle tracking analysis (NTA), microflow digital imaging (MFI), and circular dichroism (CD) spectroscopy. This study showed selective formation of submicron particles exhibiting a perturbed protein conformation. These thermally induced submicron particles were spiked into an unstressed solution at three levels, and then monitored for micron-sized particle formation upon storage at 5°C and 25°C for 12 months. The resulting particle growth effects were temperature dependent. NTA and MFI results at 5°C showed little evidence that initial submicron particle levels impacted particle growth across the range ~0.03-25 µm. In contrast, MFI results at 25°C indicated that particle growth in the 1-10 µm size range correlated strongly with initial submicron particle levels, and particle counts in the 10-25 µm size range were highest after 12 months for the samples with highest initial submicron particle content.

Calcium binding to a factor ix Fc fusion protein and effects on higher-order structure.

There is significant scope for more meaningful evaluation of higher-order structure in defining the quality of biopharmaceutical products [Bush L. 2010. Biopharm Int 23(4):14]. We have used isothermal titration calorimetry (ITC) to characterize the Ca(2+) -binding isotherm of a recombinant human factor IX Fc fusion protein (rFIXFc) and the parent recombinant human factor IX molecule (rFIX). Circular dichroism, intrinsic fluorescence, and Fourier transform infrared spectroscopies detected characteristic spectral changes that appear qualitatively consistent with the previously characterized behavior of the factor IX molecule. Sedimentation velocity and dynamic light scattering measurements were recorded in the presence and absence of Ca(2+) over the protein concentration range 1-10 mg/mL. ITC of Ca(2+) binding to rFIXFc reveals a distinctive exothermic-binding isotherm, which is interpreted as consistent with two high-affinity and approximately 14 lower-affinity Ca(2+) sites reported in the literature for human factor IX (Schmidt AE, Bajaj SP. 2003. Trends Cardiovasc Med 13(1):39-45). Analysis of accelerated degradation samples showed significant alterations in Ca(2+) binding, which correlates with significant loss of biopotency and fragmentation by gel chip capillary electrophoresis. Collectively, these data establish a close correspondence in the Ca(2+) -binding characteristics of rFIXFc and its parent rFIX molecule. The utility of ITC to provide a highly pertinent and selective biophysical signature of structure-function for a therapeutic factor protein is discussed.

Fragmentation of a highly purified monoclonal antibody attributed to residual CHO cell protease activity.

Monoclonal antibody (mAb) fragmentation can be a widespread problem across the biotechnology industry and there is a current need to better understand the underlying principles. Here, we report an example of a high-purity human IgG1 mAb prepared from CHO cells exhibiting fragmentation that can be attributed to residual proteolytic enzyme activity. The concomitant occurrence of proteolytic and non-proteolytic peptide bond cleavage is shown and the respective fragmentation patterns characterized using high-resolution LC-MS. Fragmentation rates are monitored by SE-HPLC and SDS-PAGE over the pH range 4-6 and characterized in the presence and absence of pepstatin A, an inhibitor of acidic proteases. After 20 days at 40°C, pH 4, ∼60% decrease in BIIB-mAb monomer peak occurred attributed to residual proteolytic activity. At pH 5, this value was ∼13%. These results have implications for formulation design studies and the interpretation of accelerated stability data. A simple method to screen for acidic protease activity using the proteolytic enzyme inhibitor pepstatin A is described.

Quantitation of aggregate levels in a recombinant humanized monoclonal antibody formulation by size-exclusion chromatography, asymmetrical flow field flow fractionation, and sedimentation velocity.

Size-exclusion high-performance liquid chromatography (SE-HPLC, SEC) is the long-standing biopharmaceutical industry standard for quantitation of soluble protein aggregates. Recently, sedimentation velocity analytical ultracentrifugation (SV-AUC) has emerged as a possible orthogonal technique to SEC for soluble aggregate quantitation. Moreover, asymmetrical flow field flow fractionation (AF4) has shown early promise in quantifying protein aggregates, both soluble and insoluble. We report soluble aggreg ate quantities measured by SEC, AF4, and SV-AUC analyzed by SEDFIT/c(s) for acid stressed and unstressed samples of a recombinant humanized monoclonal antibody. In equivalent antibody samples, SV-AUC, and AF4 detect markedly higher total aggregate levels than SEC. Furthermore, SEC fails to detect higher molecular weight soluble aggregates apparent in SV-AUC and AF4 analyses. Pooled fractions containing soluble dimeric aggregates were purified and re-analyzed by both SV-AUC and SEC. Reinjection of purified dimer onto the SEC column induces formation of detectable quantities of monomer and trimer. All sample types show statistically significant (p-values<0.01) antibody losses through the SEC column. This incomplete mass recovery from SEC indicates probable antibody physical adsorption to gel filtration media. Analysis of the sedimentation behavior of high molecular weight components suggests increased molecular asphericity with increasing molecular weight. We present an aggregation model based on nearly linear end-to-end assembly of monomeric subunits which is shown to be consistent with SV-AUC, SEC, AF4, and dynamic light scattering (DLS) results.

Biophysical signatures of noncovalent aggregates formed by a glucagonlike peptide-1 analog: a prototypical example of biopharmaceutical aggregation.

LY307161 is a 31 amino acid analog of glucagonlike peptide-1(7-37)OH susceptible to physical instability associated with pharmaceutical processing. Orthogonal biophysical studies were conducted to explore the origins of this physical instability and to distinguish pharmaceutically desirable states of this aggregating peptide from undesirable ones. Equilibrium sedimentation analysis established that LY307161 exists as a monomer at pH 3, and reversibly self-associates in the pH range 7.5-10.5. Causative factors for physical instability related to lyophilization conditions were investigated. Solution pH, acetonitrile content, and concentration of the peptide prior to lyophilization each impacted physicochemical properties of the resultant powders. A comparative study of two powder samples exhibiting physicochemically disparate properties established that LY307161 forms soluble noncovalent aggregates. FT-IR analyses in the solid and solution states identified a prominent band at 1657-1659 cm(-1) attributed to alpha-helix structure. Noncovalent soluble aggregate exhibited characteristic bands at 1615 and 1698 cm(-1) indicative of intermolecular beta-sheet structure. An agitation-induced, precipitated solid form of LY307161 exhibited a different FT-IR signature indicative of a conformationally distinct species. Circular dichroism and fluorescence spectroscopy, together with dynamic light scattering measurements and dye-aggregate complexation, provided additional insights into the distinctions between aggregated and native LY307161.

Hybrid insulin cocrystals for controlled release delivery.

The ability to tailor the release profile of a drug by manipulating its formulation matrix offers important therapeutic advantages. We show here that human insulin can be cocrystallized at preselected ratios with the fully active lipophilically modified insulin derivative octanoyl-N(epsilon)-LysB29-human insulin (C8-HI). The cocrystal is analogous to the NPH (neutral protamine Hagedorn) crystalline complex formed with human insulin, which is commonly used as the long-acting insulin component of diabetes therapy. The in vitro and in vivo release rates of the cocrystal can be controlled by adjusting the relative proportions of the two insulin components. We identified a cocrystal composition comprising 75% C8-HI and 25% human insulin that exhibits near-ideal basal pharmacodynamics in somatostatin-treated beagle dogs. The dependence of release rate on cocrystal ratio provides a robust mechanism for modulating insulin pharmacodynamics. These findings show that a crystalline protein matrix may accommodate a chemical modification that alters the dissolution rate of the crystal in a therapeutically useful way, yet that is structurally innocuous enough to preserve the pharmaceutical integrity of the original microcrystalline entity and the pharmacological activity of the parent molecule.