Computational tool for the early screening of monoclonal antibodies for their viscosities.

Highly concentrated antibody solutions often exhibit high viscosities, which present a number of challenges for antibody-drug development, manufacturing and administration. The antibody sequence is a key determinant for high viscosity of highly concentrated solutions; therefore, a sequence- or structure-based tool that can identify highly viscous antibodies from their sequence would be effective in ensuring that only antibodies with low viscosity progress to the development phase. Here, we present a spatial charge map (SCM) tool that can accurately identify highly viscous antibodies from their sequence alone (using homology modeling to determine the 3-dimensional structures).

Toward Computationally Designed Self-Reporting Biosensors Using Leave-One-Out Green Fluorescent Protein.

Leave-one-out green fluorescent protein (LOOn-GFP) is a circularly permuted and truncated GFP lacking the nth β-strand element. LOO7-GFP derived from the wild-type sequence (LOO7-WT) folds and reconstitutes fluorescence upon addition of β-strand 7 (S7) as an exogenous peptide. Computational protein design may be used to modify the sequence of LOO7-GFP to fit a different peptide sequence, while retaining the reconstitution activity.

Rational design of viscosity reducing mutants of a monoclonal antibody: hydrophobic versus electrostatic inter-molecular interactions.

High viscosity of monoclonal antibody formulations at concentrations ≥100 mg/mL can impede their development as products suitable for subcutaneous delivery. The effects of hydrophobic and electrostatic intermolecular interactions on the solution behavior of MAB 1, which becomes unacceptably viscous at high concentrations, was studied by testing 5 single point mutants. The mutations were designed to reduce viscosity by disrupting either an aggregation prone region (APR), which also participates in 2 hydrophobic surface patches, or a negatively charged surface patch in the variable region.

Highly viscous antibody solutions are a consequence of network formation caused by domain-domain electrostatic complementarities: insights from coarse-grained simulations.

Therapeutic monoclonal antibody (mAb) candidates that form highly viscous solutions at concentrations above 100 mg/mL can lead to challenges in bioprocessing, formulation development, and subcutaneous drug delivery. Earlier studies of mAbs with concentration-dependent high viscosity have indicated that mAbs with negatively charged Fv regions have a dipole-like quality that increases the likelihood of reversible self-association. This suggests that weak electrostatic intermolecular interactions can form transient antibody networks that participate in resistance to solution deformation under shear stress.

Concentration dependent viscosity of monoclonal antibody solutions: explaining experimental behavior in terms of molecular properties.

Purpose: Early identification of monoclonal antibody candidates whose development, as high concentration (≥100 mg/mL) drug products, could prove challenging, due to high viscosity, can help define strategies for candidate engineering and selection.

Methods: Concentration dependent viscosities of 11 proprietary mAbs were measured. Sequence and structural features of the variable (Fv) regions were analyzed to understand viscosity behavior of the mAbs.

Consequences of glycan truncation on Fc structural integrity.

Effective characterization of protein-based therapeutic candidates such as monoclonal antibodies (mAbs) is important to facilitate their successful progression from early discovery and development stages to marketing approval. One challenge relevant to biopharmaceutical development is, understanding how the stability of a protein is affected by the presence of an attached oligosaccharide, termed a glycan. To explore the utility of molecular dynamics simulations as a complementary technique to currently available experimental methods, the Fc fragment was employed as a model system to improve our understanding of protein stabilization by glycan attachment.

Agitation-induced aggregation and subvisible particulate formation in model proteins.

The kinetics of agitation-induced subvisible particle formation was investigated for a few model proteins - human serum albumin (HSA), hen egg white lysozyme (HEWL), and a monoclonal antibody (IgG2). Experiments were carried out for the first time under relatively low protein concentration and low agitation speed to investigate the details of subvisible particle formation at the initial phase of aggregation (<2%) process. Upon agitation, both soluble higher molecular mass species (HMMS) and subvisible particles (SbVPs) formed at different rates, and via different mechanisms.

On the role of aggregation prone regions in protein evolution, stability, and enzymatic catalysis: insights from diverse analyses.

The various roles that aggregation prone regions (APRs) are capable of playing in proteins are investigated here via comprehensive analyses of multiple non-redundant datasets containing randomly generated amino acid sequences, monomeric proteins, intrinsically disordered proteins (IDPs) and catalytic residues. Results from this study indicate that the aggregation propensities of monomeric protein sequences have been minimized compared to random sequences with uniform and natural amino acid compositions, as observed by a lower average aggregation propensity and fewer APRs that are shorter in length and more often punctuated by gate-keeper residues. However, evidence for evolutionary selective pressure to disrupt these sequence regions among homologous proteins is inconsistent.

Insights into the potential aggregation liabilities of the b12 Fab fragment via elevated temperature molecular dynamics.

Aggregation is a common hurdle faced during the development of antibody therapeutics. In this study, we explore the potential aggregation liabilities of the Fab (fragment antigen-binding) from a human IgG1κ antibody via multiple elevated temperature molecular dynamic simulations, analogous to accelerated stability studies performed during formulation development. Deformation and solvent exposure changes in response to thermal stress were monitored for individual structural domains (V(H), V(L), C(H)1 and C(L)), their interfaces (V(H):V(L) and C(H)1:C(L)), edge beta-strands and sequence-predicted aggregation-prone regions (APRs).

Impact of deglycosylation and thermal stress on conformational stability of a full length murine IgG2a monoclonal antibody: observations from molecular dynamics simulations.

With the rise of antibody based therapeutics as successful medicines, there is an emerging need to understand the fundamental antibody conformational dynamics and its implications towards stability of these medicines. Both deglycosylation and thermal stress have been shown to cause conformational destabilization and aggregation in monoclonal antibodies. Here, we study instabilities caused by deglycosylation and by elevated temperature (400 K) by performing molecular dynamic simulations on a full length murine IgG2a mAb whose crystal structure is available in the Protein Data bank.

Computational methods to predict therapeutic protein aggregation.

Protein based biotherapeutics have emerged as a successful class of pharmaceuticals. However, these macromolecules endure a variety of physicochemical degradations during manufacturing, shipping, and storage, which may adversely impact the drug product quality. Of these degradations, the irreversible self-association of therapeutic proteins to form aggregates is a major challenge in the formulation of these molecules.

Constraining local structure can speed up folding by promoting structural polarization of the folding pathway.

The pathway which proteins take to fold can be influenced from the earliest events of structure formation. In this light, it was both predicted and confirmed that increasing the stiffness of a beta hairpin turn decreased the size of the transition state ensemble (TSE), while increasing the folding rate. Thus, there appears to be a relationship between conformationally restricting the TSE and increasing the folding rate, at least for beta hairpin turns.

Simulating protein folding initiation sites using an alpha-carbon-only knowledge-based force field.

Protein folding is a hierarchical process where structure forms locally first, then globally. Some short sequence segments initiate folding through strong structural preferences that are independent of their three-dimensional context in proteins. We have constructed a knowledge-based force field in which the energy functions are conditional on local sequence patterns, as expressed in the hidden Markov model for local structure (HMMSTR).