Detection Of Contamination Of ATMP Production Process By Spectrometer

INTRODUCTION

ATMPs (Advanced Therapy Medicinal Products) have recently emerged to offer new treatment solutions for patients with no further therapeutic options. For some, they are based on the use of "drug" cells derived from genetic modification or tissue and cell engineering. These "living" drugs are subject to substantial manipulations that allow cells to acquire new physiological functions, biological characteristics or reconstruction properties. The development of new biological drugs is inspired by the natural processes of the body such as the use of stem cells for tissue regeneration, lymphocytes for cancer immunotherapy or apoptotic cells for antiinflammatory purposes.

However, the fabrication of these drugs requires the implementation of complex technologies of cell sorting, amplification, genetic transduction, amplification-division, activation, and this at several stages of production and in sterile clean room type environment. As for industrial products, their production is expensive, mainly because of the complex infrastructure required, the time needed to complete the production stages and the complex quality control processes.

METHODS

A major concern during the expansion phase of ATMP fabrication is the risk of contamination, which can compromise the entire production process. The optical spectroscopy method addresses this concern by not only monitoring cell growth but also detecting the presence of contaminants, such as bacteria, in the bioreactor.

The study focused on detecting Escherichia coli (E. coli) as a representative contaminant. The system's ability to differentiate between the spectral signatures of lymphocytes and E. coli was crucial for this task. Contamination was simulated by adding known concentrations of E. coli to the lymphocyte cultures, and the resulting spectra were analyzed.

The presence of contaminants, such as E. coli, alters the absorption spectrum of the culture medium. Specifically, the shape of the spectrum changes, reflecting the different optical properties of the contaminant compared to the lymphocytes. By continuously analyzing the shape of the recorded spectra, we can detect deviations from the expected lymphocyte-only spectrum.

Figure 1: Examples of absorption spectra. (a) Lymphocytes, (b) E. coli.

The study found that the system could detect E. coli contamination with a bacterial concentration threshold of 2.5 × 10⁷ cells/mL. This threshold was determined based on the drop in the R² value of the spectral fit. When the R² value fell below a pre-determined threshold (0.988), the system issued an alert, indicating possible contamination.

Figure 2: Evolution of the R2 coefficient with the concentration in E. coli for different lymphocytes concentrations. The legend corresponds to lymphocyte concentrations in cell/mL.

To enhance the sensitivity of contamination detection, a statistical technique was employed to transform complex data into components that capture the most significant variations within the data set. This method was applied to the recorded spectra, allowing for differentiation between pure lymphocyte cultures and contaminated samples.

The analysis results provided a clear separation between the spectral data of pure lymphocytes and E. coli contaminated cells. The first two components identified were sufficient to distinguish between the two conditions. When contamination occurred, the data points corresponding to contaminated samples were clearly concentrated away from those of the pure lymphocyte samples (Figure 3).

Figure 3: Blue circles: lymphocytes, green crosses: E. coli, red squares: contaminated cultures, black line: separation between pure and contaminated cultures.

BUILD SYSTEM

INTINS can provide a complete system for this application. The QE Pro, a spectrometer of Ocean Optics (Ocean Insight), is a high sensitivity spectrometer with low stray light performance. This small-footprint instrument unlocks UV-VIS signature data from 200-950 nm and entrance slit options in widths of 5 µm to 200 µm. The QE PRO spectrometer is compact, versatile, and compatible with Ocean Insight light sources and accessories.

The DH-2000 series is the world’s only balanced deuterium halogen source. It uses innovative filtering technology to produce a smooth spectrum across the entire range, balanced output from 215 to 2500 nm and eliminates problems associated with saturation. This same technology eliminates the alpha-deuterium line in the visible region. Using a combination of deuterium and halogen lamps, the DH-2000 is flexible and ideal for measuring a sample that has multiple features in different spectral regions or for analyzing a variety of different samples.

The Cuvette Holder for 1-cm pathlength cuvettes couples via SMA-terminated optical fibers to spectrometers and light sources to create small-footprint spectrophotometric systems for absorbance experiments. The Cuvette Holder has a fully integrated cover for eliminating ambient light and has 2 filter slots to enable filtering illumination light entering the cuvette holder and/or detected light leaving the cuvette holder. The unit is designed to snugly hold 1-cm square cuvettes without user adjustment, providing high data repeatability.

CONCLUSION

The ability to detect contamination early in the production process has significant implications for ATMP manufacturing. Contamination can lead to costly production halts, loss of valuable products, and delays in delivering therapies to patients. The optical spectroscopy method provides a reliable and efficient means of detecting contamination, potentially saving tens of thousands of dollars per production batch.

Moreover, by integrating this technology into existing bioreactor systems, manufacturers can enhance the overall quality control process, ensuring that only uncontaminated, high-quality products reach patients. This could also lead to a reduction in the overall cost of ATMPs, making these therapies more accessible to a broader patient population.