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Biopharmaceuticals Manufacturing is an industry that has adopted Raman spectroscopy measurements for PAT studies, particularly for cell culture and fermentation monitoring. Raman spectroscopy can be used to monitor the growth media components (including glucose) and metabolic products (such as lactate) in cell culture upon development of a multivariate statistical model.
Raman spectroscopy is a form of molecular spectroscopy – the scattering of electromagnetic radiation by atoms or molecules. Raman signal is observed as inelastically scattered light and is an invaluable tool for molecular fingerprinting.
Raman spectroscopy is a well-suited technique in the pharmaceutical industry due to its ability for non-destructive, real-time measurements, as well as its ability to be implemented for online and inline monitoring thus making this an invaluable tool for Process Analytical Technology (PAT) implementation. Raman spectroscopy as a measurement tool can be well implemented at various stages of pharmaceutical manufacturing right from analysis of incoming Raw materials for Identification and qualification for further manufacturing processes to the analysis of final products for applications like Content uniformity.
Prior to large-scale implementation of a new process or product, studies are performed in labs and at the small-scale pilot, plants to assess feasibility. B&W Tek has introduced the PTRam™, a self-calibrating portable Raman system for process development applications. The 19-inch rack-mountable system features a 785 nm laser with long-term stability together with high-throughput optics PTRam operates with Vison software, Metrohm’s proven spectroscopic software for real-time analysis, which includes instrument control and data analysis. The PTRam is self-monitoring, with self-calibration and system performance validation tests automated in the Vision software to ensure the validity of each measurement. The system testing provides a complete instrumental status diagnosis, including Raman shift accuracy and photometric precision as set forth in USP <858>.PT Ram system comes with the fiber-coupled probe, which gives the versatility and flexibility for measurements to be made in different places, including in-situ as is often the requirement for the monitoring of processes. B&W Tek offers a range of sampling probes and other accessories to optimize the system configuration tailor-made to the customer-specific application requirements.
The PTRam can aid in product and process development efforts in the chemical, petrochemical and pharmaceutical industries through the ability to increase process understanding with information-rich Raman spectral data. Predictive quantitative models can be created, or trend monitoring can be performed to reduce calibration development time before full process definition. Typical application areas where PTRam system can be used includes reaction monitoring and kinetic studies, blending and mixing, fermentation monitoring, solvent exchange, tablet coating monitoring, and other processes for real-time understanding of chemical and morphological changes as they occur.
Raman spectroscopy is a valuable technique for process analytical technology (PAT) in the pharmaceutical industry due to its capacity for non-destructive, real-time measurements, as well as its ability to be implemented for online and inline monitoring. Prior to large-scale implementation of a new process or product, studies are performed in labs and at the small-scale pilot, plants to assess feasibility. Biopharmaceuticals Manufacturing is an industry that has adopted Raman spectroscopy measurements for PAT studies, particularly for cell culture and fermentation monitoring. Raman spectroscopy can be used to monitor the growth media components (including glucose) and metabolic products (such as lactate) in cell culture Figure 1.
Figure 1. Raman spectra of glucose and lactate powders (spectra are manually offset for clarity)
Raman spectra of glucose and lactate powders (spectra are manually offset for clarity) upon development of a multivariate statistical model.Figure 1 shows the Raman spectra of pure glucose and pure lactate, which have sharp and distinct bands. In this application note, we’ll demonstrate the feasibility of monitoring glucose and lactate in a dynamic system using B&W Tek’s PTRam process development Raman analyzer.
Twenty-five aqueous solutions containing variable amounts of glucose and lactate were gravimetrically created. Glucose concentrations ranged from 0 – 4.5 g/L, and lactate concentrations ranged from 0 – 4.4 g/ L. A fibre-optic probe with an immersible fused silica shaft (RIS100-FS) was used to measure all solutions.
The laser power used was 100% of the maximum laser power (≈340 mW), and all spectra were collected with an 18 s integration time. Table 1 shows the technical specifications of the PTRam. Metrohm Vision software was used to acquire all calibration spectra.
Vision software was used to create calibration models for the prediction of glucose and lactate. Figure 2a shows the calibration data acquired by the PTRam. All spectra are relative intensity-corrected against a NIST 2241 standard.
Figure 2b shows the preprocessed calibration spectra for the glucose model. The preprocessing applied is a Savitzky-Golay first derivative and a standard normal variate (SNV) using a range of 1066-1811 cm-1.
A model for lactate was created using a Savitzky-Golay second derivative with an SNV using a range of 635-940 cm-1 and 1066-1811 cm-1. Partial least square (PLS) models were created in Vision to model the glucose and lactate constituents with cross-validation with four segments. Figure 3 shows the calculated vs. lab data plots for glucose and lactate. Each model uses three factors. Parameters for model linearity and accuracy are also shown for both constituents.
To demonstrate the performance of PTRam for monitoring the culture solution of a cell growth application, a starting aqueous solution of 5 g/L glucose was gravimetrically prepared. A fibre optic probe with an immersible fused silica shaft (RIS100-FS) was used to monitor the solution. To simulate the consumption of glucose by cells and the production of metabolic lactate, multiple volumes of aqueous lactate solutions were added to the starting solution at 10-minute intervals. To simulate the replenishing of glucose and levelling off of lactate, volumes of the monitored solution were removed and replenished with amounts of an aqueous solution containing a high concentration of glucose and a low concentration of lactate.
Finally, various amounts of aqueous lactate were again added to the solution to simulate further consumption of the glucose. The concentrations of glucose and lactate were monitored over 6.5 hours. Figure 4 shows the theoretical curves for lactate and glucose concentrations with time. A routine analysis operation method was created in the Vision software for real-time prediction of the glucose and lactate levels based on the calibration models. Spectra were collected with an integration time of 18 s and a laser power of 100% of the maximum integration time (≈ 340 mW).
Figure 5 shows the combined trend chart for glucose and lactate concentrations generated during the dynamic experiment, which matches the theoretical curve quite closely. Table 2 shows the statistics from the routine analysis prediction, including the standard error of prediction (SEP) and bias.
Raman spectroscopy is an invaluable analytical tool in the pharmaceutical manufacturing process right from the inspection of incoming raw materials, process and product development, production process monitoring and testing of finished products. It provides information critical for understanding reactions that have significant benefits in terms speed and flexibility and thus helps in consistently manufacture quality products. Also, with Raman Spectroscopy as a process development tool allows better process understanding, key process parameters can be determined which can be useful for scaling up process.
Application notes : Glucose and Lactate Monitoring with PTRam (metrohm.com) 410000058-A Glucose and Lactate Monitoring with PTRam by B&W Tek 400000420-A PT Ram flyer -2021 from B&W Tek Application note: 410000014-B Raman as a tool for PAT from B&W Tek. For more information: [email protected]
