Aggregation Onset Temperature Research Articles

Capillary mediated vitrification is a novel technique that enables storage of antibody critical reagents at ambient temperature: Impact on binding, structure, and laboratory sustainability

Antibodies and antibody conjugates are essential components of life science research, but their inherent instability necessitates cold storage or lyophilization, posing logistical and sustainability challenges. Capillary-mediated vitrification has shown promise as a tool for improving biomolecule stability. In this study, we assess the feasibility of shipping and storing CMV-stabilized antibody reagents at ambient temperature using a purified rabbit polyclonal as a model system. The conditions tested included a simulated temperature excursion, ambient shipping, and storage for approximately two months at room-temperature. Antibody function was measured by both ELISA and Octet bio-layer interferometry kinetic measurements. Yield, aggregation, and thermal stability were assessed by UV/VIS, Size Exclusion Chromatography (SEC), thermal melting, and thermal aggregation studies. Results indicate >97 % protein yield and no impact on the binding activity. No evidence of aggregation or oligomer formation was detected. Addition of the vitrification buffer to the sample matrix resulted in an increase in the aggregation on-set temperature, indicating enhanced thermostability. A slight shift in both the SEC retention time for the main peak and a difference in aggregation behavior at high temperatures were noted post-vitrification. We hypothesize that these differences are related to the interaction of the protein with the saccharide component of the vitrification matrix and the stabilization mechanism of sugars. The cumulative data supports the use of Capillary Mediated Vitrification as a viable alternative to frozen reagent storage, with the potential to significantly impact reagent stability, assay performance, laboratory operations, and sustainability initiatives.

Timing and frequency of high temperature events bend the onset of behavioural thermoregulation in Atlantic salmon (Salmo salar).

The role of temperature on biological activities and the correspondent exponential relationship with temperature has been known for over a century. However, lacking to date is knowledge relating to (a) the recovery of ectotherms subjected to extreme temperatures in the wild, and (b) the effects repeated extreme temperatures have on the temperatures that induce behavioural thermoregulation (aggregations). We examined these questions by testing the hypothesis that thermal thresholds which initiate aggregations in juvenile Atlantic salmon (AS) (Salmo salar) are not static, but are temporally dynamic across a summer and follow a hysteresis loop. To test our hypothesis, we deployed custom-made underwater camera (UWC) systems in known AS thermal refuges to observe the timing of aggregation events in a natural system and used these data to develop and test models that predict the temperatures that induce thermal aggregations. Consistent with our hypothesis our UWC observations revealed a range of aggregation onset temperatures (AOT) ranging from 24.2°C to 27.1°C, thus confirming our hypothesis that AOTs are dynamic across summer. Our models suggest it take ~ 11days of non-thermally taxing temperatures for the AOT to rebound in the study river. Conversely, we found that as the frequency of events increased, the AOT declined, from 27.1°C to 24.2°C. Integrating both model components led to more robust model performance. Further, when these models were tested against an independent data set from the same river, the results remained robust. Our findings illustrate the complexity underlying behavioural thermoregulation in AS-a complexity that most likely extends to other salmonids. The frequency of extreme heat events is predicted to increase, and this has the capacity to decrease AOT thresholds in AS, ultimately reducing their resilience to extreme temperature events.

Enhanced protein aggregation suppressor activity of N-acetyl-l-arginine for agitation-induced aggregation with silicone oil and its impact on innate immune responses

Previously, N-acetyl-l-arginine (NALA) suppressed the aggregation of intravenous immunoglobulins (IVIG) more effectively and with a minimum decrease in transition temperature (Tm) than arginine monohydrochloride. In this study, we performed a comparative study with etanercept (commercial product: Enbrel®), where 25 mM arginine monohydrochloride (arginine) was added to the prefilled syringe. The biophysical properties were investigated using differential scanning calorimetry (DSC), dynamic light scattering (DLS), size-exclusion chromatography (SEC), and flow-imaging microscopy (FI). NALA retained the transition temperature of etanercept better than arginine, where arginine significantly reduced the Tm by increasing its concentration. End-over-end rotation was applied to each formulation for 5 days to accelerate protein aggregation and subvisible particle formation. Higher monomeric content was retained with NALA with a decrease in particle level. Higher aggregation onset temperature (Tagg) was detected for etanercept with NALA than arginine. The results of this comparative study were consistent with previous study, suggesting that NALA could be a better excipient for liquid protein formulations. Agitated IVIG and etanercept were injected into C57BL/6J female mice to observe immunogenic response after 24 h. In the presence of silicone oil, NALA dramatically reduced IL-1 expression, implying that decreased aggregation was related to reduced immunogenicity of both etanercept and IVIG.

Temperature and pH-Dependent Behaviors of mAb Drugs: A Case Study for Trastuzumab

The distortions in the high-order structure of therapeutic monoclonal antibodies (mAbs) under different environmental conditions acutely affect mAb stability, resulting in altered safety, efficacy, and shelf-life profiles. The overall stability of mAbs depends on many factors, and it requires complementary techniques for an in-depth analysis. The stability of mAbs can be characterized by differential centrifugal sedimentation (DCS), differential scanning calorimetry (DSC), differential scanning fluorimetry (DSF), and size exclusion chromatography (SEC) techniques. In this report, temperature-ramped dynamic light scattering (DLS), and circular dichroism (CD) spectroscopy were employed as complementary tools to show how temperature and pH affect the aggregation of a model mAb, trastuzumab, in solution. The results showed that the aggregation onset temperature of trastuzumab defined by DLS was 75 °C, which decreases the amount of β-sheets and causes a slight increase in helix structures. Moreover, the melting temperature of trastuzumab was determined to be between 80–83 °C by temperature-ramped CD spectrophotometry, which is in line with the Tm of trastuzumab’s Fab region tested with DSC. Thus, unfolding and aggregation of trastuzumab start simultaneously at 75 °C, and unfolding triggers the aggregation. The temperature-ramped CD and DLS methods are robust tools to determine the thermal behavior of biosimilars in various solution conditions. Their complementary usage provides solid scientific background for regulatory applications and a better understanding of mAb instability and its relationship with structural changes.

Comparison of Submicron Particle Counting Methods with a Heat Stressed Monoclonal Antibody: Effect of Electrolytes and Implications on Sample Preparation

Within this study, the performance and limitations of tunable resistive pulse sensing (TRPS) was evaluated to characterize submicron particles in unstressed and heat stressed monoclonal antibody (mAb) solutions. These were compared with microfluidic resistive pulse sensing (MRPS), resonant mass measurement (RMM), and nanoparticle tracking analysis (NTA). For TRPS and MRPS measurements, an adjustment of ionic strength was required to achieve suitable measurement conditions. The addition of electrolytes is potentially critical for protein formulations and therefore the effect of salt concentration and pH on submicron particle levels was further investigated.Heat stress caused a sharp increase in particle levels between 250-900 nm, observable by all four techniques. Due to reduced colloidal stability, indicated by increased attractive forces and reduced aggregation onset temperatures in the presence of sodium chloride, protein aggregation was observed in heat stressed mAb only after the addition of sodium chloride. Achieving adequate ionic strength by replacing sodium chloride with other electrolytes similarly resulted in reduced colloidal stability and protein aggregation. It is recommended that protein samples prone for aggregation in the presence of high ionic strength should not be analyzed by RPS measurements after the addition of electrolytes. However, protein samples containing already required ionic strength can be analyzed by any of the four techniques.

Denaturation Behavior and Kinetics of Single- and Multi-Component Protein Systems at Extrusion-Like Conditions.

In this study, the influence of defined extrusion-like treatment conditions on the denaturation behavior and kinetics of single- and multi-component protein model systems at a protein concentration of 70% (w/w) was investigated. α-Lactalbumin (αLA) and β-Lactoglobulin (βLG), and whey protein isolate (WPI) were selected as single- and multi-component protein model systems, respectively. To apply defined extrusion-like conditions, treatment temperatures in the range of 60 and 100 °C, shear rates from 0.06 to 50 s⁻1, and treatment times up to 90 s were chosen. While an aggregation onset temperature was determined at approximately 73 °C for WPI systems at a shear rate of 0.06 s⁻1, two significantly different onset temperatures were determined when the shear rate was increased to 25 and 50 s⁻1. These two different onset temperatures could be related to the main fractions present in whey protein (βLG and αLA), suggesting shear-induced phase separation. Application of additional mechanical treatment resulted in an increase in reaction rates for all the investigated systems. Denaturation was found to follow 2.262 and 1.865 order kinetics for αLA and WPI, respectively. The reaction order of WPI might have resulted from a combination of a lower reaction order in the unsheared system (i.e., fractional first order) and higher reaction order for sheared systems, probably due to phase separation, leading to isolated behavior of each fraction at the local level (i.e., fractional second order).

Influence of Thermomechanical Treatment and Ratio of β-Lactoglobulin and α-Lactalbumin on the Denaturation and Aggregation of Highly Concentrated Whey Protein Systems.

The influence of thermomechanical treatment (temperature 60 °C–100 °C and shear rate 0.06 s−1–50 s−1) and mixing ratio of β-lactoglobulin (βLG) and α-lactalbumin (αLA) (5:2 and 1:1) on the denaturation and aggregation of whey protein model systems with a protein concentration of 60% and 70% (w/w) was investigated. An aggregation onset temperature was determined at approx. 80 °C for both systems (5:2 and 1:1 mixing ratio) with a protein concentration of 70% at a shear rate of 0.06 s−1. Increasing the shear rate up to 50 s−1 led to a decrease in the aggregation onset temperature independent of the mixing ratio. By decreasing the protein concentration to 60% in unsheared systems, the aggregation onset temperature decreased compared to that at a protein concentration of 70%. Furthermore, two significantly different onset temperatures were determined when the shear rate was increased to 25 s−1 and 50 s−1, which might result from a shear-induced phase separation. Application of combined thermal and mechanical treatment resulted in overall higher degrees of denaturation independent of the mixing ratio and protein concentration. At the conditions applied, the aggregation of the βLG and αLA mixtures was mainly due to the formation of non-covalent bonds. Although the proportion of disulfide bond aggregation increased with treatment temperature and shear rate, it was higher at a mixing ratio of 5:2 compared to that at 1:1.

Influence of thermomechanical treatment and pH on the denaturation kinetics of highly concentrated whey protein isolate

Abstract In this study the influence of protein concentration, temperature, shear rate, and pH on the denaturation of whey protein isolate was investigated. By decreasing the protein concentration from 70% to 60% (w/w), the aggregation onset temperature decreased from approx. 72 °C to 64 °C, respectively. Increasing the protein concentration led to lower reaction rates due to decreased molecular mobility characterized by higher values of matrix viscosity. In contrast, an increase in treatment temperature and shear rate led to higher reaction rates due to an increased molecular motion, which was characterized with lower values of matrix viscosity. Denaturation reaction was found to follow 2.687, 1.909, and 2.345 order kinetics when the pH varied from 3.0 to 6.7 and 9.0, respectively. Increasing the temperature and shear rate led to a decrease in the calculated activation energy for the denaturation reaction and it ranged from 48 to 260 kJ mol −1.

Thermodynamic Unfolding and Aggregation Fingerprints of Monoclonal Antibodies Using Thermal Profiling.

Predicting thermal protein stability is of major interest in the development of protein-based biopharmaceuticals. Therefore, this study provides a predictive tool for determining transition enthalpies, which can be used for ranking different proteins according to their thermal stability. Unfolding and aggregation profiles of eight different therapeutic monoclonal antibodies (mAbs) of type G, isotype 1 were investigated. The unfolding profiles were determined by intrinsic fluorescence (IF) spectroscopy and differential scanning calorimetry (DSC). A three-state unfolding fitting model was used to determine thermodynamic parameters for macromolecular multi-domain mAbs in IF experiments, like the van't Hoff enthalpy change (∆Hvh) and the entropy change (∆S) of the unfolding event. The derived values were compared to thermodynamic parameters obtained directly by calorimetry. Moreover, differences in the Fab enthalpies were used to predict aggregation behavior and protein thermal stabilities. To do so, the liquid-formulated mAbs were investigated exemplarily by size exclusion chromatography (SEC) after accelerated thermal-induced stress conditions. Comparing the thermodynamic parameters derived from IF spectroscopy and DSC resulted in similar values. Data generated by thermal-induced stress at 40°C show similar stability ranking as postulated through the Fab enthalpies for mAbs in two different formulations, while at 25°C a meaningful ranking is not possible, because distinct differences in the thermal stability cannot be observed. The additional consideration of Fab enthalpies to predict the 40 °C SEC ranking seems to be more reliable compared to the use of exclusively the melting temperatures or aggregation onset temperatures and times. We show that thermodynamic profiling can help predicting unfolding and aggregation properties of therapeutic mAbs at 40°C. Therefore, analyzing thermodynamic unfolding parameters is a useful and supportive tool discriminating thermal stability profiles of mAbs for further pharmaceutical development and clinical studies.

Application of interpretable artificial neural networks to early monoclonal antibodies development

The development of a new protein drug typically starts with the design, expression and biophysical characterization of many different protein constructs. The initially high number of constructs is radically reduced to a few candidates that exhibit the desired biological and physicochemical properties. This process of protein expression and characterization to find the most promising molecules is both expensive and time-consuming. Consequently, many companies adopt and implement philosophies, e.g. platforms for protein expression and formulation, computational approaches, machine learning, to save resources and facilitate protein drug development. Inspired by this, we propose the use of interpretable artificial neuronal networks (ANNs) to predict biophysical properties of therapeutic monoclonal antibodies i.e. melting temperature Tm, aggregation onset temperature Tagg, interaction parameter kD as a function of pH and salt concentration from the amino acid composition. Our ANNs were trained with typical early-stage screening datasets achieving high prediction accuracy. By only using the amino acid composition, we could keep the ANNs simple which allows for high general applicability, robustness and interpretability. Finally, we propose a novel “knowledge transfer” approach, which can be readily applied due to the simple algorithm design, to understand how our ANNs come to their conclusions.

Comparing the behavioural thermoregulation response to heat stress by Atlantic salmon parr (Salmo salar) in two rivers

AbstractClimate change is expected to increase the frequency and magnitude of extreme thermal events in rivers. The Little Southwest Miramichi River (LSWM) and the Ouelle River (OR) are two Atlantic salmon (Salmo salar) rivers located in eastern Canada, where in recent years, water temperatures have exceeded known thermal limits (~23°C). Once temperature surpasses this threshold, juvenile salmon exploit thermal heterogeneity to behaviourally thermoregulate, forming aggregations in coolwater refuges. This study aimed to determine whether the behavioural thermoregulation response is universal across rivers, arising from common thermal cues. We detailed the temperature and discharge patterns of two geographically distinct rivers from 2010 to 2012 and compared these with aggregation onset temperature. PIT telemetry and snorkelling were used to confirm the presence of aggregations. Mean daily maximum temperature in 2010 was significantly greater in the OR versus the LSWM (p = 0.005), but not in other years (p = 0.090–0.353). Aggregations occurred on 14 and 9 occasions in the OR and LSWM respectively. Temperature at onset of aggregation was significantly greater in the OR (Tonset = 28.3°C) than in the LSWM (Tonset = 27.3°C; p = 0.049). Logistic regression models varied by river and were able to predict the probability of aggregation based on the preceding number of hours >23°C (R2 = 0.61 & 0.65; P50 = 27.4°C & 28.9°C; in the OR and LSWM respectively). These results imply the preceding local thermal regime may influence behaviour and indicate a degree of phenotypic plasticity, illustrating a need for localised management strategies.

The ReFOLD assay for protein formulation studies and prediction of protein aggregation during long-term storage

The formulation of novel therapeutic proteins is a challenging task which aims at finding formulation conditions that will minimize protein degradation during long-term storage. One particularly important and difficult-to-predict protein degradation pathway is the so-called non-native aggregation. The qualitative and quantitative prediction of the latter has been a subject of extensive research over the past two decades. An increasing body of evidence shows that the widely-used short-term biophysical techniques cannot accurately rank formulation conditions in order of their effect on the aggregation during long-term storage of some therapeutic proteins, e.g. monoclonal antibodies. Here we suggest a novel approach for the selection of formulation conditions that will suppress the formation of protein aggregates during long-term storage. We postulate that conditions (i.e. pH, buffer type, ionic strength) that reduce the isothermal aggregation of various denaturant-induced partially folded protein species will be conditions that impede protein aggregation during long-term storage. To test our hypothesis, we developed an isothermal microdialysis-based unfolding/refolding assay, named ReFOLD, which we use to induce moderate aggregation of partially folded proteins. Next, we assessed the relative monomer yield after isothermal unfolding/refolding of two monoclonal antibodies, each formulated in 12 different conditions. Using the proposed approach, we were able to accurately rank the formulations in order of their effect on the amount of protein aggregates detected after storage for 12 months at 4 °C and 25 °C, while widely-used stability-indicating parameters like protein melting and aggregation onset temperatures failed to provide accurate predictive formulation rankings.

Exploiting correlated molecular-dynamics networks to counteract enzyme activity–stability trade-off

The directed evolution of enzymes for improved activity or substrate specificity commonly leads to a trade-off in stability. We have identified an activity-stability trade-off and a loss in unfolding cooperativity for a variant (3M) of Escherichia coli transketolase (TK) engineered to accept aromatic substrates. Molecular dynamics simulations of 3M revealed increased flexibility in several interconnected active-site regions that also form part of the dimer interface. Mutating the newly flexible active-site residues to regain stability risked losing the new activity. We hypothesized that stabilizing mutations could be targeted to residues outside of the active site, whose dynamics were correlated with the newly flexible active-site residues. We previously stabilized WT TK by targeting mutations to highly flexible regions. These regions were much less flexible in 3M and would not have been selected a priori as targets using the same strategy based on flexibility alone. However, their dynamics were highly correlated with the newly flexible active-site regions of 3M. Introducing the previous mutations into 3M reestablished the WT level of stability and unfolding cooperativity, giving a 10.8-fold improved half-life at 55 °C, and increased midpoint and aggregation onset temperatures by 3 °C and 4.3 °C, respectively. Even the activity toward aromatic aldehydes increased up to threefold. Molecular dynamics simulations confirmed that the mutations rigidified the active-site via the correlated network. This work provides insights into the impact of rigidifying mutations within highly correlated dynamic networks that could also be useful for developing improved computational protein engineering strategies.

Coupled molecular dynamics mediate long- and short-range epistasis between mutations that affect stability and aggregation kinetics

Multiple mutations are typically required to significantly improve protein stability or aggregation kinetics. However, when several substitutions are made in a single protein, the mutations can potentially interact in a nonadditive manner, resulting in epistatic effects, which can hamper protein-engineering strategies to improve thermostability or aggregation kinetics. Here, we have examined the role of protein dynamics in mediating epistasis between pairs of mutations. With Escherichia coli transketolase (TK) as a model, we explored the epistatic interactions between two single variants H192P and A282P, and also between the double-mutant H192P/A282P and two single variants, I365L or G506A. Epistasis was determined for several measures of protein stability, including the following: the free-energy barrier to kinetic inactivation, ∆∆G‡; thermal transition midpoint temperatures, Tm; and aggregation onset temperatures, Tagg Nonadditive epistasis was observed between neighboring mutations as expected, but also for distant mutations located in the surface and core regions of different domains. Surprisingly, the epistatic behaviors for each measure of stability were often different for any given pairwise recombination, highlighting that kinetic and thermodynamic stabilities do not always depend on the same structural features. Molecular-dynamics simulations and a pairwise cross-correlation analysis revealed that mutations influence the dynamics of their local environment, but also in some cases the dynamics of regions distant in the structure. This effect was found to mediate epistatic interactions between distant mutations and could therefore be exploited in future protein-engineering strategies.

In-Depth Comparison of Lysine-Based Antibody-Drug Conjugates Prepared on Solid Support Versus in Solution.

Antibody drug conjugates are a rapidly growing form of targeted chemotherapeutics. As companies and researchers move to develop new antibody–drug conjugate (ADC) candidates, high-throughput methods will become increasingly common. Here we use advanced characterization techniques to assess two trastuzumab-DM1 (T-DM1) ADCs; one produced using Protein A immobilization and the other produced in solution. Following determination of payload site and distribution with liquid chromatography-mass spectrometry (LC/MS), thermal stability, heat-induced aggregation, tertiary structure, and binding affinity were characterized using differential scanning calorimetry (DSC), dynamic light scattering (DLS), Raman spectroscopy, and isothermal titration calorimetry (ITC), respectively. Small differences in the thermal stability of the CH2 domain of the antibody as well as aggregation onset temperatures were observed from DSC and DLS, respectively. However, no significant differences in secondary and tertiary structure were observed with Raman spectroscopy, or binding affinity as measured by ITC. Lysine-based ADC conjugation produces an innately heterogeneous population that can generate significant variability in the results of sensitive characterization techniques. Characterization of these ADCs indicated nominal differences in thermal stability but not in tertiary structure or binding affinity. Our results lead us to conclude that lysine-based ADCs synthesized following Protein A immobilization, common in small-scale conjugations, are highly similar to equivalent ADCs produced in larger scale, solution-based methods.

Formulation of abalone hemocyanin with high antiviral activity and stability

Hemocyanin has been shown to have potential antiviral activity against herpes simplex virus type-1. However, current liquid formulations have short shelf life and high risk of bacterial contamination. The aim of our study was to develop a stable functional formulation. Analytical techniques (nano-differential scanning calorimetry and spectroscopy) and biological assays (cytotoxicity and plaque reduction) were employed to measure the effect of sugar addition on the physical properties and shelf life of the solid formulated hemocyanin. Sucrose improved thermal stability significantly by both increasing the aggregation onset temperature (70°C to>78°C) and enhancing the activation energy (18%). Lyophilisation without trehalose caused degradation and unfolding of the α-helices of hemocyanin. However, the addition of an optimal proportion of trehalose:protein (5:1 by weight) prevented the degradation and unfolding during lyophilisation, hence maintained the protein solubility. The estimated ED50 values of the formulated solid (0.43±0.1) and liquid samples (0.37±0.06) were similar in magnitude, and were significantly lower than the respective controls; thus, confirming enhanced antiviral activity of the formulation. Formulated compounds were stable for six months at 5°C storage. The enhanced shelf life and stable antiviral activity of the formulation offers its significant potential as effective therapeutic agent in future clinical applications.

Biophysical and Stabilization Studies of the Chlamydia trachomatis Mouse Pneumonitis Major Outer Membrane Protein

Native Chlamydia trachomatis mouse pneumonitis major outer membrane protein (nMOMP) induces effective protection against genital infection in a mouse challenge model. The conformation of nMOMP is crucial to confer this protective immunity. To achieve a better understanding of the conformational behavior and stability of nMOMP, a number of spectroscopic techniques are employed to characterize the secondary structure (circular dichroism), tertiary structure (intrinsic fluorescence) and aggregation properties (static light scattering and optical density) as a function of pH (3-8) and temperature (10-87.5 degrees C). The data are summarized in an empirical phase diagram (EPD) which demonstrates that the thermal stability of nMOMP is strongly pH-dependent. Three distinctive regions are seen in the EPD. Below the major thermal transition regions, nMOMP remains in its native conformation over the pH range of 3-8. Above the thermal transitions, nMOMP appears in two different structurally altered states; one at pH 3-5 and the other at pH 6-8. The EPD shows that the highest thermal transition point ( approximately 65 degrees C) of nMOMP is near pH 6. Several potential excipients such as arginine, sodium citrate, Brij 35, sucrose and guanidine are also selected to evaluate their effects on the stability of nMOMP. These particular compounds increase the aggregation onset temperature of nMOMP by more than 10(omicron)C, without affecting its secondary and tertiary structure. These results should help formulate a vaccine using a recombinant MOMP.