Temperature-Ramped Studies on the Aggregation,Unfolding, and Interaction of a Therapeutic Monoclonal Antibody

Investigations on thermal behavior are essential during the development of therapeutic proteins. Understanding the link between thermal unfolding and aggregation might help to minimize conformational and colloidal instabilities. In this study, a therapeutic monoclonal antibody and its Fab and Fc fragments were investigated. The apparent melting temperature of a protein and its onset were determined by differential scanning fluorimetry. Temperature-ramped turbidity measurements were performed to assess the temperature of aggregation, where large protein particles occurred. The formation of small aggregates was monitored and the interaction parameter k_D at low, ambient, and high temperature was calculated by temperature-ramped dynamic light scattering. Transformation of k_D into A_^*2 based on literature findings allowed the interpretation of net repulsive or attractive conditions. Repulsive net charges at low pH increased the colloidal stability, although a reduction of the conformational stability was observed. At neutral conditions and in the presence of salt, unfolding was followed by precipitation of the protein. A sharp decrease of k_D and negative A_^*2 values suggest that the aggregation was driven by hydrophobic interactions. Thus, the presented methods described and explained the thermal behavior of the protein and demonstrated their value for the development of pharmaceutical protein products.     

How Well Do Low- and High-Concentration Protein Interactions Predict Solution Viscosities of Monoclonal Antibodies?

Protein-protein interactions (PPI) and solution viscosities were measured at low and high protein concentrations under a range of formulation conditions for 4 different monoclonal antibodies. Static light scattering was used to quantify the osmotic second virial coefficient (B₂₂) and the zero-q limit static structure factor (S_q=0), versus protein concentration (c₂) from low to high c₂. Dynamic light scattering was used to measure the collective diffusion coefficient as a function of c₂ and to determine the protein interaction parameter (k_D). Static light scattering and dynamic light scattering were combined to determine the hydrodynamic factor (H_q=0), which accounts for changes in hydrodynamic PPI as a function of c₂. The net PPI ranged from strongly repulsive to attractive interactions, via changes in buffer pH, ionic strength, and choice of monoclonal antibodies. Multiple-particle tracking microrheology and capillary viscometery were used to measure monoclonal antibodies solution viscosities under the same solution conditions. In most cases, even large and qualitative changes in PPI did not result in significant changes in protein solution viscosity. This highlights the complex nature of PPI and how they influence protein solution viscosity and raises questions as to the validity of using experimental PPI metrics such as k_D or B₂₂ as predictors of high viscosity.     

Electrostatically Mediated Protein-Protein Interactions for Monoclonal Antibodies: A Combined Experimental and Coarse-Grained Molecular Modeling Approach

Electrostatically mediated protein-protein interactions (PPI) can influence key product properties such as solubility, solution viscosity, and aggregation rates. Predictive models would allow for candidates/formulations to be screened with little or no protein material. Three monoclonal antibodies that display qualitatively different experimental PPI were evaluated at a range of pH and ionic strength conditions that are typical of product formulations. PPI parameters (k_D, B₂₂, and G₂₂) were obtained from static and dynamic light scattering measurements and spanned from strongly repulsive to strongly attractive net interactions. Coarse-grained (CG) molecular simulations of PPI (specifically, B₂₂) were compared against experimental PPI parameters across multiple pH and salt conditions, using a CG model that treats each amino acid explicitly. Predicted B₂₂ values with default model parameters matched experimental B₂₂ values semiquantitatively for some cases; others required parameter tuning to account for effects such as ion binding. Experimental PPI values were also analyzed for each monoclonal antibody within the context of single-protein properties such as net charge, and domain-based and global dipole moments. The results show that PPI predicted qualitatively and semiquantitatively by CG molecular modeling of B₂₂ can be an effective computational tool for molecule and formulation assessment.     

Insulin Self-association: Effects on Lung Disposition Kinetics in the Airways of the Isolated Perfused Rat Lung (IPRL)

Purpose To characterize the kinetic dependence of pulmonary absorption and metabolism of insulin and lispro on the magnitude of their hexameric association. Methods Hexamer content by weight percent (%Hex) in various insulin-zinc and lispro-zinc solutions were determined by quantitative centrifugal ultrafiltration and zinc titration with terpyridine (QCUF-ZTT). Each of the solutions (0.1 ml) was then administered into the airways of the IPRL of normal and experimental diabetic animals. Rate constants were determined for lung absorption (k _a) and non-absorptive loss (k _nal; comprising mucociliary clearance and metabolism). Results %Hex in administered solutions ranged from 3.3 to 94.4%. Data analysis showed excellent correlations between the values for k _a or k _nal and %Hex, irrespective of insulin type, concentration, solution pH or ionic strength. The values for k _a decreased (0.22 → 0.05 h⁻¹) with increasing %Hex, as did values for k _nal. At %Hex in administered solutions ≥50%, values for k _nal approached estimates for the rate constant for mucociliary clearance, implying that lung metabolism occurred primarily with monomeric insulin. There were no differences in insulin disposition kinetics between lungs taken from experimental diabetic and sham-control animals. Conclusions The kinetics of pulmonary insulin disposition depended on the magnitude of molecular self-association. Dissociated forms of insulin (dimers or monomers) in the dosing solution showed higher rates than hexamers for both lung absorption and metabolism.     

The second virial coefficient as a predictor of protein aggregation propensity: A self-interaction chromatography study

The second osmotic virial coefficients (b₂) of four proteins – lysozyme, recombinant human lactoferrin, concanavalin A and catalase were measured by self-interaction chromatography (SIC) in solutions of varying salt type, concentration and pH. Protein aggregate sizes based on the initial hydrodynamic radius of the protein solution species present were measured using dynamic light scattering, and the relationship between b₂ and protein aggregate size was studied. A linear correlation was established between b₂ values and protein aggregate hydrodynamic size for all proteins, and for almost all solution conditions. Aggregate sizes of <∼10 nm, indicative of non-aggregated protein systems, were consistently observed to have b₂ values >0. The observed b₂ trends as a function of solution conditions were very much protein dependent, with notable trends including the existence of attractive interactions (negative b₂ values) at low ionic strengths for catalase and concanavalin A, and the highly positive b₂ values observed for lactoferrin over a wide range of solution conditions, reflecting lactoferrin’s innately high stability. It is concluded that the quantification of protein–protein interactions using SIC based b₂ data is a potentially valuable screening tool for predicting protein aggregation propensity.     

Predictive Nature of High-Throughput Assays in ADC Formulation Screening

Utilization of high-throughput biophysical screening techniques during early screening studies is warranted due to the limited amount of material and large number of samples. But the predictability of the data to longer-term storage stability is critical as the high-throughput methods assist in defining the design space for the longer-term studies. In this study, the biophysical properties of two ADCs in 16 formulation conditions were evaluated using high-throughput techniques. Conformational stability and colloidal stability were evaluated by determining T_m values, k_D, B₂₂, and T_agg. In addition, the samples were placed on stability and the extent of aggregate formation over the 8-week interval was determined. The rank order of the 16 different formulations in the high-throughput assays was compared to the rank order observed during the stability studies to assess the predictive capabilities of the screening methods. It was demonstrated that similar rank orders can be expected between high-throughput physical stability indicating assays such as T_agg and B₂₂ and traditional aggregation by SEC data, whereas conformational stability read-outs (T_m) are less predictive. In addition, the high-throughput assays appropriately identified the poor performing formulation conditions, which is ultimately what is desired of screening assays.     

Resolving Liquid-Liquid Phase Separation for a Peptide Fused Monoclonal Antibody by Formulation Optimization

Liquid-liquid phase separation (LLPS) of protein solutions has been usually related to strong protein-protein interactions (PPI) under certain conditions. For the first time, we observed the LLPS phenomenon for a novel protein modality, peptide-fused monoclonal antibody (pmAb). LLPS emerged within hours between pH 6.0 to 7.0 and disappeared when solution pH values decreased to pH 5.0 or lower. Negative values of interaction parameter (k_D) and close to zero values of zeta potential (ζ) were correlated to LLPS appearance. However, between pH 6.0 to 7.0, a strong electrostatic repulsion force was expected to potentially avoid LLPS based on the sequence predicted pI value, 8.35. Surprisingly, this is significantly away from experimentally determined pI, 6.25, which readily attributes the LLPS appearances of pmAb to the attenuated electrostatic repulsion force. Such discrepancy between experiment and prediction reminds the necessity of actual measurement for a complicated modality like pmAb. Furthermore, significant protein degradation took place upon thermal stress at pH 5.0 or lower. Therefore, the effects of pH and selected excipients on the thermal stability of pmAb were further assessed. A formulation consisting of arginine at pH 6.5 successfully prevented the appearance of LLPS and enhanced its thermal stability at 40 °C for pmAb. In conclusion, we have reported LLPS for a pmAb and successfully resolved the issue by optimizing formulation with aids from PPI characterization.     

Salt-Induced Aggregation of a Monoclonal Human Immunoglobulin G1

Physical stability is critical for any therapeutic protein's efficacy and economic viability. No reliable theory exists to predict stability de novo, and modeling aggregation is challenging as this phenomenon can involve orientation effects, unfolding, and the rearrangement of noncovalent bonds inter- and intramolecularly in a complex sequence of poorly understood events. Despite this complexity, the simple observation of protein concentration-dependent diffusivity in stable, low ionic-strength solutions can provide valuable information about a protein's propensity to aggregate at higher salt concentrations and over longer times. We recently verified this notion using two model proteins, and others have shown that this strategy may be applicable to antibodies as well. Here, we expand our previous study to a monoclonal human immunoglobulin G1 antibody and discuss both merits and limitations of stability assessments based on the diffusional virial coefficient k_D. We find this parameter to be a good predictor of relative protein stability in solutions of different chaotropic salts, and a telling heuristic for the effect of kosmotropes. Both temperature and glycosylation are seen to have a strong influence on k_D, and we examine how these factors affect stability assessments. Protein unfolding is monitored with a fluorescence assay to assist in interpreting the observed aggregation rates. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:377–386, 2013     

Specific interactions in high concentration antibody solutions resulting in high viscosity

The purpose of this work was to investigate the self‐associating behavior observed in high concentration monoclonal antibody (MAb1) solutions at pH 6.0. Zeta potential measurements over the pH range (4.0–9.0) showed lower net charges present on the molecule at pH 6.0 and 7.0. The point of zero charge or crossover from positive to negative potential was at pH 6.7 and was different from the theoretical isoelectric point (pI) of 7.8. Interaction parameter (k_D) from dynamic light scattering (DLS) studies indicated that attractive interactions of a similar magnitude span over a pH range of 5.0–8.0. Change in k_D values with increase in ionic strength corroborated that the interactions were electrostatic in origin. Comparative rheology analysis of three different monoclonal antibodies at pH 6.0, using ultrasonic shear rheometer, showed a high solution storage modulus (G′) and its sharp increase in magnitude at high concentrations in MAb1. The shape of the rheology profile was indicative of strong attractive interactions between solute molecules. High specific attractive interactions may be attributed to charge–charge and charge–dipole interactions at the surface in the Fab regions of MAb1, and gives a reasonable explanation for the pH dependency of the rheological profile observed. © 2009 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 1152–1168, 2010     

Potential Improvement in Shelf Life Using the Prodrug Approach. II. A Systematic Examination of Kinetic Requirements

The utilization time (UT) for a solution of a prodrug that is rapidly and completely converted to drug in the blood may be longer than the time for 10% loss of the initial concentration. The UT for an intravenous prodrug solution is the period during which the total prodrug and drug concentration exceeds 90% of the initial concentration. The influence of the rate of prodrug degradation (k _nc), its conversion (k _c) to drug, and the subsequent drug degradation (k _h) on the UT of a stored solution was examined by simulating the prodrug and drug concentration–time courses. The ratio of the shelf life of a prodrug solution to that of the parent drug (UT_ratio) was calculated using a wide range of values for the three rate constants. Three-dimensional plots relating the UT_ratio to the k _c, k _nc, and k _h values provide a basis for making a priori assessments of kinetic requirements for designing a prodrug to increase storage time. A parenteral prodrug intended to increase storage time may have a larger overall rate of loss than the parent drug, but it must have a smaller degradation rate (k _nc < k _h) to be successful. The UT for an oral prodrug solution depends upon the bioavailability of the prodrug relative to the drug in addition to the values for k_nc, k _c, and k _h. Two ampicillin prodrugs were used as models to calculate actual UT_ratio versus pH profiles. Intravenous solutions showed modest gains in the UT_ratio in the acid region, whereas oral solutions reached a UT_ratio as high as 22 by combining favorable rate constants with increased bioavailability. These actual UT_ratio versus pH profiles were interpreted in terms of the theory established using the simulations.     

Characterization of LLC-PK1 Kidney Epithelial Cells as an in Vitro Model for Studying Renal Tubular Reabsorption of Protein Drugs

Purpose. The purpose of this study was to assess whether LLC-PK₁ renal epithelial cells could serve as an in vitro model for studying the renal tubular reabsorption of protein drugs. Methods. The association of ¹¹¹In-labeled model protein drugs, bovine serum albumin (BSA), superoxide dismutase (SOD), soybean trypsin inhibitor (STI), and [Asu^1,7]-eel calcitonin (Asu-ECT), with the monolayers of LLC-PK₁ renal epithelial cells was characterized under various conditions. Results. The cellular association of these proteins was temperature-dependent and varied according to the protein. Saturation kinetics were observed for STI association, with the apparent Km and Vmax values determined to be 66.3 µg/ml and 250 ng/mg protein/min, respectively. The association of STI decreased with increases in medium pH from 5.4 to 8.4 and was inhibited significantly by 2,4-dinitrophenol, sodium azide, cytochalasin B, and colchicine, suggesting that the cellular association involved endocytosis. Mutual inhibition was observed in competitive binding experiments with the four protein drugs, suggesting that they shared a common binding site on the luminal membrane of LLC-PK₁ cells. Taken together, these findings show that a variety of protein drugs bind to LLC-PK₁ cells in a non-specific manner and possibly undergo endocytosis, a phenomenon that is similar to in vivo proximal tubular reabsorption. Conclusions. LLC-PK₁ renal epithelial cells would be a suitable model system for the study of the renal proximal tubular reabsorption of protein drugs.     

Protein Structural Conformation and Not Second Virial Coefficient Relates to Long-Term Irreversible Aggregation of a Monoclonal Antibody and Ovalbumin in Solution

Purpose The purpose of the study was to investigate the relationship of the second virial coefficient, B₂₂, to the extent of irreversible protein aggregation upon storage. Methods A monoclonal antibody and ovalbumin were incubated at 37°C (3 months) under various solution conditions to monitor the extent of aggregation. The B₂₂ values of these proteins were determined under similar solution conditions by a modified method of flow-mode static light scattering. The conformation of these proteins was studied using circular dichroism (CD) spectroscopy and second-derivative Fourier transform infrared spectroscopy. Results Both proteins readily aggregated at pH 4.0 (no aggregation observed at pH 7.4); the extent of aggregation varied with the ionic strength and the presence of cosolutes (sucrose, glycine, and Tween 80). Debye plots of the monoclonal antibody showed moderate attractive interactions at pH 7.4, whereas, at pH 4.0, nonlinear plots were obtained, indicating self-association. CD studies showed partially unfolded structure of antibody at pH 4.0 compared with that at pH 7.4. In the case of ovalbumin, similar B₂₂ values were obtained in all solution conditions irrespective of whether the protein aggregated or not. CD studies of ovalbumin indicated the presence of a fraction of completely unfolded as well as partially unfolded species at pH 4.0 compared with that at pH 7.4. Conclusions The formation of a structurally altered state is a must for irreversible aggregation to proceed. Because this aggregation-prone species could be an unfolded species present in a small fraction compared with that of the native state or it could be a partially unfolded state whose net interactions are not significantly different compared with those of the native state, yet the structural changes are sufficient to lead to long-term aggregation, it is unlikely that B₂₂ will correlate with long-term aggregation.     

Optimal Experimental Design for Precise Estimation of the Parameters of the Axial Dispersion Model of Hepatic Elimination

The axial dispersion model of hepatic drug elimination is characterized by two dimensionless parameters, the dispersion number, D_N , and the efficiency number, R_N , corresponding to the relative dispersion of material on transit through the organ and the relative efficiency of elimination of drug by the organ, respectively. Optimal design theory was applied to the estimation of these two parameters based on changes in availability (F) of drug at steady state for the closed boundary condition model, with particular attention to variations in the fraction of drug unbound in the perfusate (fu_B ). Sensitivity analysis indicates that precision in parameter estimation is greatest when F is low and that correlation between R_N and D_N is high, which is desirable for parameter estimation, when D_N lies between 0.1 and 100. Optimal design points were obtained using D-optimization, taking into account the error variance model. If the error variance model is unknown, it is shown that choosing Poisson error model is reasonable. Furthermore, although not optimal, geometric spacing of fu_B values is often reasonable and definitively superior to a uniform spacing strategy. In practice, the range of fu_B available for selection may be limited by such practical considerations as assay sensitivity and acceptable concentration range of binding protein. Notwithstanding, optimal design theory provides a rational approach to precise parameter estimation.     

Effects of Solution Conditions on Methionine Oxidation in Albinterferon Alfa-2b and the Role of Oxidation in its Conformation and Aggregation

Physical and chemical degradation of therapeutic proteins can occur simultaneously. In this study, our first objective was to investigate how solution conditions that impact conformational stability of albinterferon alfa-2b, a recombinant fusion protein, modulate rates of methionine (Met) oxidation. Another objective of this work was to determine whether oxidation affects conformation and rate of aggregation of the protein. The protein was subjected to oxidation in solutions of varying pH, ionic strength, and excipients by the addition of 0.02% tertiary-butyl hydroperoxide (TBHP). The rate of formation of Met-sulfoxide species was monitored by reversed-phase high-performance liquid chromatography and compared across solution conditions. Albinterferon alfa-2b exhibited susceptibility to Met oxidation during exposure to TBHP that was highly dependent on solution parameters, but there was not a clear correlation between oxidation rate and protein conformational stability. Met oxidation resulted in significant perturbation of both secondary and tertiary structure of albinterferon alfa-2b as shown by both far-ultraviolet (UV) and near-UV circular dichroism. Moreover, oxidation of the protein caused a noticeable reduction in the protein's resistance to thermal denaturation. Surprisingly, despite its negative effect on solution structure and conformational stability, oxidation actually reduced the protein's aggregation rate during agitation at room temperature as well as during quiescent incubation at 40°C. Oxidation of the protein resulted in improved colloidal stability of the protein, which is manifested by a more positive B₂₂ value in the oxidized protein. Thus, the reduced aggregation rate after oxidation suggests that increased colloidal stability of oxidized albinterferon alfa-2b counteracted oxidation-induced decreases in conformational stability.     

An Unexpected pH Effect on the Stability of Moexipril Lyophilized Powder

Because of the limited stability of moexipril (RS-10085; 1) in aqueous solution, lyophilized parenteral formulations were evaluated as a function of pH in this study. In general, the lyophilized powder of 1 showed about two orders of magnitude less reactivity at 50°C than in aqueous solution at pH values below 3 or above 6. At pH 5.1, however, the lyophilized powder had maximum reactivity, with the rate actually comparable to that observed in aqueous solution. When the distribution of the two major products, diketopiperazine (DKP) 2 and ester hydrolysis analogue 3, was compared to the observed kinetics as a function of pH, it was clear that removal of water via lyophilization suppressed the spontaneous k ₁ cyclization process, the spontaneous k ₃ hydrolysis process, and the specific base-catalyzed k ₄ hydrolysis process. The overall spontaneous k ₂ cyclization process, however, was not affected by lyophilization. The latter result is accounted for by the increased equilibrium constant for the formation of the tetrahedral intermediate, To, as a result of lyophilization. This study demonstrates that stability data in solution can not be used for predicting the stability of moexipril in lyophilized powder form.     

Imaging apomorphine stimulation of brain arachidonic acid signaling <Emphasis Type="Italic">via</Emphasis> D<Subscript>2</Subscript>-like receptors in unanesthetized rats

Rationale and objective Because of the important role of dopamine in neurotransmission, it would be useful to be able to image brain dopamine receptor-mediated signal transduction in animals and humans. Administering the D₁–D₂ receptor agonist apomorphine may allow us to do this, as the D₂-like receptor is reported to be coupled to cytosolic phospholipase A₂ activation and arachidonic acid (AA) release from membrane phospholipid. Methods Unanesthetized adult rats were given intraperitoneally apomorphine (0.5 mg/kg) or saline, with or without pretreatment with 6 mg/kg intravenous raclopride, a D₂/D₃ receptor antagonist. [1–¹⁴C]AA was injected intravenously, then AA incorporation coefficients k*—brain radioactivity divided by integrated plasma radioactivity—markers of AA signaling, were measured using quantitative autoradiography in 62 brain regions. Results Apomorphine significantly elevated k* in 26 brain regions, including the frontal cortex, motor and somatosensory cortex, caudate-putamen, thalamic nuclei, and nucleus accumbens. Raclopride alone did not change baseline values of k*, but raclopride pretreatment prevented the apomorphine-induced increments in k*. Conclusions A mixed D₁–D₂ receptor agonist, apomorphine, increased the AA signal by activating only D₂-like receptors in brain circuits containing regions with high D₂-like receptor densities. Thus, apomorphine might be used with positron emission tomography to image brain D₂-like receptor-mediated AA signaling in humans in health and disease.     

Arginine and its Derivatives Suppress the Opalescence of an Antibody Solution

Opalescence is a problem concerned with the stability of an antibody solution. It occurs when a high concentration of a protein is present. Arginine (Arg) is a versatile aggregation suppressor of proteins, which is among the candidates that suppress opalescence in antibody solutions. Here, we investigated the effect of various types of small molecular additives on opalescence to reveal the mechanism of Arg in preventing opalescence in antibody solution. As expected, Arg suppressed the opalescence of the immunoglobulin G (IgG) solution. Arg also concentration dependently inhibited the formation of microstructures in IgG molecules. Interestingly, the intrinsic fluorescence spectra of highly concentrated IgG solutions differed from those having low concentrations, even though IgG retained a distinct tertiary structure. Arginine ethylester was more effective in suppressing the opalescence of IgG solutions than Arg, whereas lysine and γ-guanidinobutyric acid were less effective. These results indicated that positively charged groups of both α-amine and guanidinium actively influence Arg as an additive for suppressing opalescence. Diols, which are the suppressors of the liquid–liquid phase separation of proteins were also effective in suppressing the opalescence. These results therefore provide insight into the control of opalescence of antibody solutions at high concentrations using solution additives.     

The effect of deuterium oxide on the conformational stability and aggregation of bovine serum albumin

Abstract

Protein aggregation is a significant problem affecting the integrity of proteins, and is a major hindrance to the development of biopharmaceutical products. Deuterium oxide (D₂O), widely used in protein characterization studies, has been shown to promote protein aggregation when used as a substitute for water in most buffered protein solutions; however, a few studies have reported minor improvements in melting point temperatures for some proteins. Our study aims to investigate the effect of D₂O on protein stability, using bovine serum albumin (BSA) as a model. We performed accelerated stability studies at high temperatures and assessed the physical and conformational stability of BSA using fluorescence spectroscopy, dynamic light scattering (DLS) and size-exclusion high performance liquid chromatography. Our findings reveal that D₂O enhances the conformational stability of monomeric BSA, reducing monomer loss and formation of small aggregates at high temperatures. There is also an increase in the formation of larger aggregates probed by thioflavin T (ThT), however, the increase is not considered significant based on DLS results. Our findings demonstrate that exchanging water with D₂O can improve the stability of proteins in solution, by maintaining the stability of the monomeric form, which may be beneficial for the long-term storage of some biological products.