Characterization and stability study of polysorbate 20 in therapeutic monoclonal antibody formulation by multidimensional ultrahigh-performance liquid chromatography-charged aerosol detection-mass spectrometry.

Polysorbate 20 is a nonionic surfactant commonly used in the formulation of therapeutic monoclonal antibodies (mAb) to prevent protein denaturation and aggregation. It is critical to understand the molecular heterogeneity and stability of polysorbate 20 in mAb formulations as polysorbate can gradually degrade in aqueous solution over time by multiple pathways losing surfactant functions and leading to protein aggregation. The molecular heterogeneity of polysorbate and the interference from proteins and the excipient in the formulation matrix make it a challenge to study polysorbate in protein formulations. In this work, the characterization and stability study of polysorbate 20 in the presence of mAb formulation sample matrix is first reported using two-dimensional liquid chromatography (2DLC) coupled with charged aerosol detection (CAD) and mass spectrometry (MS) detection. A mixed-mode column that has both anion-exchange and reversed-phase properties was used in the first dimension to separate protein and polysorbate in the formulation sample, while polysorbate 20 esters were trapped online and then analyzed using an reversed-phase ultrahigh-performance liquid chromatography (RP-UHPLC) column in the second dimension to further separate the ester species. The MS served as the third dimension to further resolve as well as to identify the polysorbate ester subspecies. Another 2DLC method using a cation-exchange column in the first dimension and the same RP-UHPLC method in the second dimension was developed to analyze the degradation products of polysorbate 20. Stability samples of a protein drug product were studied using these two 2DLC-CAD-MS methods to separate, identify, and quantify the multiple ester species in polysorbate 20 and also to monitor the change of their corresponding degradants. We found different polysorbate esters degrade at different rates, and importantly, the degradation rates for some esters are different in the protein formulation compared to a placebo that has no protein. The multidimensional UHPLC-CAD-MS approach provides insights into the heterogeneous stability behaviors of polysorbate 20 subspecies in real-time stability samples of a mAb formulation.

DNA hydrodynamic degradation controlled by Kolomogorov length scales in pipe flow.

Strict US Food and Drug Administration regulations on contamination levels for DNA therapeutics acceptable for human use complicate the manufacturing process. This study aims to improve therapeutic production through the investigation of the molecular effects of hydrodynamic forces encountered during processing. Results suggest that the strain rate and residence time were not solely responsible for degradation within the system. Instead, turbulent flows at the entrance or developing flow regions dominate especially when the Kolmogorov length scale approaches the stretched molecular length scale. We specifically suggest this for linear genomic DNA and supercoiled plasmid DNA when the ratio of the molecular length to the Kolmogorov length scale must remain smaller than unity to minimize loss of the desired structure. These findings suggest that bioprocessing systems should design expansions and contractions to minimize recirculation and turbulent mixing zones, although, not always possible, careful attention should be paid to pipe surface roughness to ensure that turbulent eddies are not generated in low Reynolds number flows.

In vitro stability and compatibility of tenecteplase in central venous access devices.

Central venous access devices (CVADs) aid in the delivery of nutritional support, infusion therapy, and hemodialysis. Maintaining continuous flow through these devices is challenging, because they are susceptible to complications such as thrombi occlusion. Therefore, CVADs may require treatment with anticoagulant or thrombolytic agents. Using these agents as locking solutions has been widely investigated; however, few publications have described the compatibility of the therapeutic with the CVAD itself. The objective of this investigation was to evaluate the in vitro stability and compatibility of a thrombolytic biologic agent, tenecteplase, with various CVAD materials. Tenecteplase was reconstituted to 1 mg/mL with either sterile water for injection or bacteriostatic water for injection (0.9% benzyl alcohol) then incubated in glass vials, polysulfone/silicone vascular access ports, and polyurethane or silicone catheters for up to 96 hours. Biochemical assays including protein monomer, protein one-chain, and in vitro bioactivity were used to assess tenecteplase's compatibility with the investigated diluents and materials every 24 hours. Antimicrobial testing was also performed for up to 28 days on bacteriostatic water for injection-reconstituted samples only. Our results showed tenecteplase to be compatible with both types of diluents (in glass vials) and catheters for up to 72 hours. Furthermore, tenecteplase was compatible with the polysulfone/silicone vascular access ports for up to 24 hours. Finally, bacteriostatic water for injection-reconstituted tenecteplase effectively met USP criteria for the inhibition of growth of micro-organisms. This study serves as an example of a best practice to evaluate the in vitro stability and compatibility of a biologic agent with CVAD materials.

Rationale for the selection of an aerosol delivery system for gene delivery.

Genetic therapeutics show great promise toward the treatment of illnesses associated with the lungs; however, current methods of delivery such as jet and ultrasonic nebulization decrease the activity and effectiveness of these treatments. Extremely low transfection rates exhibited by non-complexed plasmid DNA in these nebulizers have been primarily attributed to poor translocation and loss of molecular integrity as a consequence of shear-induced degradation. Current research focusing on methods to increase transfection rates via the pulmonary delivery route has largely concentrated on the incorporation of carbon dioxide in the air stream to increase breath depth as well as the addition of cationic agents that condense DNA into compact, ordered complexes. The purpose of this study was to examine the impact of several classic as well as the latest atomization devices on the structure of non-complexed DNA. Various sizes of plasmid and cosmid DNA were processed through an electrostatic spray, ultrasonic nebulizer, vibrating mesh nebulizer, and jet nebulizer. Results varied dramatically based upon atomization device as well as DNA size. This may explain the inefficiency experienced by genetic therapeutics during pulmonary delivery. More importantly, this suggests that the selection of an atomization device should consider DNA size in order to achieve optimal gene delivery to the lungs.

The stability of lyophilized lipid/DNA complexes during prolonged storage.

It is well known that excipients are required to protect nonviral vectors during the lyophilization process. The goal of this study is to describe the stability of lyophilized nonviral vector preparations on pharmaceutically relevant timescales and provide insight into the factors that govern long-term stability of vectors in the dried state. Lipid/DNA complexes were lyophilized in glucose, sucrose, or trehalose and stored for a period of up to 2 years at five different temperatures (-20, 4, 22, 40, 60 degrees C). We evaluated simultaneously the physico-chemical characteristics (size, zeta potential, ethidium bromide (EtBr) accessibility, supercoiled DNA content) and the ability of vector formulations to transfect COS-7 cells at different time intervals. In addition, a fluorescence assay was utilized to assess levels of ROS in the dried cake after storage. The physical state of each formulation was evaluated by determination of the glass transition temperature and residual moisture content, before and after storage. Results from our stability study show that a progressive degradation of lipid/DNA complexes occurs in terms of transfection rates, particle size, dye accessibility, and supercoil content, even when samples are stored at low temperatures (e.g., -20 degrees C). Furthermore, our preliminary results on the quantification of free radicals in rehydrated formulations emphasize the importance of developing strategies to prevent the formation of reactive oxygen species (ROS) during prolonged storage in the dried state.