Development of stable liquid formulations for oligonucleotides.

Oligonucleotide-based therapeutics have been implemented as a new therapeutic modality in biotech industry, which offers the opportunity to develop formulation platforms for robust parenteral formulations. The aim of this study was to gain a better understanding of stabilizing/de-stabilizing effects of different formulation parameters on unconjugated and N-acetylgalactosamine (GalNAc) conjugated single stranded oligonucleotides with locked nucleic acid modifications (LNA SSO), as model oligonucleotides. Various buffer systems, pH levels and different excipients were evaluated to optimize conditions for LNA SSO in liquid formulations. LNA SSO were exposed to different temperature conditions, mechanical stress as well as oxidative conditions, and the maximum feasible LNA SSO concentrations regarding handling and processing were determined. Finally, options for terminal sterilization of LNA SSO were evaluated. Results show that the tested LNA SSO were most stable under slightly alkaline conditions. A decrease in viscosity was best accomplished in the presence of spermine and lysine. Heat treatment and gamma irradiation caused high levels of degradation of the LNA SSO. Crucial formulation parameters, as identified in this study, should contribute to a significant increase in future productivity in drug product development for single-stranded oligonucleotides.

Individual second virial coefficient determination of monomer and oligomers in heat-stressed protein samples using size-exclusion chromatography-light scattering.

The objective of this work was to determine the second virial coefficient (B(22)) of monomer and oligomer protein species in heat-stressed samples individually and simultaneously. A high-performance size-exclusion chromatography equipped with a flow-mode detector system enabling measurement of light scattering (LS) and ultraviolet transmission in the same cell was used to separate and analyze different species. The folded/unfolded nature of the protein was analyzed by extrinsic fluorescence spectroscopy using 4,4'-Bis(1-anilinonaphthalene 8-sulfonate). The B(22) of each species was calculated from the concentration and LS data as described in an earlier publication. Upon heat exposure, monomer and formed oligomers yielded more negative B(22) values, reflecting stronger attractive forces as compared with those for the initial monomer. The increased attractive forces are attributed to partial unfolding of the protein species, and were reflected in the extent of aggregation. On the basis of the B(22) values, the monomer appeared to be the most reactive species after heat stress. In summary, the presented B(22) determination technique can be used to analyze and follow the nature of intermolecular interactions for all present protein species (monomer and oligomers) and provide further understanding of the mechanism of protein aggregation and growth of aggregates.

Simultaneous detection and analysis of protein aggregation and protein unfolding by size exclusion chromatography with post column addition of the fluorescent dye BisANS.

For development and optimization of protein formulation sensitive analytical tools are required to follow both aggregation and changes in protein structure. The latter can be seen as the beginning of physical instability leading to aggregation. The focus of this work laid on the development of a novel analysis simultaneously detecting changes in protein conformation and the formation of oligomers. By adding the extrinsic fluorescent dye 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt (BisANS) after size exclusion chromatography (SEC) and UV detection, it was possible to separate protein monomer and oligomers by size, analyze the amount of formed oligomers quantitatively using UV detection, and observe changes in protein structure of different protein species by fluorescence detection. This enabled us to distinguish between native-like and denatured oligomers and monomers formed under different stress conditions. Correspondingly, increased fluorescence reflecting partial unfolding was assigned specifically to monomer, oligomer, or both. The unfolding of monomer is not traceable by commonly used detection methods, but its monitoring may provide important information about activity and long-term stability. By adding the dye after SEC and UV detection, interferences with prior detectors are precluded, excipients are separated avoiding interferences with the protein-dye interaction and, in addition, the dye-protein interaction cannot impact the aggregation formation, as added after the separation of monomer and aggregates.

Behaviour of polysorbate 20 during dialysis, concentration and filtration using membrane separation techniques.

During formulation development of a therapeutic protein, combinations of buffers, pH and excipients need to be tested. As the protein bulk solution used for formulation development usually contains a buffer component at a defined pH and potentially one or more excipients already, this bulk requires to be processed. In case low concentrations of non-ionic surfactants, for example polysorbate 20, are already present in the bulk, the surfactant needs to be removed in lab-scale for further development use. The scope of the work was to study the behaviour of low concentrations of polysorbate 20 during membrane separation processes. The first part focuses on evaluating the behaviour of polysorbate 20 during a dialysis process, whereas the second part analyses concentration changes of polysorbate during a membrane concentration process using a stirred cell. The third part analyses potential membrane absorption of polysorbate at sterilizing-grade filters. In conclusion, it was found that polysorbate could not be significantly reduced during a dialysis process and accumulated during a membrane concentration process in unreproducable manner. During sterile filtration, no significant influence on the concentration of polysorbate was measurable. In any case, it is recommendable to quantify the concentration of polysorbate during critical membrane process steps in pharmaceutical industry.