Demystifying Fogging in Lyophilized Products: Impact of Pharmaceutical Processing.

A commonly encountered challenge with freeze-dried drug products is glass vial fogging. Fogging is characterized by a thin layer of product deposited upon the inner surface of the vial above the lyophilized cake. While considered to be a routine cosmetic defect in many instances, fogging around the shoulder and neck of the vial may potentially impact container closure integrity and reject rates during inspection. In this work, the influence of processing conditions i.e. vial pre-treatment, lyophilization cycle modifications and filling conditions on fogging was evaluated. A battery of analytical techniques was employed to investigate factors affecting glass vial fogging. A fogging score was used to quantify its severity in freeze-dried products. Additionally, a dye-based method was used to study solution upcreep (Marangoni flow) following product filling. Our lab-scale results indicate measurable improvement in fogging following the addition of an annealing step in the lyophilization cycle. Pre-freeze isothermal holding of the vials (at 5°C on the lyophilizer shelf) for an extended duration indicated a reduction in fogging whereas an increase in the freezing time exhibited no effect on fogging. Vial pre-treatment conditions were critical determinants of fogging for Type 1 vials whereas they had no impact on fogging in TopLyo® vials. The headspace relative humidity (RH) investigation also indicated sufficient increase in the water vapor pressure inside the vial to be conducive to the formulation of a hydration film - the precursor to Marangoni flow.

Designing Robust Monoclonal Antibody Drug Products: Pitfalls of Simplistic Approaches for Stability Prediction.

Thermal stability attributes including unfolding onset (Tonset) and mid-point (Tm) are often utilized for efficient development of monoclonal antibody (mAb) products during lead selection and formulation screening workflows. An assumption of direct correlation between thermal and kinetic physical stability underpins this basic approach. While literature reports have substantiated this general approach under specific conditions, clear exceptions have been highlighted alongside. Herein, a set of mAbs formulated under diverse solution conditions to generate a broad array of thermal and kinetic stability profiles were systematically analyzed. Sequence modifications in the Fc region were purposefully engineered to generate a set of low-melting mAbs. A diverse set of excipients were subsequently utilized and shown to modulate the Tm over a wide range. While a general correlation between high Tm and low aggregation rate was observed under accelerated conditions, the predictive utility of Tm under relevant product storage conditions was inadequate at best. Critically, Tm data did not correlate with long-term aggregation rates under refrigerated or room temperature conditions. Even under accelerated conditions, Tm appeared to be a poor predictor of aggregation once it exceeded the solution storage temperature (40°C) by ∼15°C, similar to conditions routinely encountered in the development of canonical mAbs (Tm > 60°C). Pitfalls of simplistic correlative approaches are discussed in the context of practical biologics product development.

Freeze Drying and Vial Breakage: Misconceptions, Root Causes and Mitigation Strategies for the Pharmaceutical Industry.

Vial breakage during or following freeze drying (lyophilization) is a well-known and documented phenomenon in the pharmaceutical industry. However, the underlying mechanism and probable root causes are not well characterized. Mostly, the phenomenon is attributed to the presence of crystallizing excipients, such as mannitol in the formulation, while other potential factors are often underestimated or not well studied. In this work we document a systematic multipronged approach to characterize and identify potential root cause(s) of vial breakage during lyophilization. Factors associated with formulation, product configuration, primary container and production process stress conditions were identified and their impact on vial breakage was studied in both lab and manufacturing scale conditions. Studies included: 1) strain gauge and lyophilization analysis for stress on glass vials with different formulation conditions and fill volumes, 2) manufacturing fill-finish process risk assessment (ex. loading and frictive force impact on the vials), and 3) glass vial design and ruggedness (ex. glass compression resistance or burst strength testing). Importantly, no single factor could be independently related to the extent of vial breakage observed during production. However, a combination of formulation, fill volume, and vial weakening processes encountered during at-scale production, such as vial handling, shelf loading and unloading, were identified to be the most probable root causes for the low levels of vial breakage observed. The work sheds light on an often-encountered problem in the pharmaceutical industry and the results presented in this paper argue against the simplistic root-cause explanations reported in literature. The work also provides insight into the possibility of implementing mitigative approaches to minimize or eliminate vial breakage associated with lyophilized drug products.

Succinate Buffer in Biologics Products: Real-world Formulation Considerations, Processing Risks and Mitigation Strategies.

The succinic acid/succinate system has an excellent buffering capacity at acidic pH values (4.5-6.0), promising to be a buffer of choice for biologics having slightly acidic to basic isoelectric points (pI 6 - 9). However, its prevalence in drug products is limited due to the propensity (risk) of its components to crystallize during freezing and the consequent shift in the pH which might affect the product stability. Most of these previous assessments have been performed under operational conditions that do not simulate typical drug product processing conditions. In this work, we have characterized the physicochemical behavior of succinate formulations under representative pharmaceutical conditions. Our results indicate that the pH increases by ∼ 1.2 units in 25 mM and 250 mM succinate buffers at pharmaceutically relevant freezing conditions. X-ray diffractometry studies revealed selective crystallization of monosodium succinate, which is posed as the causative mechanism. This salt crystallization was not observed in the presence of 2% w/v sucrose, suggesting that this pH shift can be mitigated by including sucrose in the formulation. Additionally, three monoclonal antibodies (mAbs) that represent different IgG subtypes and span a range of pIs (5.9 - 8.8) were formulated with succinate and sucrose and subjected to freeze-thaw, frozen storage and lyophilization. No detrimental impact on quality attributes (QA) such as high molecular weight (HMW) species, turbidity, alteration in protein concentration and sub-visible particles, was observed of any of the mAbs tested. Lastly, drug formulations lyophilized in succinate buffer with sucrose demonstrated acceptable QA profiles upon accelerated kinetic storage stability, supporting the use of succinate buffers in mAb drug products.

A Systematic Approach to Evaluating Closed System Drug-Transfer Devices During Drug Product Development.

The use of Closed System Drug-Transfer Devices (CSTDs) has increased significantly in recent years due to NIOSH and USP recommendations to use them during preparation of hazardous drugs. Mechanistic and material differences between CSTDs and traditional in-use components warrant an assessment of their impact on product quality and dosing accuracy. Using a combination of prevalent CSTDs with biologic molecules, we performed an extensive assessment of the effect of using CSTDs for dose preparation and observed no negative impact on product quality attributes. Additionally, we found that the CSTD hold-up volume is 2 to 4-fold higher than conventional in-use components and exhibited a strong dependence on the CSTD brand used. We also found that the CSTD brand and dosing volume have a major influence on dosing accuracy with suboptimal protein recovery at very low dosing volumes. We identified entrapment of product in the CSTD spike as the root cause for this sub-optimal recovery and found that flushing the CSTD spike with a brand-new syringe and not the dosing syringe aided in complete protein recovery. Taken together we present a systematic approach to evaluate the risks and impact of CSTD to drug product quality, dose preparation, and dosing accuracy.

Predicting High-Concentration Interactions of Monoclonal Antibody Solutions: Comparison of Theoretical Approaches for Strongly Attractive Versus Repulsive Conditions.

Nonspecific protein-protein interactions of a monoclonal antibody were quantified experimentally using light scattering from low to high protein concentrations (c2) and compared with prior work for a different antibody that yielded qualitatively different behavior. The c2 dependence of the excess Rayleigh ratio (Rex) provided the osmotic second virial coefficient (B22) at low c2 and the static structure factor (Sq=0) at high c2, as a function of solution pH, total ionic strength (TIS), and sucrose concentration. Net repulsive interactions were observed at pH 5, with weaker repulsions at higher TIS. Conversely, attractive electrostatic interactions were observed at pH 6.5, with weaker attractions at higher TIS. Refined coarse-grained models were used to fit model parameters using experimental B22 versus TIS data. The parameters were used to predict high-c2 Rex values via Monte Carlo simulations and separately with Mayer-sampling calculations of higher-order virial coefficients. For both methods, predictions for repulsive to mildly attractive conditions were quantitatively accurate. However, only qualitatively accurate predictions were practical for strongly attractive conditions. An alternative, higher resolution model was used to show semiquantitatively and quantitatively accurate predictions of strong electrostatic attractions at low c2 and low ionic strength.

Predicting Protein-Protein Interactions of Concentrated Antibody Solutions Using Dilute Solution Data and Coarse-Grained Molecular Models.

Protein-protein interactions for solutions of an IgG1 molecule were quantified using static light scattering (SLS) measurements from low to high protein concentrations (c2). SLS was used to determine second osmotic virial coefficients (B22) at low c2, and excess Rayleigh profiles (Rex/K vs. c2) and zero-q structure factors (Sq=0) as a function of c2 at higher c2 for a series of conditions (pH, sucrose concentration, and total ionic strength [TIS]). Repulsive (attractive) interactions were observed at low TIS (high TIS) for pH 5 and 6.5, with increasing repulsions when 5% w/w sucrose was also present. Previously developed and refined coarse-grained antibody models were used to fit model parameters from B22 versus TIS data. The resulting parameters from low-c2 conditions were used as the sole input to multiprotein Monte Carlo simulations to predict high-c2Rex/K and Sq=0 behavior up to 150 g/L. Experimental results at high-c2 conditions were quantitatively predicted by the simulations for the coarse-grained models that treated antibody molecules as either 6 or 12 (sub) domains, which preserved the basic shape of a monoclonal antibody. Finally, preferential accumulation of sucrose around the protein surface was identified via high-precision density measurements, which self-consistently explained the simulation and experimental SLS results.

Coarse-Grained Antibody Models for "Weak" Protein-Protein Interactions from Low to High Concentrations.

So-called "weak" protein-protein interactions are important for the control of solution properties and stability at elevated protein concentrations (c2) but are not practical to capture in atomistic simulations. This report focuses on a series of coarse-grained models for predicting second osmotic virial coefficients (B22) and high-concentration Rayleigh scattering (osmotic compressibility) as a function of c2 for monoclonal antibodies (MAbs) that are of interest in biotechnology. B22 and molecular volume along with c2-dependent osmotic compressibility were calculated for a series of models with increasing structural detail. Models were refined to include contributions from sterics, short-ranged van der Waals and hydrophobic attractions, screened electrostatics, and the flexibility of the mAb hinge region. The results highlight shortcomings for spherical models of MAbs and a useful balance between numerical accuracy and computational burden offered by models based on 6 or 12 spherical, partly overlapping domains. The results provide bounds for realistic values of effective charges on variable domains in order for MAbs to be stable in solution and more generally illustrate semiquantitative bounds for the space of model parameters that can reproduce experimental behavior and provide a basis for future development of computationally efficient and accurate CG mAb models to predict both low- and high-c2 behavior.

Relating Protein-Protein Interactions and Aggregation Rates From Low to High Concentrations.

At low protein concentrations (c2), non-native protein aggregation rates are known to be sensitive to changes in conformational stability and "weak" or "colloidal" protein-protein interactions. Protein-protein interactions are also known to be strong functions of c2. In the present work, protein-protein interactions and rates of aggregation were quantified systematically for a monoclonal antibody (MAb) across a broad range of c2 at pH 5.1 and 6.5, with or without 5 wt/wt % sucrose or 100 mM NaCl present. Aggregation rates were determined from initial-rate analysis with size-exclusion chromatography, and interactions were quantified with static and dynamic laser light scattering. A number of hypotheses were tested regarding whether changes in protein-protein interactions can be predictive of changes in aggregation rates versus c2. Hypotheses were based on (i) changes in thermodynamic activity; (ii) statistical mechanical fluctuation theory; and (iii) surface-contact probabilities. Arguments based on (i) and (ii) were qualitatively inconsistent with experimental rates and scattering. Hypothesis (iii) was reasonably successful and resulted in a semiquantitative correlation between rates and protein-protein interactions across almost 2 orders of magnitude in c2. However, (iii) requires one to assume that the concentration-dependent protein-protein Kirkwood-Buff integral is a reasonable surrogate for contact probabilities.

Significance of unfolding thermodynamics for predicting aggregation kinetics: a case study on high concentration solutions of a multi-domain protein.

PURPOSE: To enable aggregation rate prediction over a broad temperature range for complex multi-domain proteins at high concentrations. METHODS: Thermal unfolding, non-isothermal kinetics and storage stability studies were conducted on a model multi-domain protein (MDP) at moderate to high concentrations (25-125 mg/mL) over a broad temperature range (4-40°C). RESULTS: Storage stability studies indicated the aggregation of MDP in solution to be a second order process. Application of Arrhenius kinetics to accelerated stability data resulted in underestimation of the aggregation rate under refrigerated conditions. Additional studies undertaken to understand the mechanism of the aggregation process highlighted the association of the monomer (or the aggregation competent species) to be the rate-limiting step for aggregation over the temperature range studied. Thermal unfolding studies in the presence of urea were used to calculate the heat capacity change upon unfolding (Δcp,un). The resulting value of Δcp,un when used in the extended Lumry-Eyring model resulted in a more accurate and a conservative estimate of the aggregation rate under refrigerated condition. Some complicating factors for the aggregation rate prediction for multi-domain proteins at high concentration are discussed. CONCLUSIONS: The work highlights (i) the significance of incorporating unfolding thermodynamics in protein aggregation rate prediction, (ii) the advantages and challenges associated with the use of the extended Lumry-Eyring (ELE) model for rate prediction and (iii) the utility of using the Arrhenius and the ELE models in tandem during product development.

Effective charge measurements reveal selective and preferential accumulation of anions, but not cations, at the protein surface in dilute salt solutions.

Specific-ion effects are ubiquitous in nature; however, their underlying mechanisms remain elusive. Although Hofmeister-ion effects on proteins are observed at higher (>0.3 M) salt concentrations, in dilute (<0.1 M) salt solutions nonspecific electrostatic screening is considered to be dominant. Here, using effective charge (Q*) measurements of hen-egg white lysozyme (HEWL) as a direct and differential measure of ion-association, we experimentally show that anions selectively and preferentially accumulate at the protein surface even at low (<100 mM) salt concentrations. At a given ion normality (50 mN), the HEWL Q* was dependent on anion, but not cation (Li(+), Na(+), K(+), Rb(+), Cs(+), GdnH(+), and Ca(2+)), identity. The Q* decreased in the order F(-) > Cl(-) > Br(-) > NO(3)(-) ∼ I(-) > SCN(-) > ClO(4)(-) ≫ SO(4)(2-), demonstrating progressively greater binding of the monovalent anions to HEWL and also show that the SO(4)(2-) anion, despite being strongly hydrated, interacts directly with the HEWL surface. Under our experimental conditions, we observe a remarkable asymmetry between anions and cations in their interactions with the HEWL surface.

Diffusion and sedimentation interaction parameters for measuring the second virial coefficient and their utility as predictors of protein aggregation.

The concentration-dependence of the diffusion and sedimentation coefficients (k(D) and k(s), respectively) of a protein can be used to determine the second virial coefficient (B2), a parameter valuable in predicting protein-protein interactions. Accurate measurement of B2 under physiologically and pharmaceutically relevant conditions, however, requires independent measurement of k(D) and k(s) via orthogonal techniques. We demonstrate this by utilizing sedimentation velocity (SV) and dynamic light scattering (DLS) to analyze solutions of hen-egg white lysozyme (HEWL) and a monoclonal antibody (mAb1) in different salt solutions. The accuracy of the SV-DLS method was established by comparing measured and literature B2 values for HEWL. In contrast to the assumptions necessary for determining k(D) and k(s) via SV alone, k(D) and ks were of comparable magnitudes, and solution conditions were noted for both HEWL and mAb1 under which 1), k(D) and k(s) assumed opposite signs; and 2), k(D) ≥k(s). Further, we demonstrate the utility of k(D) and k(s) as qualitative predictors of protein aggregation through agitation and accelerated stability studies. Aggregation of mAb1 correlated well with B2, k(D), and k(s), thus establishing the potential for k(D) to serve as a high-throughput predictor of protein aggregation.

Ion-specific modulation of protein interactions: anion-induced, reversible oligomerization of a fusion protein.

Ions can significantly modulate the solution interactions of proteins. We aim to demonstrate that the salt-dependent reversible heptamerization of a fusion protein called peptibody A or PbA is governed by anion-specific interactions with key arginyl and lysyl residues on its peptide arms. Peptibody A, an E. coli expressed, basic (pI = 8.8), homodimer (65.2 kDa), consisted of an IgG1-Fc with two, C-terminal peptide arms linked via penta-glycine linkers. Each peptide arm was composed of two, tandem, active sequences (SEYQGLPPQGWK) separated by a spacer (GSGSATGGSGGGASSGSGSATG). PbA was monomeric in 10 mM acetate, pH 5.0 but exhibited reversible self-association upon salt addition. The sedimentation coefficient (s(w)) and hydrodynamic diameter (D(H)) versus PbA concentration isotherms in the presence of 140 mM NaCl (A5N) displayed sharp increases in s(w) and D(H), reaching plateau values of 9 s and 16 nm by 10 mg/mL PbA. The D(H) and sedimentation equilibrium data in the plateau region (>12 mg/mL) indicated the oligomeric ensemble to be monodisperse (PdI = 0.05) with a z-average molecular weight (M(z)) of 433 kDa (stoichiometry = 7). There was no evidence of reversible self-association for an IgG1-Fc molecule in A5N by itself or in a mixture containing fluorescently labeled IgG1-Fc and PbA, indicative of PbA self-assembly being mediated through its peptide arms. Self-association increased with pH, NaCl concentration, and anion size (I(-) > Br(-) > Cl(-) > F(-)) but could be inhibited using soluble Trp-, Phe-, and Leu-amide salts (Trp > Phe > Leu). We propose that in the presence of salt (i) anion binding renders PbA self-association competent by neutralizing the peptidyl arginyl and lysyl amines, (ii) self-association occurs via aromatic and hydrophobic interactions between the ..xxCTRWPWMC..xxxCTRWPWMCxx.. motifs, and (iii) at >10 mg/mL, PbA predominantly exists as heptameric clusters.

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.

Anion binding mediated precipitation of a peptibody.

PURPOSE: Understand the underlying mechanism governing the salt-induced precipitation of a basic (pI = 8.8) protein, Peptibody A (PbA), in acidic solutions. METHODS: The rate, extent, and reversibility of PbA precipitation was monitored over 4-weeks as a function of pH (3.7-5.0), salt concentration (0-400 mM), and ion identity using a series of monovalent, Hofmeister anions (F(-), Cl(-), Br(-), I(-), ClO(4) (-), SCN(-)) and cations (Li+, Na+, K+, Rb+, Cs+). The effects of salt on conformational stability and reduced valence were determined using Fourier-transform infrared spectroscopy, circular dichroism, and capillary electrophoresis/analytical ultracentrifugation. RESULTS: PbA precipitation occurred upon salt addition and could be modulated with solution pH, salt identity & concentration. The precipitation was sensitive to anions, but not cations, and increased with anion size. A reverse Hofmeister effect (SCN(-) approximately ClO(4) (-)>I(-)>Cl(-)>Br(-)>F(-)) was observed with "salting-in" anions being the more effective precipitants. An increase in the precipitation rate below pH 4.3 indicated that protonation of aspartyl and glutamyl side-chains was also important for precipitation. The reversibility of precipitation was excellent (100%) at 4 degrees C but decreased upon storage at 25 degrees C and 37 degrees C; the loss in reversibility correlated with an increase in intermolecular beta-sheet content of the precipitate. CONCLUSION: Salts, employed as buffering, tonicifying, and viscosity modifying agents, may adversely affect the solubility of basic proteins formulated under acidic conditions.

Nature and consequences of protein-protein interactions in high protein concentration solutions.

High protein concentration solutions are becoming increasingly important in the pharmaceutical industry. The solution behavior of proteins at high concentrations can markedly differ from that predicted based on dilute solution analysis due to thermodynamic non-ideality in these solutions. The non-ideality observed in these systems is related to the protein-protein interactions (PPI). Different types of forces play a key role in determining the overall nature and extent of these PPI and their relative contributions are affected by solute and solvent properties. However, individual contributions of these forces to the solution properties of concentrated protein solutions are not fully understood. The role of PPI, driven by these intermolecular forces, in governing solution rheology and physical stability of high protein concentration solutions is discussed from the point of view of pharmaceutical product development. Investigation of protein self-association and aggregation in concentrated protein solutions is crucial for ensuring the safety and efficacy of the final product for the duration of the desired product shelf life. Understanding rheology of high concentration protein solutions is critical for addressing issues during product manufacture and administration of final formulation to the patient. To this end, analysis of solution viscoelastic character can also provide an insight into the nature of PPI affecting solution rheology.

Ultrasonic rheology of a monoclonal antibody (IgG2) solution: implications for physical stability of proteins in high concentration formulations.

The purpose of this work was to investigate if physical stability of a model monoclonal antibody (IgG(2)), as determined by extent of aggregation, was related to rheology of its solutions. Storage stability of the model protein was assessed at 25 degrees C and 37 degrees C for three months in solutions ranging from pH 4.0 to 9.0 and ionic strengths of 4 mM and 300 mM. The rheology of IgG(2) solutions has been characterized at 25 degrees C in our previous work and correlation of solution storage modulus (G') with protein-protein interactions established. The extent of aggregation was consistent with solution rheology as understood in terms of changes in G' with protein concentration. Thermodynamic stability of native IgG(2) conformation increased with increasing pH. The correlation between rheology and aggregation was also assessed at increased ionic strengths. The decrease in aggregation was consistent with change in solution rheology profile at pH 7.4 and 9.0. The results provide evidence of a relationship between solution rheology and extent of aggregation for the model protein studied. The implications of this relationship for formulation and physical stability assessment in high concentration protein solutions are discussed.

Ultrasonic storage modulus as a novel parameter for analyzing protein-protein interactions in high protein concentration solutions: correlation with static and dynamic light scattering measurements.

The purpose of this work was to establish ultrasonic storage modulus (G') as a novel parameter for characterizing protein-protein interactions (PPI) in high concentration protein solutions. Using an indigenously developed ultrasonic shear rheometer, G' for 20-120 mg/ml solutions of a monoclonal antibody (IgG(2)), between pH 3.0 and 9.0 at 4 mM ionic strength, was measured at frequency of 10 MHz. Our understanding of ultrasonic rheology indicated decrease in repulsive and increase in attractive PPI with increasing solution pH. To confirm this behavior, dynamic (DLS) and static (SLS) light scattering measurements were conducted in dilute solutions. Due to technical limitations, light scattering measurements could not be conducted in concentrated solutions. Mutual-diffusion coefficient, measured by DLS, increased with IgG(2) concentration at pH 4.0 and this trend reversed as pH was increased to 9.0. Second virial coefficient, measured by SLS, decreased with increasing pH. These observations were consistent with the nature of PPI understood from G' measurements. Ultrasonic rheology, DLS, and SLS measurements were also conducted under conditions of increased ionic strength. The consistency between rheology and light scattering analysis under various solution conditions established the utility of ultrasonic G' measurements as a novel tool for analyzing PPI in high protein concentration systems.

Application of high-frequency rheology measurements for analyzing protein-protein interactions in high protein concentration solutions using a model monoclonal antibody (IgG2).

The purpose of this work was to explore the utilization of high-frequency rheology analysis for assessing protein-protein interactions in high protein concentration solutions. Rheology analysis of a model monoclonal immunoglobulin G2 solutions was conducted on indigenously developed ultrasonic shear rheometer at frequency of 10 MHz. Solutions at pH 9.0 behaved as most viscous and viscoelastic whereas those at pH 4.0 and 5.4 exhibited lower viscosity and viscoelasticity, respectively. Intrinsic viscosity, hydrophobicity, and conformational analysis could not account for the rheological behavior of IgG2 solutions. Zeta potential and light scattering measurements showed the significance of electroviscous and specific protein-protein interactions in governing rheology of IgG2 solutions. Specific protein-protein interactions resulted in formation of reversible higher order species of monomer. Solution storage modulus (G'), and not loss modulus or complex viscosity, was the more reliable parameter for predicting protein-protein interactions. Predictions about the nature of protein-protein interactions made on the basis of solution G' were found to be consistent with observed effect of pH and ionic strength on zeta potential and scattered intensity of IgG2 solutions. Results demonstrated the potential of high-frequency storage modulus measurements for understanding behavior of proteins in solutions and predicting the nature of protein-protein interactions.

Application of ultrasonic shear rheometer to characterize rheological properties of high protein concentration solutions at microliter volume.

The purpose of this work was to conduct preliminary rheological analysis on high protein concentration solutions by using the technique of ultrasonic shear rheometry at megahertz frequencies. The work was aimed at establishing the viability of the technique for analyzing protein solution rheology as well as obtaining an initial understanding of the effect of solution conditions on solution rheology of a model protein. Bovine serum albumin (BSA) was used for this study, and rheological analysis was conducted at 20 microL sample volume between pH 2.0 and 9.0 at different ionic strengths at 25 degrees C using 5 and 10 MHz quartz crystals. Significant differences in storage modulus among solutions at pH 5.0, 7.0, and 9.0 could only be detected at 10 MHz, and the errors associated with measurements were smaller as compared to those at 5 MHz for all the solutions studied. Solutions at pH 2.0 and 3.0 showed a time-dependent change in solution rheology. For solutions at pH 5.0, 7.0, and 9.0, which did not show time dependence in solution rheology, loss modulus data at lower concentrations correlated well with the dilute solution data in the literature. At higher concentrations, pH 5.0 solutions exhibited a higher loss modulus than pH 7.0 and pH 9.0 solutions. Storage modulus decreased with increasing ionic strength, unlike loss modulus, which did not show any change, except at pI of protein when no effect was observed. The results show the potential of high frequency rheometry for analyzing subtle differences in rheology of pharmaceutically relevant protein solutions at microliter volume.