Dilutions For The Enzymatic Assays Biology Essay Example

employed for analyzing these metabolites and substrates in mammalian cell culture. Nevertheless, it is important to note that these assays are expensive and time-consuming.

The purpose of this survey is to compare the HPLC method for glucose, lactate, ammonium hydroxide, glutamine, and glutamate with commercially available enzymatic check kits in order to obtain quantitative results. If the HPLC method produces similar results as the enzymatic checks, then the analysis of metabolites/substrates can be exclusively done using HPLC. However, if the enzymatic check proves to be more accurate than HPLC, the next step is to condense the check into a 96-well format in order to reduce costs.

Cell Culture

Cell culture was conducted in a 125ml shake flask (Belco Glass) with a working volume of 50ml that was specifically prepared for batch operation.

CHO320 cell line was cultured using EXCELL CHO DHFR medium (Sigma Aldrich) supplemented with 4mM L-Glutamine (Sigma) and 1AµM Methotrexate (Sigma) with a starting cell density of 0.3 * 106 cells/ml. The cell culture work was carried out in a strictly sterile clean room environment and the culture flask was maintained in a 37°C incubator for 7 days with continuous agitation at a speed of 100rpm (no CO2 supplemented).

Sampling

Cell culture samples of 2.5 milliliters were collected daily for 7 days from the cell culture flask in 15ml extractor tubes and were spun down in the extractor for 5 minutes at 200g speed. The spun sample was filtered through a 0.22 Aµm sterile filter using a syringe and aliquoted into 1.5ml micro-centrifuge tubes according to table 1 below and stored in a -80°C freezer for analysis.

All checks were carried out in

accordance with the established operating procedures of each kit. These checks operate based on an enzyme reaction, where the substrate of each reaction is measured using a spectrophotometer at a specific wavelength. The measurement of optical density obtained from this reaction is then utilized to calculate the concentration of unknown substances in the sample, such as Ammonia. Table 2 below provides information on all the dilutions performed on the samples for the respective checks.

HPLC Analysis

The HPLC analysis for metabolite sensing was done using the Agilent HPLC 1100 series and Supelcogel C-610H (saccharide) column with a guard column. An isocratic elution/additive gradient was performed using a diluted H2SO4 Mobile stage solution (1.35ml H2SO4 in 5L ultra-pure H2O). The method for analyzing samples via HPLC was established using the Agilent Chemstation - online package, while the Agilent Chemstation - offline package was used to analyze the data and obtain results for both sample and standard analysis. To create ammonium, glucose, and lactate standards, different concentrations (2g/L, 1g/L, 0.5g/L, and 0.25g/L) were prepared utilizing ultra-pure water according to standard operating procedure. These standards were then filtered through a syringe with a 0.22Aµm filter before being transferred into HPLC vials. Similar filtration and transfer methods were used for the cell culture samples.

A 30 g/L isopropanol solution was prepared as an internal criterion to minimize background errors in the results. The HPLC procedures, including column equilibration, sensor stabilization, sample and standard loading, and sequence table creation, were performed according to the specified operating procedure for HPLC metabolite detection at the Laboratory of Integrated Bioprocessing (See Pages 011-014, Lab Manual 1197). After completing the HPLC run for standards and samples, data

was collected using Agilent Chemstation - offline software and analyzed based on the peak retention time, peak area, and peak height of each component (See Pages 015-021, Lab Manual 1197).

Enzymatic Assay Results

Figure 1-3 below displays the Standard Curves generated for the respective enzymatic tests, with excellent regression values of approximately 0.99.

The computations for metabolites and substrates in the cell culture samples were performed using the criterion curve equations. Figure 1 presents the standard curve for glucose using the Sigma Aldrich Kit, while Figure 2 displays the standard curve for glutamine using the same kit. Additionally, Figure 3 illustrates the standard curve for lactate with the utilization of the Sigma Aldrich Kit. A diagrammatic representation of the concentration of various metabolites and substrates in the CHO320 cell culture samples throughout a 7-day culture period is shown in Figures 4, A, and 5.

Figure 4 shows the correlation between glucose and lactate concentrations in a 7-day CHO320 batch cell culture. Figure 5, on the other hand, illustrates the relationship between L-glutamine and ammonium concentrations. The glucose concentration decreased gradually from 35mM to 13mM (equivalent to 5.25 g/L to 2.48 g/L) over time due to its consumption through glycolysis. As a result, there was an increase in lactate concentration from 0mM to 9.31mM.

Enzymatic tests revealed that only about 0.6 moles of lactate were produced per mole of glucose, indicating incomplete anaerobic glycolysis occurred. This could be attributed to the presence of oxygen at the beginning of the culture and potential air exchange during sampling (refer to Table 3).

Figure 5 shows that the concentrations of Glutamine, A, and Ammonium in a 7-day CHO320 batch cell culture are correlated. As the

culture progresses, the ammonium concentration increases as anticipated. This increase is thought to be due to the non-enzymatic breakdown of glutamine into pyroglutamate and ammonium during the cell culture process. Theoretical assumptions suggest that under anaerobic conditions, one mole of L-Glutamine decomposes into two moles of ammonium and no moles of pyroglutamate.

According to the results obtained from CHO320 cell culture samples using enzymatic tests, it is observed that approximately 1.5 moles of ammonium were formed from 1 mole of Glutamine (Refer Table 3). This could be attributed to the formation of small amounts of pyroglutamate due to the presence of air introduced into the cell culture flask during routine sampling as described above.

HPLC Results

Duplicate sets of standards for glucose, lactate, and ammonium were prepared at four different concentrations (2 g/L, 1 g/L, 0.5 g/L, and 0.25 g/L) using glucose, calcium lactate, and ammonium chloride chemical powders (Sigma Aldrich) with ultra-pure water. An internal standard of 30 g/L isopropanol solution was prepared and used to remove/minimize background errors from the results. The analysis of the results from standard sets 1 and 2 obtained from Agilent Chemstation - offline software are provided in Table 4A and 4B in terms of Peak Area and Height, which are further used for preparing the standard curves for glucose, lactate, and ammonium respectively.

HPLC analysis was performed on glucose, lactate, and ammonium using two parameters: Peak Area and Peak Height. Standard curves for each substance were prepared by comparing the results from standard sets 1, A, and 2 from Table 4A, A, and 4B. The standard curves were compared to each other using area and height data, as shown in Figures 6,

7, and 8. These figures display graphs from two duplicate sets of prepared standards. Figure 6 shows the standard curves for glucose, Figure 7 shows the standard curves for lactate, and Figure 8 shows the standard curves for ammonium. The figures demonstrate that similar equations and regression values were obtained for both area and height resonances using the duplicate sets of standards for glucose, lactate, and ammonium. This indicates the effectiveness and specificity of the experiment conducted for profiling cell culture constituents.

The Table 5 below displays the numerical data acquired from HPLC Chemstation - offline package for profiling of CHO320 cell culture samples. The profiling was done for lactate, ammonium, and glucose over a 7-day period using two parameters - Peak Area and Peak Height. Moreover, the actual concentrations of lactate, ammonium, and glucose were determined using the standard curve equations from figures 6-8 above. These concentrations were reported in g/L units for both resonance area and resonance height in two duplicate sets. HPLC analysis of CHO320 cell culture samples was conducted to profile lactate and ammonium metabolites as well as glucose substrate using Peak Area and Height parameters. The data obtained from HPLC Chemstation - offline package for profiling CHO320 cell culture samples for lactate, ammonium, and glucose using Peak Area and Peak Height parameters is graphically represented in Figures 9-11 below. These figures clearly show that the results obtained from both methods (peak area and peak height) yield almost identical concentrations. Hence, it can be concluded that both methods, peak area and peak height, are reliable as they provide consistent results.

Figure 9. HPLC analysis of CHO320 cell culture samples for

glucose profiling using two parameters in duplicate sets: Peak Area and Peak Height
Figure 10. HPLC analysis of CHO320 cell culture samples for lactate profiling using two parameters in duplicate sets: Peak Area and Peak Height
Figure 11. HPLC analysis of CHO320 cell culture samples for ammonium profiling using two parameters in duplicate sets: Peak Area and Peak Height

Comparison - HPLC vs. Enzymatic Assays

The quantitative comparison of HPLC for profiling glucose, lactate, and ammonium to commercially available enzymatic kits in CHO320 cell culture samples is presented below in Table 6. The table shows the components (glucose, lactate, and ammonium) in similar units for both methods (HPLC and enzymatic checks) for ease of comparison.

Table 6 shows a comparison between the HPLC and enzymatic methods for analyzing glucose, lactate, and ammonium in CHO320 cell culture samples. The findings suggest that both methods are similar in profiling glucose and to some extent lactate in the samples. Hence, using HPLC can efficiently profile these substances while saving time and cost. However, when compared to HPLC, the enzymatic analysis does not produce consistent results for ammonium concentration in the cell culture samples.

The HPLC method cannot accurately profile it, so the data in table 6 is shown in figures 12 and 13. Figure 12 compares glucose and lactate concentrations using both HPLC and enzymatic methods. Figure 13 contrasts ammonium concentrations obtained from the two methods.

The CHO320 cell culture showed a decrease in glucose concentration from 35mM to 13mM according to the enzymatic assay and from 32mM to 14mM using HPLC analysis (see Table 6; A; Figure 12). Additionally, glycolysis led to

an increase in lactate concentration from 0mM to 11.88mM based on the enzymatic assay and from 0mM to 8.2mM as determined by HPLC analysis. Figure 12 illustrates a comparison of glucose; A; lactate profiling between CHO320 cell culture samples obtained through both HPLC and enzymatic assays. It is suggested that L-glutamine undergoes non-enzymatic breakdown in the CHO320 cell culture system, resulting in the production of pyroglutamate and ammonium. As expected, there was an increase in ammonium concentration detected using enzymatic assays, rising from1.02µM/mlto6.02µM/ml, while only a slight increase was observed with HPLC analysis, going up from0.08µM/mlto0.103µM/ml

The Supelcogel saccharide column used in this study is sensitive for detecting glucose and lactate concentrations, but it lacks specificity for measuring ammonium concentrations in samples.

Decision

Commercial enzymatic test kits are highly sensitive and specific for analyzing metabolites and substrates in mammalian cell cultures. However, these tests can be costly and time-consuming. To improve analysis efficiency, an alternative method involving HPLC with a Supelcogel carbohydrate column was compared to enzymatic tests. The results showed that both methods reliably profile glucose and lactate in CHO320 cell culture samples. However, for cost and time-saving purposes, HPLC should be preferred. On the other hand, the measurement of ammonium concentration using both enzymatic tests and HPLC yielded inconsistent results, making it unreliable to profile ammonium using HPLC.

Due to HPLC dislocation, it was not possible to perform glutamine profiling using the HPLC method. However, a new Waters Column has been obtained for accurately measuring the concentrations of ammonium and glutamine-glutamate in cell culture samples. Additionally, in the meantime, the enzymatic checks for profiling ammonium and glutamine could be downsized to a 96-well format, reducing costs and

sample quantity requirements.