In Situ Monitoring of Osmolality and pH in Bioreactors Using Near Infrared Spectroscopy

Robert Mattes, Denise Root FOSS NIRSystems, Inc. Misa A. Sugui, Fan Chen, Xiao Shi, Jonathan Liu, Philippe-Alexandre Gilbert, MedImmune
Abstract Two important cell culture process parameters that are often monitored in an effort to improve mammalian cell culture are osmolality and pH. Proper control of pH and osmolality can help optimize cell viability and growth by maintaining the isotonicity of the culture medium and regulating the transport of water and nutrients through cell membranes. Samples are typically withdrawn from the bioreactor and passed through a biomedical analyzer such as the NOVA BioProfile 400. While in situ pH measurement is common, it has some inherent technical challenges that reduce the quality of the test results with time. Near infrared spectroscopy can perform in situ real-time monitoring of pH and osmolality and provide closed-loop feedback to the bioreactor controllers. With this input the controllers can adjust culture media conditions to optimize cell performance and productivity. This poster discusses how near infrared spectroscopy can measure ionic concentrations in a bioreactor cell culture process. Introduction The fundamental absorption bands of functional groups occur in the mid-infrared region of the electromagnetic spectrum. These are very strong absorptions that require dilutions to lower the absorbances within the linear range of a mid-Infrared detector. The majority of the overtone absorptions and combination bands of these fundamental absorptions are detected in the near infrared (NIR) spectral region. Overtone absorptions occur at frequencies that are approximately integer multiples of the fundamental or first excited vibrational state of the molecular bond (1). NIR absorption in solutions of cell culture media are relatively weak and direct measurement without sample preparation or dilution is possible. The NIR absorptions are due to the change of dipole moment in covalent bonds as found in organic compounds. The OH, CH, NH and SH bonds as well as C=O and C=C have strong absorbance in the NIR region. NIR probes can be sterilized in place and real-time measurements can be acquired throughout a mammalian cell culture process (2, 3). NIR has been used to acquire real-time estimates of the level of glucose, glutamine and glutamate as well as other culture media nutrients like amino acids and metabolites like lactate and ammonia. Studies have found that analytes that are not considered to have characteristic near infrared spectra such as Osmolality and pH, have been correlated to NIR spectral absorbances. Osmolality measurement is performed to determine the concentration of ionic salts present in the solution, ensuring cell viability and optimal cell membrane transport. During metabolism, mammalian cells produce lactic and carbonic acids that tend to drive the pH below 7.0. Most mammalian cells thrive in the range 7.0 to 7.5 pH and it is important to keep the culture medium buffered at the optimum pH. Commonly the pH is controlled by the addition of sodium bicarbonate and CO2 (4). Sodium hydroxide solution was used in this study and is also commonly used to control the pH. Near infrared spectroscopists usually state that inorganic compounds have no characteristic spectra in the NIR because they have no change in dipole moment. This is true for solids, but aqueous ionic species have special interaction with the solvent. When salts are dissolved in water and become ionized, spheres of hydration around the positively and negatively charged ions are formed. These spheres of hydration have a radius inversely proportional to the ions non-hydrated radius and directly proportional to the ions charge (5,6). These hydrated radii are predicted by the Debye-Hckel Equation, Eq 1: -log () = Az+z- 1+Ba Eq. 1 Experimental A cell line was adapted to serum free growth condition and inoculated into bioreactor at 2x105 cells/mL with 4g/L of microcarrier. The culture was grown at 37C with pH controlled at 7.100.05 with CO2 and base for 3 days. Dissolved oxygen (DO) was allowed to float down from the initial 100% to the set point of 50% of air saturation by sparging pure oxygen. The agitation rate for cultures in Applikon bioreactor was set to 125 rpm. Before infection, ~80% of growth medium was removed from the culture and replaced with equal volume of infection medium. Cells were infected with a virus at the MOI of 0.01 FFU/cell and cultured at 30C for 9 days. Agitation, pH and DO were controlled as described above. A inch (19 mm) diameter and 12 inch long interactance immersion probe was installed in a 3 liter Applikon bioreactor (Applikon, Foster City, CA) with a 1.5 liter working volume. The adjustable pathlength probe was set to a 1.0 mm gap (and 2.0 mm pathlength) prior to being autoclaved. A FOSS XDS Process Analytics NearInfrared Spectrophotometer (FOSS Laurel, MD) with a 3 meter microbundle (40 illumination/40 collection) optical fiber was set up near the reactor. After the culture was prepared and the run was initiated the optical fibers were inserted into the probe that was already in place and sterilized in the bioreactor. NIR data acquisition and analysis were accomplished using Vision software supplied by FOSS. All sample spectra were collected over the range from 800 nm to 2200 nm in the interactance immersion mode. 32 spectra were averaged for each sample resulting in a scan time of approximately 16 seconds per spectrum. The XDS Process instrument uses a standardized internal reference loop that compensated for lamp variations throughout the twelve day cell culture. Scans were collected every 15 minutes for the duration of approximately 12 days. Laboratory samples were collected daily during the culture process. The lab pH values were determined by withdrawing a sample from the bioreactor and analyzing it on the Nova BioProfile 400 (Nova Biomedical, Waltham, MA). The Nova values were checked against a pH probe (Mettler Toledo 405 DPAS-SC-K88/325, Columbus, OH) in the bioreactor to be within 0.05 and recorded to correlate with the NIR spectra. The lab osmolality was also determined by analyzing it on the Nova BioProfile 400. The Nova BioProfile 400 calculates a first-order approximation of the osmolality based upon the measured ions concentrations of sodium, potassium, ammonium, glutamate and lactate by the formula in Eq 2: Osm =1.86 ([Na+] + [K+] + [NH4+]) + [Glu]/0.18 + [Lact]/0.096 + Const. Results and Discussion Osmolality: The NIR spectra were mathematically pre-treated before regression with the second derivative and the standard normal variate (SNV). The second derivative enhances absorption bands and normalizes the spectra and the SNV removes the baseline offset due to scattering. Because of the complexity of the growth media a partial least squares (PLS) model was developed. The strong absorption bands for water near 1400 nm and past 1770 nm were removed because the 2 mm pathlength caused them to exceed the dynamic range of the detector in those regions. Figure 2 shows a process trend plot of the osmolality over the course of a culture run. The lab osmolality data are superimposed over the NIR predicted values. Figure 4. The calibration set plotted against the lab data. Osmolality model using 3 actors, R2=0.9905, SEC=1.8825 mOsm/Kg. pH: Conclusion The model for pH was also developed with the second derivative and the SNV mathematic pre-treatments with the strong water absorption bands removed. Figure 5 shows the trend plot of NIR predicted pH values over part of a culture run with the Nova pH values (lab data) superimposed. The real-time in situ near infrared probe was shown to track the laboratory osmolality and pH of the mammalian cell culture media. Although these ionic analytes would not be expected to have signatures in the near infrared, NIR spectroscopy is very sensitive to the aqueous ionic concentrations most likely due to the perturbation of water absorbance bands. Near infrared spectroscopy can monitor a bioreactor in situ real-time, and is capable of replacing or supplementing osmolality and pH measurements off-line or current pH on-line methods. NIR spectroscopy may be used to improve cell viability and productivity with closed-loop feed back. Figure 7. The NIR pH calibration set plotted against the lab data from the Nova BioProfile 400. pH model using 3 factors, R2=0.8729, SEC=0.0134. Eq. 2

Figure 3. PLS loadings from the osmolality model. The first principal component (dark blue) shows where the strongest correlated variance occurs in the NIR spectrum.

Figure 6. PLS loadings from the pH model. These areas of high correlated variance occur near water bands due to clustering of water molecules around hydronium ions.

Where is the activity coefficient, z is the charge of the ion, is the ionic strength of the aqueous solution, and a is the effective diameter. A and B are constants with values of 0.5085 and 0.3281 at 25 C. The hydrogen ion, H+, is commonly denoted as associating with one water molecule and is referred to as the hydronium ion, H3O+. Actually, more molecules of water are probably associated with the hydronium ion and the sphere of hydration or effective diameter is increased with a hydrated ionic formula of H9O4+ (7). The hydronium ion is thought to be at the center with strong hydrogen bonding to three other water molecules (8). In the context of near infrared analysis of aqueous ions, although these ions may not be detected directly by near infrared absorbance, the ions may be detected and quantitated indirectly with the perturbation of the water absorbance caused by the hydrated form. The Debye-Hckel Equation tells us that radius and charge of the ion will determine the number of water molecules and tightness (diameter) of the hydrated ion. Also, the positive or negative charge will determine whether the water molecules are attached to the oxygen or hydrogen end (see Figure 1). These binding differences lead to unique absorbances for each ion due to the perturbed absorbance of the associated water molecules.

Osmolality Trend Chart

440 430 420 NIR Predicted mOsm/Kg 410 400 390 380 370 360 350 340 0 200 400 600 Sample # 800 1000 1200 NIR Predicted pH 7.14 7.12 7.10 7.08 7.06 7.04 7.02 7.00 0 200 400 7.16

pH Trend Chart

References

1) Infrared Spectral Interpretation, Smith B, CRC Press, 1999: pp 14.

NIR Predicted Lab pH
600 Sample # 800 1000 1200

NIR Predicted Lab Osmolality

Figure 2. Osmolality predicted by NIR in situ in the reactor with the lab data superimposed.

Figure 5. pH values predicted by NIR in situ in the reactor with the lab data superimposed.

Figure 3 shows the partial least squares model loadings indicating where correlated variance is modeled between the lab data and the spectral absorbances. There is a large loading band adjacent to the 1st overtone of the OH stretch at 1400 nm and smaller loadings near 1150 nm and 930 nm where OH is known to absorb. Figure 4 shows the calibration set with the NIR osmolality predictions plotted against the lab data from the Nova BioProfile 400. The osmolality model using 3 factors has an R2 value of 0.9905 with a standard error of 1.8825 mOsm/Kg. Figure 1. Spheres of hydration around positively (Na+) and negatively (Cl-) charged ions.

Figure 6 shows the PLS loadings for the pH model. Again there are strong loadings around the 1st overtone OH absorption band but the character of the loadings is significantly different from the osmolality loadings indicating no cross correlation. Figure 7 shows the calibration set with the NIR predictions plotted against the lab pH data. The pH model using 3 factors has an R2 value of 0.8729 with a standard error of 0.0134.

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