A Streamlined Bioanalytical Approach to Select a Compatible Primary Container System Early in Drug Development: A Toolbox for the Biopharmaceutical Industry.

To ensure drug efficacy and patient safety, the importance of interaction between primary container and a biological drug product must not be ignored. The United States Food and Drug Administration guidance on development and manufacturing of combination products (e.g., the biologic and the primary container) encourages careful selection and stability testing of the drug and its performance in the marketed primary container. With various options available for primary container type (vials, prefilled syringes, and cartridges), material (e.g., glass or plastic), and lubricants/coatings, we designed a platform consisting of several bioanalytical methods that can simplify selection of a compatible primary container. We tested the stability of a commercially available monoclonal antibody (mAb) in 3 syringe types under 3 conditions: cold storage, high temperature, and agitation induced stress, respectively. Under each condition, dynamic fluid imaging was sensitive enough to differentiate mAb stability as measured by aggregate formation in different syringe systems, followed by size exclusion-high performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis but only at high temperature. With this platform, we identified a primary container that provided higher mAb stability under cold storage as well as stress conditions. We recommend that such an approach should be applied early in drug development stage to identify a superior primary container system to maintain drug stability and quality.

Stability assessment of antibody-drug conjugate Trastuzumab emtansine in comparison to parent monoclonal antibody using orthogonal testing protocol.

Antibody-drug conjugates (ADC) represent an emerging, novel class of biopharmaceuticals. The heterogeneity originating from the sophisticated structure requires orthogonal analytical techniques for quality and stability assessment of ADC to ensure safety and efficacy. In this study, the stability of Trastuzumab (recombinant humanized IgG1 mAb, targeting HER2 receptor) and its ADC with DM1 (anti-tubulin anticancer drug), Trastuzumab emtansine (T-DM1) were studied. SE-HPLC was used to monitor formation of aggregates and/or fragments of the monoclonal antibodies (mAb). Correlation with the results of reducing and non-reducing sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE) and dynamic light scattering (DLS) were performed to interpret the obtained results. RP-HPLC was used for assessment of the stability of DM1 in ADC while spectrophotometry was employed to determine drug antibody ratio (DAR) . The studied drugs were subjected to several stress conditions including pH, temperature, mechanical agitation and repeated freeze and thaw to generate possible degradation products and ensure suitability of the assay protocol. The degradation pattern and extent were demonstrated under the indicated stress conditions. The correlation between the results of SE-HPLC and those of SDS-PAGE and DLS ensured the validity of the orthogonal assay protocol and indicated aggregates that were not detected using SE-HPLC. Results showed clearly that T-DM1 is relatively less stable than its parent mAb. This was attributed to the presence of the drug-linker part that is attached to the mAb. RP-HPLC showed that the cytotoxic drug moiety is liable for degradation under the studied conditions resulting in alteration of DAR as well as formation of degradation products. This confirmed the need for more robust coupling chemistries for production of safe and effective ADC and highlighted the importance of orthogonal testing protocols for quality assessment. The assay protocol should be applicable for quality and stability assessment of various ADC.

A critical evaluation of self-interaction chromatography as a predictive tool for the assessment of protein-protein interactions in protein formulation development: a case study of a therapeutic monoclonal antibody.

The aim of this study was to establish and evaluate a screening method for the physical characterization of protein-protein interactions of therapeutic proteins based on the determination of the osmotic second virial coefficient (B(22)). B(22) of an IgG1 was measured by self-interaction chromatography (SIC) and was compared to data obtained from static light scattering (SLS). As assessed by Fourier transform infrared spectroscopy (FTIR), the protein coupling to chromatography particles had no relevant influence on the three-dimensional native structure of the IgG1. B(22) variations could be measured for physiological relevant excipient concentrations. Significant positive B(22) values were observed for the following solution conditions of the investigated antibody: (i) acidic pH conditions, (ii) low buffer concentrations, (iii) low salt concentrations and (iv) high amino acid concentrations. B(22) was compared to IgG1 stability data derived from a study conducted for 12weeks at 40 degrees C. A concentration of 5mM histidine, which was the most promising buffer candidate according to B(22), showed a slightly better physical stability (as assessed by turbidity and size exclusion chromatography) compared to the other tested formulations. This is confirmed in a stress study investigating the colloidal stability. Thus, measuring protein-protein interactions with SIC appeared as a promising screening tool for physical characterization of protein formulations for cases in which the protein stability is governed by interparticle interactions.

Comparison of cleanup methods for fipronil and its degradation products in sediment extracts.

Gel permeation chromatography (GPC) and solid phase extraction (SPE) were compared for cleaning extracts containing fipronil, fipronil-sulfide, and fipronil-sulfone at sub-ppb concentrations in sediment. With both methods, analytes were extracted using accelerated solvent extraction, and analyzed with gas chromatography equipped with an electron capture detector. The GPC was performed with a Waters Envirogel GPC column with dichloromethane as the mobile phase, while SPE was conducted with dual-layer cartridges containing graphitized carbon black and primary and secondary amines with a mixture of acetone and hexane as the eluting solvent. Method detection limits for fipronil, fipronil-sulfide, and fipronil-sulfone from three sediments with varying organic carbon content ranged from 0.12 to 0.52 microg/kg dry weight, while percent recoveries were 72-119% from sediment aged from 0.24 to 14d. Although both methods were effective at analyzing fipronil and its degradation products, SPE was the less expensive and less labor-intensive method.