Biology subjects

Blech, M.

Publications and source records attributed to Blech, M..

5 recordsLinked to original sources

bioRxiv2025

Predicting the Dynamic Viscosity of High-Concentration Antibody Solutions with a Chemically Specific Coarse-Grained Model

The viscosity of high-concentration protein solutions is a critical parameter in bio-pharmaceutical formulation development. Conventionally, the viscosity is measured and optimized in labor-intensive experimental workflows that require a lot of material. While predicting the viscosity with atomistic molecular dynamics (MD) simulations is feasible, they are computationally prohibitively expensive due to the large system sizes and the long simulation times involved. Coarse-grained MD (CG-MD) simulations significantly reduce computational demands, but evaluating their accuracy and predictive power requires rigorous validation. Here, we assess the capability of the Martini 3 CG force field to predict the viscosity of high-concentration antibody solutions. We show that a refined Martini 3 force field, with optimized protein-protein interactions, can predict the elevated viscosities observed in concentrated solutions of F(ab)2 fragments of the therapeutic monoclonal antibody (mAb) omalizumab. Furthermore, we show that our previously developed Martini 3-exc model for arginine excipients successfully captures the trend of lowering viscosity, as observed in our rheology experiments. These findings open the way to physics-based computational prediction of the properties of dense biopharmaceutical solutions via large-scale MD simulations.

biophysics↗

bioRxiv2024

Optimized Protein-Excipient Interactions in theMartini 3 Force Field

The high doses of drugs required for biotherapeutics, such as monoclonal antibodies (mAbs), and the small volumes that can be administered to patients by subcutaneous injections pose challenges due to high concentration formulations. The addition of excipients, such as arginine and glutamate, to high concentration protein formulations can increase solubility and reduce the tendency of protein particle formation. Molecular dynamics (MD) simulations can provide microscopic insights into the mode of action of excipients in mAb formulations but require large system sizes and long time scales that are currently beyond reach at the fully atomistic level. Computationally efficient coarse-grained models such as the Martini 3 force field can tackle this challenge but require careful parametrization, testing, and validation. This study extends the popular Martini 3 force field towards realistic protein-excipient interactions of arginine and glutamate excipients, using the Fab domains of the therapeutic mAbs trastuzumab and omalizumab as model systems. A novel all-atom to coarse-grained mapping of the amino acid excipients is introduced, which explicitly captures the zwitterionic character of the backbone. The Fab-excipient interactions of arginine and glutamate are characterized concerning molecular contacts with the Fabs at the single-residue level. The Martini 3 simulations are compared with results from all-atom simulations as a reference. Our findings reveal an overestimation of Fab-excipient contacts with the default interaction parameters of Martini 3, suggesting a too strong attraction between protein residues and excipients. Therefore, we reparametrized the protein-excipient interaction parameters in Martini 3 against all-atom simulations. The excipient interactions obtained with the new Martini 3 mapping and Lennard-Jones (LJ) interaction parameters, coined Martini 3-exc, agree closely with the all-atom reference data. This work presents an improved parameter set for mAb-arginine and mAb-glutamate interactions in the Martini 3 coarse-grained force field, a key step towards large-scale coarse-grained MD simulations of high-concentration mAb formulations and the stabilizing effects of excipients.

biophysics↗

bioRxiv2024

Competitive Adsorption of a Monoclonal Antibody and Amphiphilic Polymers to the Air-Water Interface

Understanding structure and self-organization of monoclonal antibodies (mAbs) at the air-water interface is important for the stability and effectiveness of protein drug formulations used in pharmaceutical industry. This paper investigates the competitive adsorption of a mAb and the amphiphilic surfactants poloxamer 188 (P188) and polysorbate 20 (PS20), both of which are commonly used to prevent mAb surface adsorption. Firstly, it is studied whether these surfactants prevent mAb adsorption, and secondly, whether it is possible to desorb mAb molecules from the air-water interface by surfactant addition. For surface pressure and surface tension data, Langmuir film balance measurements and drop shape tensiometry were used. Infrared Reflection-Absorption Spectroscopy (IRRAS) provided information on the surface composition, including the amount of adsorbed molecules. P188 exists in different self-assembled phases depending on its surface concentration. Our experiments show that the phase state of P188 has a significant impact on mAb adsorption. The presence of P188 in the brush phase ([≥] 0.3 mg/L) consistently inhibits mAb adsorption. On the contrary, addition of P188 after mAb film formation could not cause desorption of mAb. However, addition of PS20 leads to desorption of freshly formed interfacial mAb layers of up to two hours age. Interestingly, an aged mAb layer of 17 hours could not be desorbed by PS20. This suggests a time dependent reorganization of mAb at the air-water interface, which increases its resistance to desorption. These findings are discussed with respect to possible inter-molecular interactions within the interfacial film.

biophysics↗

bioRxiv2023

Viscosity Prediction of High-ConcentrationAntibody Solutions with AtomisticSimulations

The computational prediction of the viscosity of dense protein solutions is highly desirable, for example in the early development phase of high-concentration biophar-maceutical formulations where the material needed for experimental determination is typically limited. Here, we use large-scale atomistic molecular dynamics (MD) simulations with explicit solvent to de novo predict the dynamic viscosities of solutions of a monoclonal IgG1 antibody (mAb) from the pressure fluctuations using a Green-Kubo approach. The viscosities at simulated mAb concentrations of 200 mg/ml and 250 mg/ml are compared to the experimental values, which we measured with rotational rheometry. The computational viscosity of 24 mPa s at a mAb concentration of 250 mg/ml matches the experimental value of 23 mPa s obtained at a concentration of 213 mg/ml, indicating slightly different effective concentrations (or activities) in the MD simulations and in the experiments. This difference is assigned to a slight underestimation of the effective mAb-mAb interactions in the simulations, leading to a too loose dynamic mAb network that governs the viscosity. Taken together, the present study demonstrates the feasibility of all-atom MD simulations for predicting the properties of dense mAb solutions and provides detailed microscopic insights into the underlying molecular interactions. At the same time, it also shows that there is room for further improvements and highlights challenges, such as the massive sampling required for computing collective properties of dense biomolecular solutions in the high-viscosity regime with reasonable statistical precision. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/555069v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@4dd23corg.highwire.dtl.DTLVardef@12579eaorg.highwire.dtl.DTLVardef@167151dorg.highwire.dtl.DTLVardef@9b3306_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2019

Thermal and Chemical Unfolding of a recombinant monoclonal IgG1 antibody: Application of the Multi-State Zimm-Bragg Theory

The thermal unfolding of a recombinant monoclonal antibody IgG1 (mAb) was measured with differential scanning calorimetry (DSC). The DSC thermograms reveal a pre-transition at 72{degrees}C with an unfolding enthalpy of {Delta}Hcal [~] 200-300 kcal/mol and a main transition at 85 {degrees}C with an enthalpy of [~]900 - 1000 kcal/mol. In contrast to single-domain molecules, mAb unfolding is a complex reaction that is analysed with the multi-state Zimm-Bragg theory. For the investigated mAb, unfolding is characterised by a cooperativity parameter {sigma} [~]10-4 and a Gibbs free energy of unfolding of gnu [~]100 cal/mol per amino acid. The enthalpy of unfolding provides the number of amino acid residues v participating in the unfolding reaction. On average, v[~]220{+/-}50 amino acids are involved in the pre-transition and v[~]850{+/-}30 in the main transition, accounting for [~]90% of all amino acids. Thermal unfolding was further studied in the presence of guanidineHCl. The chemical denaturant reduces the unfolding enthalpy {Delta}Hcal and lowers the midpoint temperature T0. Both parameters depend linearly on the concentration of denaturant. The guanidineHCl concentrations needed to unfold mAb at 25 {degrees}C are predicted to be 2-3 M for the pre-transition and 5-7 M for the main transition, varying with pH. GuanidineHCl binds to mAb with an exothermic binding enthalpy, which partially compensates the endothermic mAb unfolding enthalpy. The number of guanidineHCL molecules bound upon unfolding is deduced from the DSC thermograms. The bound guanidineHCl-to-unfolded amino acid ratio is 0.79 for the pre-transition and 0.55 for the main transition. The pre-transition binds more denaturant molecules and is more easily destabilised than the main transition.\n\nOverall, the current study shows the strength of the Zimm-Bragg model for the quantitative description of unfolding events of large, therapeutic proteins, such as a monoclonal antibody.\n\nStatement of significanceFirst quantitative thermodynamic study of an antibody with differential scanning calorimetry and analyzed with the multi-state Zimm-Bragg theory.

biophysics↗