Quantitative H NMR optimization for high-throughput metabolite analysis in industrial bioprocess monitoring.

Quantitative H NMR (H qNMR) is an ideal tool for bioprocess monitoring because it can comprehensively detect and quantify diverse metabolites that significantly influence bioprocess performance. However, the long experiment time associated with the H qNMR, due to the long longitudinal relaxation time (T1) of some metabolites, does not meet the requirements for high-throughput analysis. We developed a high-throughput H qNMR method for bioprocess analysis using a short relaxation delay (D1) to reduce analytical time and a correction factor (k) to compensate for incomplete relaxation. A total of 27 metabolites were quantified using spectral deconvolution via a peak fitting algorithm and MCR-ALS. Methodological validation results indicated that the precision and accuracy of the developed qNMR method were consistently high across different D1 values, with LOQs ranging from 0.008 to 0.13 mM and LODs ranging from 0.024 to 0.38 mM. Notably, a longer D1 value generally resulted in lower LODs and LOQs for most metabolites. A D1 value of 4 s was optimal for balancing analysis time and performance. The method is broadly applicable for bioprocess monitoring and control, offering valuable guidance for optimizing CHO cell culture processes and improving yield.