Residue-Specific Impact of EDTA and Methionine on Protein Oxidation in Biotherapeutics Formulations Using an Integrated Biotherapeutics Drug Product Development Workflow.

The rational design and selection of formulation composition to meet molecule-specific and product-specific needs are critical for biotherapeutics development to ensure physical and chemical stability. This work, based on three antibody-based (mAb) proteins (mAbA, mAbB, and mAbC), evaluates residue-specific impact of EDTA and methionine on protein oxidation, using an integrated biotherapeutics drug product development workflow. This workflow includes statistical experimental design, high-throughput experimental automation and execution, structure-based in silico modeling, inferential statistical analysis, and enhanced interactive data visualization of large datasets. This oxidation study evaluates the impact of formulation parameters including pH, protein concentration, and the presence of polysorbate 80 on the oxidation of specific conserved and variable residues of mAbs A, B, and C in the presence of stressors (iron, peroxide) and/or protectants (EDTA, L-methionine). Residue-specific analysis by automated high-throughput peptide mapping demonstrates differential residue-specific effects of EDTA and methionine in protecting against oxidation, highlighting the need for molecule-specific and product-specific selection of these excipients during formulation development. Computational modeling based on a homology model and the two-shell water coordination method (WCN) was employed to gain mechanistic understanding of residue-specific oxidation susceptibility of methionine residues. The computational determinants of local solvent exposure of methionine residues showed good correlation of WCN with experimentally determined oxidation for corresponding residues. The rapid generation of high-resolution data, statistical data analysis and interactive visualization of the high-throughput residue-level data containing ∼200 unique formulations facilitate residue-specific, molecule-specific and product-specific oxidation (global and local) assessment for oxidation protectants during early development for mAbs and related mAb-based modalities.

Adapting to Biology: Maintaining Container-Closure System Compatibility with the Therapeutic Biologic Revolution.

Many pharmaceutical companies are transitioning their research and development drug product pipeline from traditional small-molecule injectables to the dimension of evolving therapeutic biologics. Important concerns associated with this changeover are becoming forefront, as challenges develop of varying complexity uncommon with the synthesis and production of traditional drugs. Therefore, alternative measures must be established that aim to preserve the efficacy and functionality of a biologic that might not be implemented for small molecules. Conserving protein stability is relative to perpetuating a net equilibrium of both intrinsic and extrinsic factors. Key to sustaining this balance is the ability of container-closure systems to maintain their compatibility with the ever-changing dynamics of therapeutic biologics. Failure to recognize and adjust the material properties of packaging components to support compatibility with therapeutic biologics can compromise patient safety, drug productivity, and biological stability. This review will examine the differences between small-molecule drugs and therapeutic biologics, lay a basic foundation for understanding the stability of therapeutic biologics, and demonstrate potential sources of container-closure systems' incompatibilities with therapeutic biologics at a mechanistic level. LAY ABSTRACT: Many pharmaceutical companies are transitioning their research and development drug product pipeline from traditional small-molecule injectables to recombinantly derived therapeutic biologics. Concerns associated with this transformation are becoming prominent, as therapeutic biologics are uncharacteristic to small-molecule drugs. Maintaining the stability of a therapeutic biologic is a combination of balancing intrinsic factors and external elements within the biologic's microenvironment. An important aspect of this balance is relegated to the overall compatibility of primary, parenteral container-closure systems with therapeutic biologics. A packaging component within a container-closure system that is incompatible with the therapeutic biologic can potentially compromise patient safety, drug productivity, and product quality. Thus, each primary, parenteral packaging component within a container-closure system should be optimized to preserve the efficacy and therapeutic activity of a biologic. This review will examine the differences between small-molecule drugs and therapeutic biologics, lay a basic foundation for understanding the stability of therapeutic biologics, and demonstrate potential sources of container-closure system incompatibilities with therapeutic biologics at a mechanistic level.

A homozygous missense mutation in HERC2 associated with global developmental delay and autism spectrum disorder.

We studied a unique phenotype of cognitive delay, autistic behavior, and gait instability segregating in three separate sibships. We initiated genome-wide mapping in two sibships using Affymetrix 10K SNP Mapping Arrays and identified a homozygous 8.2 Mb region on chromosome 15 common to five affected children. We used exome sequencing of two affected children to assess coding sequence variants within the mapped interval. Four novel homozygous exome variants were shared between the two patients; however, only two variants localized to the mapped interval on chromosome 15. A third sibship in an Ohio Amish deme narrowed the mapped interval to 2.6 Mb and excluded one of the two novel homozygous exome variants. The remaining variant, a missense change in HERC2 (c.1781C>T, p.Pro594Leu), occurs in a highly conserved proline residue within an RCC1-like functional domain. Functional studies of truncated HERC2 in adherent retinal pigment epithelium cells suggest that the p.Pro594Leu variant induces protein aggregation and leads to decreased HERC2 abundance. The phenotypic correlation with the mouse Herc1 and Herc2 mutants as well as the phenotypic overlap with Angelman syndrome provide further evidence that pathogenic changes in HERC2 are associated with nonsyndromic intellectual disability, autism, and gait disturbance. Hum Mutat 33:1639-1646, 2012. © 2012 Wiley Periodicals, Inc.