Application of quantitative NMR in pharmaceutical analysis: Method qualification and potency determination for two new chemical entities in early-stage clinical studies.

Quantitative NMR (qNMR), being a well-established analytical tool featuring efficiency, simplicity as well as versatility, has been extensively employed in pharmaceutical and medicinal testing. In this study, two 1H qNMR methods were developed to determine the %wt/wt potency of two new chemical entities (compound A and compound B) used in early clinical phase process chemistry and formulation development. The qNMR methods were demonstrated to be significantly more sustainable and efficient than the LC-based approach by substantially reducing the cost, hands-on-time, and materials consumed for testing. The qNMR methods were achieved on a 400 MHz NMR spectrometer equipped with 5 mm BBO S1 broad band room temperature probe. The methods with CDCl3 (for compound A) and DMSO-d6 (compound B) as solvent as well as commercially certified standards for quantitation were phase-appropriately qualified in terms of specificity, accuracy, repeatability/precision, linearity, and range. Both qNMR methods were demonstrated to be linear over the range of 0.8-1.2 mg/mL (80% to 120% of the nominal sample concentration of 1.0 mg/mL) with a correlation coefficient greater than 0.995. The methods were also demonstrated to be accurate with average recoveries ranging from 98.8% to 98.9% and 99.4-99.9% for compound A and compound B respectively as well as precise with %RSD of 0.46% and 0.33% for compound A and compound B respectively. The potency results of compound A and compound B determined by qNMR were compared with those determined by the conventional LC-based method and the qNMR results were demonstrated to be consistent with the LC-based method with absolute difference of 0.4% and 0.5% for compound A and B respectively.

Human-Relevant Sensitivity of iPSC-Derived Human Motor Neurons to BoNT/A1 and B1.

The application of botulinum neurotoxins (BoNTs) for medical treatments necessitates a potency quantification of these lethal bacterial toxins, resulting in the use of a large number of test animals. Available alternative methods are limited in their relevance, as they are based on rodent cells or neuroblastoma cell lines or applicable for single toxin serotypes only. Here, human motor neurons (MNs), which are the physiological target of BoNTs, were generated from induced pluripotent stem cells (iPSCs) and compared to the neuroblastoma cell line SiMa, which is often used in cell-based assays for BoNT potency determination. In comparison with the mouse bioassay, human MNs exhibit a superior sensitivity to the BoNT serotypes A1 and B1 at levels that are reflective of human sensitivity. SiMa cells were able to detect BoNT/A1, but with much lower sensitivity than human MNs and appear unsuitable to detect any BoNT/B1 activity. The MNs used for these experiments were generated according to three differentiation protocols, which resulted in distinct sensitivity levels. Molecular parameters such as receptor protein concentration and electrical activity of the MNs were analyzed, but are not predictive for BoNT sensitivity. These results show that human MNs from several sources should be considered in BoNT testing and that human MNs are a physiologically relevant model, which could be used to optimize current BoNT potency testing.

Possibility of Immediate Introduction of a Single-Dose Antibody Induction Test as a Refinement of the NIH Test for Inactivated Rabies Vaccine Potency Determination.

Antibody induction test (AIT) is a promising candidate as a refinement of the troublesome National institutes of Health (NIH) test in the sense of animal welfare 3R approach for determination of potency of inactivated rabies vaccines for veterinary and human use. In this study, we initially try to develop AIT as a suitable alternative to NIH test, to achieve a reduction of test duration and diminish animal suffering by omitting intracerebral CVS infection and measuring humoral immunity upon vaccination. Designs of both multi-dose and single-dose AIT were examined. Biological reference preparation, batch 5 with assigned titer of 10 IU/vial, was taken as both standard and test vaccine. Six consecutive AITs were performed and eight pools of sera in each AIT were tested in triplicate by rapid fluorescent focus inhibition test. We estimated the upper detection limit and calculated test variability for individual dilutions. For multi-dose AIT, we estimated the dose-response function and performed calculations of final test results and statistical validity parameters for both linear and sigmoidal model using CombiStats program. Sigmoidal 4-parameter dose-response model was found optimal. Presented design of multi-dose AIT showed a satisfactory detection limit for testing of inactivated rabies vaccines for both veterinary and human use. However, due to nonconformity of obtained results with statistical validity criteria, we concluded that the presented model of multi-dose AIT was unsuitable for introduction in routine practice. However, we concluded that there was a realistic option for introduction of two versions of single-dose AIT. The first version would be with two standard vaccine controls and could be introduced immediately, while the second version would include testing of the sample only and rely on comparison of the induced rabies antibody level with absolute cut-off limits set in advance.

Experience with the active immunization method for determining the potency of typhoid vaccine

The study was done to gain experience with the active immunization method for vaccine potency testing. It also determined the relative potency of locally prepared vaccines compared with foreign prepared vaccinesWhite Swiss mice equally distributed as to sex were used as control and test groups. Granular mucin type 1201-w, strain of Salmonella typhosa, and three vaccines (vaccines A,B, and C) were used. The procedure used was the mucin technique of RakeThe LD50 dose was found to be 0.5 ml of a dilution of 10-5-752. It was felt necessary to enhance virulence of the organism, thus another determination was done after two passages. Results of potency determination showed that vaccine A gave a protection to approximately 1,500 LD50 dose. However, vaccines B and C failed to protect immunized animals against the minimal number of LD50 doses measurable in the range of the tests conductedFurther studies involving V: antigen are deemed needed to supplement this initial experience