Machine learning aided UV absorbance spectroscopy for microbial contamination in cell therapy products

Abstract

We demonstrate the feasibility of machine-learning aided UV absorbance spectroscopy for in-process microbial contamination detection during cell therapy product (CTP) manufacturing. This method leverages a one-class support vector machine to analyse the absorbance spectra of cell cultures and predict if a sample is sterile or contaminated. This label-free technique provides a rapid output (< 30 minutes) with minimal sample preparation and volume (< 1 mL). Spiking of 7 microbial organisms into mesenchymal stromal cells supernatant aliquots from 6 commercial donors showed that contamination events could be detected at low inoculums of 10 CFUs with mean true positive and negative rates of 92.7% and 77.7% respectively. The true negative rate further improved to 92% after excluding samples from a single donor with anomalously high nicotinic acid. In cells spiked with 10 CFUs of E. coli, contamination was detected at the 21-hour timepoint, demonstrating comparable sensitivity to compendial USP < 71 > test (~ 24 hours). We hypothesize that spectral differences between nicotinic acid and nicotinamide in the UV region are the underlying mechanisms for contamination detection. This approach can be deployed as a preliminary test during different CTP manufacturing stages, for real-time, continuous culture monitoring enabling early detection of microbial contamination, assuring safety of CTP.

Fig. 1

Resultant mean absorbance spectra of E. coli spiked samples at 3 hour intervals between 9-hour and 24-hour timepoints. a. Resultant mean absorbance spectra of positive control E. coli spiked samples (orange) after subtraction of mean absorbance spectra of negative control samples of the same timepoint. Rapid increase in absorbance can be observed from 9-hour to 18-hour after which the rate of increase of absorbance becomes gradual. b. 10 CFUs (dark blue) E. coli spiked samples after subtraction of mean absorbance spectra of negative control samples of the same timepoint. Increase in absorbance is more significant for 21-hour and 24-hour timepoints.

Fig. 2

Confusion matrix and classification report for performance of one-class SVM model (trained on PBS spiked sterile Donor A samples) in correctly assessing sterility status of 80 Donor A test samples. 100% true positive rate (detecting contaminated samples correctly) and 100% true negative rate (detecting sterile samples correctly) were achieved.

Fig. 3

LC-MS measurements illustrating concentration of NA in Donor A MSC culture samples inoculated with different microbial species at 10 and 100 CFUs in a 24 h incubation period. P. paraeruginosa spiked samples have the highest NA concentration, approximately 41 times higher than C. albicans and B. spizizenii infected samples at 10 CFUs. No NA was detected in C. sporogenes spiked samples due to possible antimicrobial activity of MSCs and no NA was detected in C.acnes spiked samples due to its slow-growing nature.

Fig. 4

Confusion matrix and classification report summarizing SVM model (trained on PBS spiked Donor A and B samples) performance in assessing the sterility status of 418 test samples from 6 commercial MSC donors (donors B-G). Model achieved a 92.7% true positive accuracy and 77.7% true negative accuracy.

Fig. 5

Absorbance spectra of NA and NAM measured using UV-Vis spectrometer. a. Absorbance spectra of varying concentrations (6.25 ug/ml – 100 ug/ml) of NA and NAM dissolved in PBS respectively. NA spiked samples have stronger absorbance than NAM spiked samples. Three resonant peaks are clearly observed within region of interest (255–272 nm) b. Resultant NAM spectra (dashed lines) after spectrum of 6.25 ug/ml NAM spiked in DMEM and PBS were subtracted from higher concentrations of NAM spiked in DMEM and PBS. Resultant NA spectra (solid lines) after spectrum of 6.25 ug/ml NA spiked in DMEM and PBS were subtracted from higher concentrations of NA spiked in DMEM and PBS spectra. Peaks of NA and NAM could be seen after background subtraction. (i) Spectra of varying concentrations of NA and NAM (6.25 ug/ml–100 ug/ml) spiked in DMEM and PBS, where peaks of NA and NAM can no longer be observed.

Fig. 6

LC-MS analysis for culture mediums spiked with 5 CFUs and 10 CFUs C. sporogenes spiked samples. Representative examples of Extracted Ion Chromatograms (EIC) of C.sporogenes contaminated MSC cultures with highlighted regions of NA and NAM. Identification of NAM peak at ~ 1.98 min and NA peak at ~ 1.74 min. A high concentration of NAM and low concentration of NA was observed in these samples.