Abstract
Biopharmaceuticals are emerging as viable alternatives to chemically synthesized drugs for potential treatment to various diseases. However, many of these human-derived components cannot withstand terminal sterilization procedures, and the duration of conventional sterility testing methods often exceeds their limited shelf life. Consequently, biopharmaceuticals are now frequently administered to patients before sterility confirmation. Here we present a nanoparticle-based enrichment and rapid sterility test that can determine product sterility within a single day, mitigating clinical risks of biopharmaceuticals and maintaining therapeutic efficacies during delivery. The assay incorporates synthetic beta-2-glycoprotein I peptides for selective isolation and purification of a broad spectrum of microorganisms and a microfluidic chip designed to automatically monitor their metabolic activities via fluorescence imaging, which are inferred from the reduction of a non-toxic dye as they grow. Compared with conventional approaches, the turnaround time was substantially reduced by >58 h with 100% accuracy and a limit of detection down to a concentration of 1 colony forming unit per millilitre. We validate our approach using various forms of clinical-grade biopharmaceutical products.
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Data availability
The data supporting the results in this study are available within the Article and its Supplementary Information. Source data are provided with this paper.
Code availability
All codes (python, ver. 3.9.12) to reproduce figures are available to download via GitHub at https://github.com/phisoart/NEST. No additional custom code was generated in this study.
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Acknowledgements
This work was supported by the Korea-US Collaborative Research Fund (KUCRF) funded by the Ministry of Science and ICT and Ministry of Health and Welfare, Republic of Korea (grant no. RS-2024-00468338 to S.K.), the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health &Welfare (grant no. RS-2024-00438476 to E.J.L.), the National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Science and ICT) (grant nos. NRF-2020R1A2C1007242 to E.J.L. and RS-2024-00347997 to T.H.K.), the BK21 FOUR Program of the Education and Research Program for Future ICT Pioneers (Seoul National University in 2024), the Korea University grant (to T.H.K.) and by the Korea Research-Driven Hospital through the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health and Welfare (grant no. HI14C1277 to E.J.L.). The funders played no role in the study design, data collection and analysis, decision to publish or manuscript preparation.
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Contributions
J.K., H.K., H. Jang, T.H.K., S.K. and E.J.L. conceived and designed the study. J.K. and H. Jang optimized the protocol for microorganism enrichment. J.K., H.K., H. Jang, H. Joo and G.Y.L. evaluated the capture performance of β2GPI nanoparticles. J.K., H.K. and T.H.K. designed and fabricated the NEST chip and the automated imaging system. J.K., H.K., H. Jang, H. Joo, S.L. and T.H.K. conducted the resazurin assay experiments and analysed the data. H.K., H. Joo and Y.K. carried out the MSC culture and cell staining. E.J.L., H.J.K. and Y.K. collected the clinical-grade biopharmaceutical products and provided advice on the clinical data during assay validation. J.K., H.K., T.H.K., S.K. and E.J.L. cowrote the manuscript. All the authors discussed the results and commented on the manuscript.
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Extended data
Extended Data Fig. 1 Optimization of the amount of β2GPI nanoparticles, as well as incubation and magnetic separation times for microbial enrichment.
a-b, Capture efficiency based on the amount of β2GPI nanoparticles injected. Varying volumes of β2GPI nanoparticle solution (1.9 × 1010 particles/μL) were mixed with 5 mL of MSC culture medium containing 5 CFU/mL of P. aeruginosa (a) or either 10 CFU/mL of C. albicans (b) (N = 3). The samples were incubated for 60 mins for microbial cell binding and magnetically separated. The resuspended particles were streaked onto TSA plates, overnight cultured, and quantified to calculate the capture efficiency. Above a particle solution volume of 30 μL, the overall efficiency was saturated. c-d, Capture efficiency based on the incubation time for nanoparticle-to-microorganism binding. The enrichment performance was again tested over different incubation times for P. aeruginosa (c) and C. albicans (d), using the optimized β2GPI nanoparticle solution volume of 30 μL (N = 3). The total counts of the microorganisms after the enrichment procedure started to saturate around 15 mins which, in result, was chosen for our final protocols. e-f, Capture efficiency based on the magnetic separation time. Utilizing the optimized nanoparticle solution volume of 30 μL and an incubation time of 15 min, the enrichment performance was reassessed using P. aeruginosa (e) and C. albicans (f) by varying the duration of magnetic separation (N = 3). The capture efficiency plateaued after 5 min, which was established as the optimal time for magnetic isolation. g, Micrograph illustrating the aggregation of β2GPI nanoparticles in solution as a function of magnetic separation time.
Extended Data Fig. 2 Characterization of microorganism enrichment in a clinical-grade biopharmaceutical product using synthetic β2GPI nanoparticles.
a-c, The capture efficiency of β2GPI nanoparticles was re-evaluated in clinical-grade MSC products, containing 5 × 106 MSCs/mL. In all experiments, microorganisms were spiked into 5 mL of clinical-grade MSC products and subjected to microbial capture, followed by colony enumeration via agar plating. a, Capture efficiency for six microbial species at a concentration of 10 CFU/mL (N = 10). Compared to pure MSC culture medium (grey bar), similar capture efficiency was observed regardless of MSC presence, suggesting minimal impact on enrichment performance. b, Capture efficiency of multi-drug resistant (MDR) strains. Reference strains and MDR strains of S. aureus and P. aeruginosa were tested at a concentration of 10 CFU/mL (N = 5). The average capture efficiency between MDR and reference strains differed by less than 3% within each species. c, Microbial suspensions of S. aureus and P. aeruginosa were prepared using Bioball® products at 1 CFU/μL, and 3 μL (equivalent to 3 CFU) were spiked into 5 mL of clinical-grade MSC products to achieve a theoretical concentration of 0.6 CFU/mL (N = 10), followed by microbial capture. An identical 3 μL aliquot of the same microbial suspension was directly streaked onto agar plates (N = 10) to validate the precision of ultra-low inoculation. This approach yielded mean colony counts of 1.8 ± 1.17 CFU for S. aureus and 1.9 ± 1.14 CFU for P. aeruginosa, with one sample per species showing no colony formation. Samples with average colony counts deviating by more than ±2 CFU from the expected 3 CFU were excluded from analysis. Under these validated conditions, positivity rates following microbial capture were 80% and 90% for S. aureus and P. aeruginosa, respectively, supporting reliable microbial detection by NEST even at ultra-low inoculum levels. Bar plots display the mean, with SD shown as an upper error bar. All data points represent biologically independent experimental replicates.
Extended Data Fig. 3 Complete operational workflow of the NEST.
The schematic illustrates the overall testing procedure of NEST, wherein a 10 mL sample of biopharmaceutical product is divided into two 5 mL aliquots for aerobic and anaerobic microbial enrichment, followed by time-sequential fluorescence imaging and contaminant detection.
Extended Data Fig. 4 Defining the detection threshold for positive contamination.
a-d, Thresholds defined based on the TSB (a, b) and FTM (c, d) culture media for aerobic and anaerobic microorganism detection respectively. For an anaerobic condition using FTM, incubation was done under 5 % carbon dioxide. Fluorescence intensity curves were obtained from 30 repeated experiments and overlaid (grey). The red solid line indicates the mean intensity value over time. Three standard deviations (±3σ) above and below the mean intensity curves, which confers to the 99.7 % confidence interval (6σ), were plotted in light and dark blue lines respectively. The detection thresholds were defined based on these upper and lower limits (red dotted line) for aerobic and anaerobic culture conditions.
Extended Data Fig. 5 Differential fluorescence intensity curves based on the amount of anaerobic microbial cell input.
The differential intensity signals were measured over time using NEST, from samples spiked with varying amount of C. sporogenes in FTM media (N = 3). Unlike aerobic species, anaerobic microbes utilize alternative molecules instead of oxygen during their metabolic processes which is less efficient in generating reducing equivalents. As a result, the conversion of resazurin to resorufin mainly occurred due to the influence of medium components, causing delays in the increase of fluorescence intensity signals from wells containing microorganisms compared to controls (negative changes in differential intensity). We utilized this effect to detect the presence of anaerobic contaminants which revealed a limit of detection of 10 CFU using NEST. The colored areas in the graph indicate the standard deviation associated with each measurement condition. The gray bar depicted below represents the range of the detection time.
Extended Data Fig. 6 Performance of NEST compared to the conventional sterility test.
a, Contaminant detection time from the MSC products spiked with different types of bacteria and fungi at a concentration of 10 CFU/mL, using NEST and the conventional filtration-based sterility test (N = 10). When all species combined, the average detection time using NEST was 10.94 h (with a range between 3.5 and 17 h), whereas the convention test required 67.25 h (with a range between 48-76 h), resulting in a difference of 56.31 h for sterility determination. b, Final detection time clustered based on bacterial and fungal species. Dashed lines indicate the maximum detection time for each group.
Extended Data Fig. 7 Evaluation of NEST performance under polymicrobial contamination across diverse microbial species combinations.
A total of 45 pairwise combinations were generated using 10 microbial species: Staphylococcus epidermidis (ATCC 12228), Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 6538), Streptococcus mitis (ATCC 49456), Pseudomonas aeruginosa (ATCC 9027), Klebsiella pneumoniae (ATCC 13883), Acinetobacter baumannii (ATCC 19606), Enterococcus faecalis (ATCC 29212), Enterococcus faecium (ATCC 19434), and Candida parapsilosis (ATCC 22019). To mimic polymicrobial conditions, MSC solutions were prepared by spiking two microbial species at a concentration of 10 CFU/mL each (total 20 CFU/mL, N = 1), followed by the NEST assay under aerobic conditions. For comparison, single-species samples (10 CFU/mL) were processed in parallel using NEST and served as controls (N = 1). NEST demonstrated 100% detection sensitivity across all tested conditions. Consistent with our previous observations, the detection time for polymicrobial samples was predominantly determined by the faster-growing species. In 44 out of 45 combinations, samples tested positive within one hour of the detection time observed for the faster-growing species. A moderate delay (2.5 h) was observed for the E. faecium/C. parapsilosis combination, potentially due to interspecies growth interference, although contaminant detection remained unaffected.
Supplementary information
Source data
Source Data Fig. 2
Source data for microbial capture efficiency.
Source Data Fig. 3
Raw data for microbial growth rate, differential fluorescence intensity and detection time.
Source Data Fig. 4
Raw data for differential fluorescence intensity and MSC purification efficiency.
Source Data Fig. 5
Source data for detection time.
Source Data Extended Data Fig./Table 1
Source data for microbial capture efficiency.
Source Data Extended Data Fig./Table 2
Source data for microbial capture efficiency.
Source Data Extended Data Fig./Table 4
Raw data for differential fluorescence intensity.
Source Data Extended Data Fig./Table 5
Raw data for differential fluorescence intensity.
Source Data Extended Data Fig./Table 6
Source data for detection time.
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Kang, J., Kim, H., Jang, H. et al. One-day rapid sterility test for human-derived biopharmaceuticals. Nat. Biomed. Eng (2025). https://doi.org/10.1038/s41551-025-01524-3
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DOI: https://doi.org/10.1038/s41551-025-01524-3
