A brand-new technology for online monitoring of glucose and lactic acid.

The fed-batch cultivation process for animal cells is widely used in many areas of biopharmaceutical production, including antibody drugs, AAV, and CAR-T therapies.

A brand-new technology for online monitoring of glucose and lactic acid.

2023-04-24

The fed-batch cultivation process for animal cells is widely used in many areas of biopharmaceutical production, including: Antibody drugs, AAV 、CAR-T Wait The development of cultivation processes has a significant impact on expression levels, cell viability, and product quality. The advantages of fed-batch or perfusion cultures lie in their ability to continuously replenish various nutrients in the culture medium. Optimization of feeding strategies—such as the timing of feed additions, the composition of the feed medium, and the total amount of feed added—can generally increase cell expression levels, maintain high cell viability, produce highly specific products, and extend the culture duration. Currently, a variety of process analytical technologies have been developed. PAT ), so that the entire metabolic process can be monitored in a convenient and efficient manner during the cultivation of animal cells, thereby achieving the goal of quality control.

So far, many parameters can be monitored and controlled in real time, such as rotational speed, pH 、 DO And some offline parameters, such as live cell density. VCD Osmotic pressure and metabolic products such as glucose, lactic acid, and ammonium ions can be obtained through manual sampling and offline analysis.

Although the offline testing technologies for some critical parameters have become highly mature, excessive manual sampling—not only increases the risk of contamination and labor costs—also renders offline testing, which provides non-real-time results, unsuitable for scenarios that require frequent sampling and analysis, demand a deeper understanding of cell culture processes, or necessitate stringent parameter control.

In response to this type of situation, many companies are currently working on developing a range of online monitoring methods for critical parameters, such as: Incyte Arc Smart live cell density monitoring sensor Hamilton ) It can measure the density of living cells in solution in real time online, using near-infrared ( NIR )、mid-infrared ( MIR ) Raman spectroscopy ( Raman ) and fluorescence spectroscopy ( Fluorescent ) Spectroscopic analysis methods have also made progress in the monitoring and control of biological processes.

For the two important parameters of the conventional type Glucose, lactic acid online monitoring, United States 908devices The German company acquired by the firm Trace The brand offers a new possibility. (Figure 1 ).

Figure 1: Trace Sensor

First, Trace The primary technical principles employed are dialysis membranes and biological (enzyme) sensors, among which... nm The pore-size-selective dialysis membrane ensures that the sterile environment inside the container remains isolated from the non-strictly sterile transport buffer system, allowing only small molecules such as glucose and lactic acid to pass through. These molecules are transported via the buffer solution to the measurement unit, achieving the effect of analysis without sample collection. This approach is particularly suitable for fermentation processes involving small volumes. / This sampling method, which does not cause sample loss, enables high-frequency sampling while also reducing the risk of contamination. It is currently in use. Trace The reactor can be as small as 60ml。

And Trace Another core component of the technology—the measurement unit—utilizes biosensors (enzymes) that exhibit high specificity toward the analyte and can maintain effective measurement accuracy even at low concentrations. Compared to conventional enzyme-membrane sensors, it boasts a significantly higher... 5000 Sensors with a second-life lifespan don't even require low-temperature storage. 5-25 Simply store at ℃. In addition, calibration can be set freely and is easy to operate; under conditions where the culture environment remains largely unchanged, calibration is required only once per day.

Selective dialysis + Biosensors, enabling Trace Online monitoring of key parameters—glucose and lactic acid—was achieved without consuming the culture volume within the reactor, and the highest... 2 The measurement frequency of once per minute makes it possible, from a process perspective, to strictly control glucose concentration in order to minimize lactate accumulation. 。

Trace The results are compared with those from conventional offline biochemical analyzers as follows (Figure: 2、3、4 ):

Figure 2 University of Hanover, Germany: 240 hours Cell Fed-batch Trace Online Monitoring vs. YSI Offline results

Figure 3 Four-channel parallel fermentation Trace Online Monitoring vs. Nova Offline results (Amgen, USA)

Figure 4 17 Tian cell culture Trace Online Monitoring vs. ABL800 Offline results ( Sartorius Stedim Biotech )

As you can see, Trace On the premise of achieving online monitoring, the results show an extremely high degree of agreement with those obtained from conventional offline biochemical analyzers using sampled measurements—this further confirms... Trace Even when providing large volumes of data, it still maintains excellent data accuracy.

Trace The applicable scenarios include:

① Attempting to maintain low concentrations of nutrients / Metabolites (such as those that, by controlling glucose at lower concentrations, can effectively reduce lactate accumulation);

② Operate within strict ranges or process parameters (e.g., cells with high environmental sensitivity);

③ The process of attempting to maintain a stable state over an extended period (e.g., perfusion);

The following is Trace An academic case study of successful application in a relevant scenario ( Zhang A, et al. Biotechnol Bioeng. 2015 Dec;112(12):2495-504 ):

In previous batches, cell viability was affected by excessive lactic acid accumulation. HEK 293 Cultivation process, through Trace High-frequency, non-sampling analysis of the technology keeps glucose concentration strictly controlled at: 0.2-0.5g/L (Traditional offline sampling does not support high-frequency analysis), ultimately achieving lower lactate levels (Figure: 🛡️). 5 ), enhancing cell viability and extending the culture duration, titer Improved 32% (Figure 6 ).

Figure 5

Figure 6

Clearly, the conclusion is that controlling glucose and lactate at a high sampling frequency leads to a more efficient and robust process.

In addition, beyond innovations in measurement technology, for different types of reactors, Trace A variety of probes are also available, ensuring flexible integration with most upstream systems (see figure). 7 ).

Figure 7

In the biopharmaceutical sector, traditional manual sampling is still widely used today, which demands a large workforce and is more prone to human error. This poses significant challenges for quality control. However, as companies now... / The demand for online monitoring technologies is steadily increasing in research institutes, and the biotechnology industry has also seen a surge in more practical and stable online monitoring technologies. Online monitoring VCD 、 TCD electrodes, Raman spectroscopy (online measurement of glucose, glutamate, ammonium ions, lactic acid, VCD and TCD ) and the aforementioned use of dialysis + of biosensors Trace Wait—biological cultivation processes may soon undergo a major transformation. Perhaps very soon, engineered bacteria... / Cell culture / Fermentation will achieve fully automated process monitoring and control, which is crucial for... PAT , for the final GMP In terms of production, this is undoubtedly a leap-forward advancement.

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