Algorithm-Based Liquid Formulation Development Including a DoE Concept Predicts Long-Term Viral Vector Stability.

Specifically tailored amino acid-based formulations were previously shown to have a high potential to avoid stress-mediated degradation of complex molecules such as monoclonal antibodies and viral vectors. By using adenovirus 5 (Ad5) as a model, we studied whether such formulations may also efficiently protect viral vectors in thermal stress experiments and during long-term liquid storage. Algorithm-based amino acid preselection using an excipient database and subsequent application of design of experiments (DoE) in combination with a 37°C challenging model enabled the prediction of long-term storage stability of Ad5. By statistical analysis of the Ad5 infectivity, amino acids with significant influence on Ad5 stability were detected after 2 and 3 weeks of liquid storage at 37°C. Ad5 formulations comprising positively selected amino acids did not reveal any loss of infectivity after 24 months in liquid storage at 5°C. By contrast, a 2 log reduction after 3 months and complete loss of infectivity after 18 months was observed with a standard viral vector formulation. By an optimization round, we designed a simple and well-balanced formulation avoiding MgCl2, previously considered essential in Ad5 formulations. This work demonstrates the efficacy of an algorithm-based development approach in the formulation development for viral vectors.

Amino Acid-Based Advanced Liquid Formulation Development for Highly Concentrated Therapeutic Antibodies Balances Physical and Chemical Stability and Low Viscosity.

To develop highly concentrated therapeutic antibodies enabling convenient subcutaneous application, well stabilizing pharmaceutical formulations with low viscosities are considered to be key. The purpose of this study is to select specific amino acid combinations that reduce and balance aggregation, fragmentation and chemical degradation, and also lower viscosity of highly concentrated liquid antibodies. As a model, the therapeutically well-established antibody trastuzumab (25->200 mg mL-1 ) in liquid formulation is used. Pre-testing of formulations based on a stabilizing and protecting solutions (SPS®) platform is conducted in a thermal unfolding model using differential scanning fluorimetry (DSF) and accelerated aging at 37 and 45 °C. Pre-selected amino acid combinations are further iteratively adjusted to obtain stable highly concentrated antibody formulations with low viscosity. Size exclusion chromatography (SE-HPLC) reveals significantly lower aggregation and fragmentation at specific amino acid:sugar and protein:excipient ratios. Dynamic viscosities <20 mPa * s of highly concentrated trastuzumab (≥200 mg mL-1 ) are measured by falling ball viscosimetry. Moreover, less chemical degradation is found by cationic exchange chromatography (CEX-HPLC) even after 6 months liquid storage at 25 °C. In conclusion, specifically tailored and advanced amino acid-based liquid formulations avoid aggregation and enable the development of stable and low viscous highly concentrated biopharmaceuticals.

Bacillus halodurans Strain C125 Encodes and Synthesizes Enzymes from Both Known Pathways To Form dUMP Directly from Cytosine Deoxyribonucleotides.

Analysis of the genome of Bacillus halodurans strain C125 indicated that two pathways leading from a cytosine deoxyribonucleotide to dUMP, used for dTMP synthesis, were encoded by the genome of the bacterium. The genes that were responsible, the comEB gene and the dcdB gene, encoding dCMP deaminase and the bifunctional dCTP deaminase:dUTPase (DCD:DUT), respectively, were both shown to be expressed in B. halodurans, and both genes were subject to repression by the nucleosides thymidine and deoxycytidine. The latter nucleoside presumably exerts its repression after deamination by cytidine deaminase. Both comEB and dcdB were cloned, overexpressed in Escherichia coli, and purified to homogeneity. Both enzymes were active and displayed the expected regulatory properties: activation by dCTP for dCMP deaminase and dTTP inhibition for both enzymes. Structurally, the B. halodurans enzyme resembled the Mycobacterium tuberculosis enzyme the most. An investigation of sequenced genomes from other species of the genus Bacillus revealed that not only the genome of B. halodurans but also the genomes of Bacillus pseudofirmus, Bacillus thuringiensis, Bacillus hemicellulosilyticus, Bacillus marmarensis, Bacillus cereus, and Bacillus megaterium encode both the dCMP deaminase and the DCD:DUT enzymes. In addition, eight dcdB homologs from Bacillus species within the genus for which the whole genome has not yet been sequenced were registered in the NCBI Entrez database.