Dilutions For The Enzymatic Assays Biology Essay

Published:

This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.

With the advent of biotechnology, culturing of mammalian cells for producing commercially beneficial products has become increasingly popular. The cell culture samples containing various metabolites - lactate & ammonium and substrates - glucose & glutamine are still being quantified using commercially available enzymatic assays involving time and cost. This study quantitatively compares the enzymatic assays to HPLC method for profiling of cell culture components. The study is based on batch culturing of Chinese Hamster Ovary (CHO320) cells producing human interferon-γ. Samples were collected daily from culture flask incubated in 37°C shaking incubator (no CO2 supplemented), 100rpm for 7 days. HPLC analysis and enzymatic assays were carried out on samples collected to profile the cell culture metabolites and substrates. Results were analysed to quantitatively compare Agilent HPLC method to enzymatic assays. It was concluded that enzymatic assays and HPLC method are directly comparable for profiling glucose and lactate in the CHO320 cell culture samples, thus they can be reliably profiled using HPLC from further on to save cost and time. But, the ammonium concentration detected in cell culture samples using enzymatic assays and HPLC results are not concurrent, thus it can not be profiled reliably using HPLC. Glutamine profiling using HPLC method could not be carried out due to HPLC breakdown, but a new Waters Column has been received to accurately profile ammonium and glutamine-glutamate concentrations in cell culture samples.

AIM & INTRODUCTION

HPLC application has been set up to detect the main cell culture metabolites - lactate and ammonia, and substrates - glucose, glutamate and glutamine. Also, enzymatic assays are used for mammalian cell culture metabolite and substrate analysis. However, assays are expensive and time consuming. Thus, the aim of this study is to quantitatively compare HPLC method for glucose, lactate, ammonia, glutamine and glutamate to commercially available enzymatic assay kits. If the HPLC method detection level is comparable to the enzymatic assays, the future metabolite/substrate analysis can be done with HPLC solely. If the enzymatic assay is found more accurate than HPLC, the next aim is to scale down the assay for 96-well format to reduce costs.

MATERIALS & METHODS

Cell Culture. Cell culture was carried out in a 125ml shake flask (Belco Glass) with a working volume of 50ml, specially conditioned for batch operation. CHO320 cell line was cultured using EXCELL CHO DHFR medium (Sigma Aldrich) supplemented with 4mM L-Glutamine (Sigma) and 1µM Methotrexate (Sigma) with a starting cell density of 0.3 * 106 cells/ml. The cell culture work was carried out in strictly sterile clean room environment and the culture flask was maintained in 37°C incubator for 7 days with continuous agitation at the speed of 100rpm (no CO2 supplemented).

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

Table 1. Volume of cell culture samples aliquoted in micro-centrifuge tubes for enzymatic assays and HPLC analysis

 

Enzymatic Kits

HPLC

Glutamine-Glutamate

600 µL

500 µL

Ammonia

100 µL

500 µL

Glucose

100 µL

Lactate

100 µL

Enzymatic Assays

Glucose Assay 3. Lactate Assay

Ammonia Assay 4. Glutamine - Glutamate Assay

All assays were performed in accordance with the standard operating procedures of each kit. These assays work on the basis of an enzyme reaction where the substrate of each reaction is measured using a spectrophotometer using a specific wavelength. This absorbance reading is then used to calculate the concentration of unknown substance in the sample e.g. Ammonia. Table 2 below details all the dilutions done on the samples for the respective assays.

Table 2. Dilutions for the Enzymatic Assays

 

Ammonia

Glucose

Lactate

Gln-Glu

D0

1:5 

1:100

1:100

 1:2

D1

 1:5

 1:100

 1:100

1:2

D2

 1:10

 1:100

 1:100

 1:2

D3

 1:10

 1:100

 1:100 

 1:2

D4

 1:10

 1:80

1:200

 1:2

D5

 1:10

 1:70

 1:300

 -

D6

 1:15

 1:70

 1:400

 -

D7

 1:15

 1:70

 1:400

 -

HPLC Analysis

Metabolite detection using HPLC method was carried out using Agilent HPLC 1100 series and Supelcogel C-610H (carbohydrate) column equipped with a guard column. A diluted H2SO4 mobile phase solution (1.35ml H2SO4 in 5L ultra-pure water) was used for isocratic elution / linear gradient. Agilent Chemstation - online software was used to set up the method for HPLC analysis of samples and the Agilent Chemstation - offline software was used to analyse the data and obtain the results of sample and standard analysis.

Ammonium, Glucose and Lactate standards of different concentrations (2g/L, 1g/L, 0.5 g/L and 0.25 g/L) were prepared using ultra pure water according to the standard operating procedure, filtered through 0.22µm filter using syringe and transferred into HPLC vials. The cell culture samples were filtered in similar way and transferred to vials. An internal standard of 30 g/L isopropanol solution was prepared and used to remove or minimize the background error from the results.

The HPLC a) lines flushing, b) equilibration of column, c) stabilization of detector, d) sample and standard loading and e) sequence table creation steps were carried out in accordance with the standard operating procedure set-up for HPLC metabolite detection at the Laboratory of Integrated Bioprocessing (Refer Pages 011-014, Lab Manual 1197). Once the HPLC run of standards and samples was finished, the data was collected using Agilent Chemstation - offline software and was analysed on the basis of component's peak retention time, peak area and the peak height (Refer Pages 015-021, Lab Manual 1197).

RESULTS & DISCUSSION

Enzymatic Assay Results

1. Glucose Assay 3. Lactate Assay

2. Ammonium Assay 4. Glutamine-Glutamate Assay

Figure 1-3 below shows the Standard Curves prepared for the respective enzymatic assays with excellent regression values of around 0.99. The corresponding standard curve equations were used for the calculations for metabolites and substrates in the cell culture samples.

Figure 1. Standard Curve for Glucose using Sigma Aldrich Kit

Figure 2. Standard Curve for Glutamine using Sigma Aldrich Kit

Figure 3. Standard Curve for Lactate using Sigma Aldrich Kit

Figure 4 & 5 below graphically shows the concentration of different metabolites and substrates in the CHO320 cell culture samples varying over a 7-day culture period.

Figure 4. Relationship between Glucose & Lactate concentrations within a 7-day CHO320 batch cell culture

Figure 4 shows the relationship between the glucose and lactate concentrations within the culture; on the other hand, contrast between L-glutamine and ammonium concentrations is shown in Figure 5. As expected, there was a decrease in glucose concentration from 35mM to 13mM (5.25 g/L to 2.48 g/L) in the CHO320 cell culture with time due to consumption of glucose as carbon and energy source via the glycolysis pathway in the batch culture and its conversion to lactate and thus increase in lactate concentration from 0mM to 9.31mM was observed.

Theoretically, according to the process of anaerobic glycolysis (absence of air) 1 mole of glucose gets converted into pyruvate and then pyruvate to 1 mole of lactate using lactate dehydrogenase. This phenomenon is not observed in the above CHO320 cell culture study, as 1 mole of glucose is converted to ~0.6 moles of lactate (Refer Table 3 below) according to the results using enzymatic assays. The reason for this incomplete anaerobic glycolysis could be the air which was present in the cell culture flask at the time of starting the culture; also, air could be exchanged within the flask during everyday sampling.

Figure 5. Relationship between Glutamine & Ammonium concentrations within a 7-day CHO320 batch cell culture

As expected, there was an increase in ammonium concentration during the 7-day CHO320 cell culture period. This can be justified due to the non-enzymatic breakdown of glutamine into pyroglutamate and ammonium during cell culture. Theoretically, it is assumed that in anaerobic conditions, 1 mole of L-Glutamine is non-enzymatically broken down to 2 moles of ammonium and 0 moles of pyroglutamate. According to our results obtained from CHO320 cell culture samples using enzymatic assays, it is observed, that 1 mole of Glutamine was converted to ~1.5 moles of ammonium (Refer Table 3). This could be due to formation of small quantities of pyroglutamate due to the influence of air which could have been introduced into the cell culture flask during everyday sampling as described above.

Table 3 below numerically shows the varying concentrations of cell culture metabolites - lactate and ammonia, and substrates - glucose, glutamate and glutamine quantitatively measured through the enzymatic assays over 7-day cell culture period.

Table 3. Numerical data set showing varying concentrations of cell culture metabolites and substrates quantitatively measured using enzymatic assays

Samples (Days)

Glucose Concentration (mM)

Lactate Concentration (mM)

Glutamine Concentration (µM/ml)

Ammonium Concentration (µM/ml)

0

29.1966849

-0.276995305

3.902079882

1.022748092

1

34.91444426

2.962441315

1.840603718

1.67519084

2

32.62734052

5.098591549

1.811154059

2.036679389

3

26.87948769

10.49765258

1.538744708

2.98889313

4

16.88123311

11.88732394

0.434382477

3.932290076

6

15.69795786

6.685446009

-0.044174489

5.730916031

7

13.7810038

9.314553991

0.220872446

6.427442748

HPLC Results

Duplicate sets of standards of glucose, lactate and ammonium were prepared at four different concentrations of 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 30g/L isopropanol solution was prepared and used to remove / minimize the background error from the results.

The results analysis of standard sets 1 & 2 from Agilent Chemstation - offline software are detailed below in Table 4A, 4B giving the data in the form of Rapport Area and Height which are further used for preparing the standard curves for glucose, lactate and ammonium respectively.

Table 4A. HPLC analysis of glucose, lactate and ammonium standards set 1 of varying concentrations using two parameters: Peak Area and Peak Height

STANDARD SET 1

Glucose Concentration (g/L)

Time (Mins)

Area

Height

Rapport Area (X / Isopropanol)

Rapport Height (X / Isopropanol)

0.25

13.128

118624.2

2579.7

0.04036971

0.033950613

Isopropanol

28.667

2938445.7

75983.9

1

1

0.5

13.129

117353.4

4255.9

0.040727627

0.056264311

Isopropanol

28.669

2881420

75641.2

1

1

1

13.127

206717.1

7745.4

0.072060502

0.102352601

Isopropanol

28.662

2868660.3

75673.7

1

1

2

13.129

396490.9

15572.6

0.137577453

0.205554456

Isopropanol

28.651

2881946.8

75759

1

1

Lactate Concentration (g/L)

Time (Mins)

Area

Height

Rapport Area (X / Isopropanol)

Rapport Height (X / Isopropanol)

0.25

18.069

87239.8

3045.9

0.029689097

0.040086124

Isopropanol

28.667

2938445.7

75983.9

1

1

0.5

18.072

164532.1

6119.4

0.057101047

0.080900356

Isopropanol

28.669

2881420

75641.2

1

1

1

18.071

297415.5

11421.2

0.10367749

0.15092694

Isopropanol

28.662

2868660.3

75673.7

1

1

2

18.073

591810.3

22986.6

0.205350876

0.303417416

Isopropanol

28.651

2881946.8

75759

1

1

Ammonium Concentration (g/L)

Time (Mins)

Area

Height

Rapport Area (X / Isopropanol)

Rapport Height (X / Isopropanol)

0.25

7.995

6209.9

550.7

0.002113328

0.007247588

Isopropanol

28.667

2938445.7

75983.9

1

1

0.5

8.538

115894.2

8327.8

0.04022121

0.110096085

Isopropanol

28.669

2881420

75641.2

1

1

1

8.552

370537.4

26158.1

0.129167403

0.345669632

Isopropanol

28.662

2868660.3

75673.7

1

1

2

8.601

918870.6

64842.4

0.318836767

0.855903589

Isopropanol

28.651

2881946.8

75759

1

1

Table 4B. HPLC analysis of glucose, lactate and ammonium standards set 2 of varying concentrations using two parameters: Peak Area and Peak Height

STANDARD SET 2

Glucose Concentration (g/L)

Time (Mins)

Area

Height

Rapport Area (X / Isopropanol)

Rapport Height (X / Isopropanol)

0.25

13.122

65938.5

2026.4

0.022943297

0.026865569

Isopropanol

28.617

2873976.7

75427.4

1

1

0.5

13.121

114127.3

3761.9

0.039117978

0.049629157

Isopropanol

28.594

2917515.3

75800.2

1

1

1

13.119

195857.8

7448.9

0.067689556

0.098519086

Isopropanol

28.559

2893471.5

75608.7

1

1

2

13.122

433494.5

15590.7

0.146646206

0.205007265

Isopropanol

28.533

2956056.7

76049.5

1

1

Lactate Concentration (g/L)

Time (Mins)

Area

Height

Rapport Area (X / Isopropanol)

Rapport Height (X / Isopropanol)

0.25

18.067

80619.4

2834

0.028051515

0.037572553

Isopropanol

28.617

2873976.7

75427.4

1

1

0.5

18.068

132572.8

5212.4

0.04544031

0.06876499

Isopropanol

28.594

2917515.3

75800.2

1

1

1

18.071

278317.2

10913.8

0.096187987

0.144345823

Isopropanol

28.559

2893471.5

75608.7

1

1

2

18.076

604462.2

22894.2

0.204482614

0.301043399

Isopropanol

28.533

2956056.7

76049.5

1

1

Ammonium Concentration (g/L)

Time (Mins)

Area

Height

Rapport Area (X / Isopropanol)

Rapport Height (X / Isopropanol)

0.25

8.002

2144.8

160.7

0.000746283

0.002130526

Isopropanol

28.617

2873976.7

75427.4

1

1

0.5

8.536

88862.7

6693.1

0.030458349

0.088299239

Isopropanol

28.594

2917515.3

75800.2

1

1

1

8.553

362655.2

25864.3

0.125335674

0.342081004

Isopropanol

28.559

2893471.5

75608.7

1

1

2

8.605

927195.1

65838.3

0.313659444

0.865729558

Isopropanol

28.533

2956056.7

76049.5

1

1

Using the results of standard sets 1 & 2 from Table 4A & 4B, standard curves were prepared for glucose, lactate and ammonium. The standard curves were compared to each other using rapport area and rapport height data and are illustrated in Figures 6, 7 and 8 below showing graphs from two duplicate sets of standards prepared.

Figure 6. Standard Curves for glucose prepared using data obtained from HPLC analysis of duplicate sets of standards prepared

Figure 7. Standard Curves for lactate prepared using data obtained from HPLC analysis of duplicate sets of standards prepared

Figure 8. Standard Curves for ammonium prepared using data obtained from HPLC analysis of duplicate sets of standards prepared

The figures 6-8 presented above shows that almost similar equations and regression values are obtained for both area and height rapports using two duplicate sets of standards prepared for glucose, lactate and ammonium, thus showing the efficacy and specificity of experimentation carried out for profiling cell culture components.

The Table 5 below shows the numerical data obtained from HPLC Chemstation - offline software for profiling of CHO320 cell culture samples for lactate, ammonium and glucose profiling over 7-day period using two parameters - Peak Area and Peak Height. Furthermore, using the standard curve equations obtained in figures 6-8 above, the actual concentrations of the cell culture metabolites - lactate and ammonium and substrates - glucose were obtained and reported in g/L units in two duplicate sets of rapport area and rapport height.

Table 5. HPLC analysis of CHO320 cell culture samples for metabolite: lactate and ammonium & substrate: glucose profiling using 2 parameters: Peak Area and Height

CHO320 Cell Culture Sample - Day 0

Time (Mins)

Area

Height

Rapport Area (X / Isopropanol)

Rapport Height (X / Isopropanol)

STD 1:Compound Concentrations (g/L) - using Rapport Area

STD 1:Compound Concentrations (g/L) - using Rapport Height

STD 2:Compound Concentrations (g/L) - using Rapport Area

STD 2:Compound Concentrations (g/L) - using Rapport Height

Glucose

13.131

1027040.3

40593.1

0.27250314

0.418002924

4.35389983

4.173810208

3.809366385

4.094847746

Lactate

18.104

40149.9

763.9

0.010652916

0.007866175

0.052634427

0.028517213

0.12832113

0.081409975

Ammonium

8.605

771866.9

56074.3

0.204798345

0.577418857

1.388148823

1.444665008

1.416794867

1.441712575

Isopropanol

28.376

3768911.8

97112

1

1

-

-

-

-

 

 

CHO320 Cell Culture Sample - Day 1

Time (Mins)

Area

Height

Rapport Area (X / Isopropanol)

Rapport Height (X / Isopropanol)

STD 1:Compound Concentrations (g/L) - using Rapport Area

STD 1:Compound Concentrations (g/L) - using Rapport Height

STD 2:Compound Concentrations (g/L) - using Rapport Area

STD 2:Compound Concentrations (g/L) - using Rapport Height

Glucose

13.131

984375.8

38855.8

0.34626274

0.515256476

5.614747689

5.162159308

4.851168642

5.045517848

Lactate

18.064

75939.5

2215.5

0.026712379

0.029379159

0.213550891

0.172320581

0.284998818

0.223035937

Ammonium

8.577

743204.6

52268

0.261428675

0.693112109

1.698622121

1.681498688

1.727779654

1.672406997

Isopropanol

28.742

2842858

75410.6

1

1

-

-

-

-

 

 

CHO320 Cell Culture Sample - Day 2

Time (Mins)

Area

Height

Rapport Area (X / Isopropanol)

Rapport Height (X / Isopropanol)

STD 1:Compound Concentrations (g/L) - using Rapport Area

STD 1:Compound Concentrations (g/L) - using Rapport Height

STD 2:Compound Concentrations (g/L) - using Rapport Area

STD 2:Compound Concentrations (g/L) - using Rapport Height

Glucose

13.136

939211.2

36252.9

0.326974329

0.4795687

5.285031272

4.799478664

4.578733466

4.696663739

Lactate

18.072

160369.3

4980.6

0.055830514

0.065885484

0.505315774

0.416346819

0.569078188

0.463367243

Ammonium

8.588

798434.6

56527.2

0.277964762

0.747765719

1.789280495

1.793379159

1.818587382

1.781387277

Isopropanol

28.722

2872431

75594.8

1

1

-

-

-

-

 

 

CHO320 Cell Culture Sample - Day 3

Time (Mins)

Area

Height

Rapport Area (X / Isopropanol)

Rapport Height (X / Isopropanol)

STD 1:Compound Concentrations (g/L) - using Rapport Area

STD 1:Compound Concentrations (g/L) - using Rapport Height

STD 2:Compound Concentrations (g/L) - using Rapport Area

STD 2:Compound Concentrations (g/L) - using Rapport Height

Glucose

13.133

840990.1

31890.8

0.289938868

0.420226777

4.651946468

4.196410336

4.055633734

4.116586286

Lactate

18.07

225429.1

7388.3

0.077718701

0.097356024

0.724636283

0.626711392

0.782621473

0.670546572

Ammonium

8.586

814020.3

57271.9

0.280640788

0.754674889

1.80395169

1.807522803

1.833282747

1.795164285

Isopropanol

28.717

2900577.3

75889.5

1

1

-

-

-

-

 

 

CHO320 Cell Culture Sample - Day 4

Time (Mins)

Area

Height

Rapport Area (X / Isopropanol)

Rapport Height (X / Isopropanol)

STD 1:Compound Concentrations (g/L) - using Rapport Area

STD 1:Compound Concentrations (g/L) - using Rapport Height

STD 2:Compound Concentrations (g/L) - using Rapport Area

STD 2:Compound Concentrations (g/L) - using Rapport Height

Glucose

13.135

767333.8

29029.8

0.266958763

0.383321757

4.259124146

3.821359324

3.731055968

3.755833406

Lactate

18.073

223872.1

7858.8

0.077886076

0.103770919

0.726313388

0.669591705

0.784254401

0.712777611

Ammonium

8.59

806629.8

57116.6

0.280630012

0.754191744

1.803892608

1.806533765

1.833223567

1.794200885

Isopropanol

28.72

2874353.3

75732.2

1

1

-

-

-

-

 

 

CHO320 Cell Culture Sample - Day 6

Time (Mins)

Area

Height

Rapport Area (X / Isopropanol)

Rapport Height (X / Isopropanol)

STD 1:Compound Concentrations (g/L) - using Rapport Area

STD 1:Compound Concentrations (g/L) - using Rapport Height

STD 2:Compound Concentrations (g/L) - using Rapport Area

STD 2:Compound Concentrations (g/L) - using Rapport Height

Glucose

13.131

598122.6

22773.5

0.205588136

0.299924405

3.210053601

2.973825253

2.864239204

2.940610018

Lactate

18.072

204781.5

6925.9

0.070387989

0.091213315

0.651182251

0.585650503

0.711102329

0.630107408

Ammonium

8.589

817861.1

57358.4

0.28111718

0.755403604

1.806563488

1.809014543

1.835898848

1.796617357

Isopropanol

28.666

2909324.5

75930.8

1

1

-

-

-

-

 

 

CHO320 Cell Culture Sample - Day 7

Time (Mins)

Area

Height

Rapport Area (X / Isopropanol)

Rapport Height (X / Isopropanol)

STD 1:Compound Concentrations (g/L) - using Rapport Area

STD 1:Compound Concentrations (g/L) - using Rapport Height

STD 2:Compound Concentrations (g/L) - using Rapport Area

STD 2:Compound Concentrations (g/L) - using Rapport Height

Glucose

13.125

495248.9

18879.3

0.169125818

0.247803419

2.586766117

2.444140442

2.349234716

2.43111847

Lactate

18.068

169262.9

6083.3

0.057802706

0.079847375

0.525077211

0.509674965

0.58831908

0.555282256

Ammonium

8.585

801200.6

56946.7

0.273607285

0.747463465

1.765390815

1.792760419

1.794658345

1.780784576

Isopropanol

28.663

2928286.8

76186.6

1

1

-

-

-

-

The data obtained from the HPLC Chemstation - offline software for profiling of CHO320 cell culture samples for lactate, ammonium and glucose using two parameters - Peak Area and Peak Height have been graphically represented in Figures 9-11 below. The figures clearly indicate that the results obtained via both methods, that is, using peak area and peak height give almost exactly same concentration of the component. Thus, it can be concluded that both methods, that is, data analysis using peak area and peak heigh,t are reliable as both ways provides concurrent 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 assays) for the ease of comparison.

Table 6. Numerical comparison of HPLC and enzymatic assays for glucose, lactate and ammonium profiling of CHO320 cell culture samples

HPLC

ENZYMATIC ASSAYS

Sample

Glucose Concentration- mM

Lactate Concentration - mM

Ammonium Concentration - µM/ml

Sample

Glucose Concentration -mM

Lactate Concentration - mM

Ammonium Concentration - µM/ml

0

24.18833333

0.561797753

0.081620675

0

29.1966849

-0.276995305

1.022748092

1

31.16666667

2.359550562

0.099236648

1

34.91444426

2.962441315

1.67519084

2

29.38888889

5.730337079

0.105108639

2

32.62734052

5.098591549

2.036679389

3

25.83333333

8.08988764

0.105695838

3

26.87948769

10.49765258

2.98889313

4

23.66666667

8.202247191

0.105695838

4

16.88123311

11.88732394

3.932290076

6

17.83333333

7.303370787

0.106283037

6

15.69795786

6.685446009

5.730916031

7

14.38888889

5.842696629

0.103934241

7

13.7810038

9.314553991

6.427442748

The table above clearly indicates that the HPLC and enzymatic methods are directly comparable to each other for profiling glucose and to some extent lactate as well in the CHO320 cell culture samples. Thus they can be reliably profiled using HPLC from further on to save cost and time. But, the ammonium concentration detected in the cell culture samples using the enzymatic assays does not correspond to the HPLC results. Therefore, it can not be profiled reliably using HPLC.

The data presented above in the table 6 is graphically represented in the figures 12 and 13. The Figure 12 shows the relationship between the glucose and lactate concentrations within the culture comparing both the HPLC and enzymatic methods. On the other hand, contrast between ammonium concentrations using two methods is shown in Figure 13.

A decrease in glucose concentration from 35mM to 13mM was detected using enzymatic assay and a decrease from 32mM to 14mM was detected using HPLC method in the CHO320 cell culture with time (refer Table 6 & Figure 12). Also, due to consumption of glucose as carbon and energy source via the glycolysis pathway in the batch culture and its conversion to lactate, an increase in lactate concentration from 0mM to 11.88mM was detected using enzymatic assay, whereas, an increase from 0mM to 8.2mM was detected using HPLC analysis.

Figure 12. Graphical comparison of HPLC and enzymatic assays for glucose & lactate profiling of CHO320 cell culture samples

Non-enzymatic breakdown of L-glutamine in CHO320 cell culture system is assumed to produce pyroglutamate and ammonium. Thus, as expected, there was an increase in ammonium concentration detected in samples from 1.02µM/ml to 6.02µM/ml using enzymatic assays, whereas, only a slight increase from 0.08µM/ml to 0.103µM/ml was detected using HPLC analysis. This shows that the Supelcogel carbohydrate column used in this study is quite sensitive to detect glucose & lactate concentrations, but not specific for detecting correct ammonium concentrations in samples.

Figure 13. HPLC and assay comparison for ammonium profiling of CHO320 samples

CONCLUSION

Commercially available enzymatic assay kits are very sensitive & specific for mammalian cell culture metabolites and substrates analysis. However, assays are expensive & time consuming. Thus, an alternative method of HPLC - Supelcogel carbohydrate column for profiling cell culture components has been compared to enzymatic assays for improving analysis efficiency. It has been concluded that enzymatic assays & HPLC method are directly comparable for profiling glucose & lactate in the CHO320 cell culture samples thus they can be reliably profiled using HPLC from further on to save cost and time. But, the ammonium concentration detected in cell culture samples using enzymatic assays and HPLC results are not concurrent, thus it can not be profiled reliably using HPLC. Glutamine profiling using HPLC method could not be carried out due to HPLC breakdown, but a new Waters Column have been received to accurately profile ammonium and glutamine-glutamate concentrations in cell culture samples. Also, in the meantime, the enzymatic assays for profiling ammonium and glutamine could be scaled down to 96-well format to reduce costs and sample quantity requirements.

Writing Services

Essay Writing
Service

Find out how the very best essay writing service can help you accomplish more and achieve higher marks today.

Assignment Writing Service

From complicated assignments to tricky tasks, our experts can tackle virtually any question thrown at them.

Dissertation Writing Service

A dissertation (also known as a thesis or research project) is probably the most important piece of work for any student! From full dissertations to individual chapters, we’re on hand to support you.

Coursework Writing Service

Our expert qualified writers can help you get your coursework right first time, every time.

Dissertation Proposal Service

The first step to completing a dissertation is to create a proposal that talks about what you wish to do. Our experts can design suitable methodologies - perfect to help you get started with a dissertation.

Report Writing
Service

Reports for any audience. Perfectly structured, professionally written, and tailored to suit your exact requirements.

Essay Skeleton Answer Service

If you’re just looking for some help to get started on an essay, our outline service provides you with a perfect essay plan.

Marking & Proofreading Service

Not sure if your work is hitting the mark? Struggling to get feedback from your lecturer? Our premium marking service was created just for you - get the feedback you deserve now.

Exam Revision
Service

Exams can be one of the most stressful experiences you’ll ever have! Revision is key, and we’re here to help. With custom created revision notes and exam answers, you’ll never feel underprepared again.