Defi Ning Hydrolysates: Generation Of A Chemically Defi Ned Alternative
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Defining Hydrolysates: Generation of a Chemically Defined Alternative Zachary W. Deeds, Ashley D. Smith, Benjamin J. Cutak, C. Steven Updike, Mindy S. Wilson, Daiva Dailide, Dennis W. Cooley, Barry R. Drew, and Matthew V. Caple SAFC Biosciences, 2909 Laclede Avenue, Saint Louis, MO 63103 USA Correspondence to: [email protected]
Results
Abstract Protein hydrolysates are commonly utilized in cell culture processes either as a component of a complete medium formulation or as part of a fed-batch bioreactor process. It is well documented that hydrolysates can have a substantial positive impact on cell growth and/or protein production. Given the undefined nature and the lot-to-lot variability associated with hydrolysates, there exists a need to mitigate these risks with a chemically defined (CD) alternative that can maintain the desired performance. In order to create a CD alternative to protein hydrolysates, a two-pronged approach was taken to capture the associated effects. The first experimental path utilized standard analytical techniques to identify the nutritive components supplied by hydrolysates (i.e. amino acids, vitamins). A “basal” supplement was designed to supply these nutrients, as this is a significant portion of the functionality of hydrolysates. The second path was based upon Reverse Phase HPLC fractionation of multiple different hydrolysate types. Cell culture screening with Chinese Hamster Ovary (CHO) cells led to the identification of “bioactive” fractions. Subsequent identification of the components contained within the fractions led a greater understanding of the effects of hydrolysates. The learnings from both approaches were utilized to generate a chemically defined supplement, EX-CELLTM CD Hydrolysate Fusion, which is capable of replacing the functions of hydrolysates in many CHO cell systems.
Overlaid Traces 062804.001, Chan A 062904.002, Chan A
Screen Hydrolysates
750
750
500
500
RP-HPLC Fractionate & Lyophilize
Screen Fractions (+ Base) with CHO-IgG1
m V o l t s
m V o l t s
ID Fraction Components & Source CD Versions 250
250
Screen Components with Multiple CHO Lines
Materials and Methods
0
Sigma-Aldrich Corporation (St. Louis, MO, USA) supplied all chemicals, media, and solutions unless otherwise stated.
Combine and Optimize with Base Supplement for Final Product
Cell Lines Cell lines CHO-IgG1 and CHO-IgG2 are proprietary Chinese Hamster Ovary (CHO) clones expressing recombinant antibodies.
0
Figure 1. Process Flow Chart
Flow Rate (mL/min)
0 2 10 10.5 15.1 15.3
95 95 60 0 0 95
5 5 40 100 100 5
1.2 1.2 1.2 1.2 1.2 1.2
For fed-batch assays the TPP tubes were fed with a one-time bolus on/around Day 2, depending on the cell density (0.8 to 1.2e6 viable cells/mL). The feed included Glucose (6g/L) to prevent carbon source limitation and a “basal” supplement to provide much of the nutritive function of hydrolysates. It was designed to contain only compounds seen in the hydrolysates or in a standard medium formulation (amino acids, trace elements, and vitamins). The basal supplement is added to help elucidate the unknown positive effectors by supplying known nutritive compounds. This supplement continually evolved over the course of the project as more was learned about the action of hydrolysates. As new compounds were identified they were titrated into the “Base” supplement. This process continued until the final product goals were achieved.
300
350
100.0%
80.0%
1
2
3
4
5
6
7
8
Day
-40.0%
Figure 3. Growth Curve for Fraction Screening. Two fractions (of 66) showed a positive response for growth (Fractions A & D) when fed in addition to the nutritive “Base” supplement with the CHO-IgG1 cell line.
Fraction D
0
0.0%
-20.0%
Fraction C
Feed
0.00E+00
7
Fraction B
1.00E+06
4
20.0%
Fraction A
2.00E+06
40.0%
Yeast Extract 2g/L
3.00E+06
Base Supplement
4.00E+06
60.0%
No Feed
5.00E+06
% Change from Base Control
No Feed Base Supplement Yeast Extract 2g/L Base + Fraction A Base + Fraction B Base + Fraction C Base + Fraction D
6.00E+06
Figure 4. Normalized IgG productivity from Fraction Screening. Four fractions (of 66) showed a positive response for IgG production when fed in addition to the nutritive “Base” supplement with the CHO-IgG1 cell line. Data is normalized to the “Base” supplement.
1.00E+07
70.0%
9.00E+06 8.00E+06 7.00E+06 6.00E+06
Hydrolysate 2g/L
5.00E+06
Base Supplement
4.00E+06
Base + Compound
3.00E+06 2.00E+06
% Change from Base Control
%B
250
200
7.00E+06
Viable Cells/mL
%A
150
8.00E+06
Product quality was measured by Cation Exchange HPLC. MAb charge variants were separated by HPLC by injecting 50ug of purified MAb (1mg/mL) into a 4.6mm × 250mm ProPac SCX-10 strong cation-exchange column (Dionex, CA, USA) connected to an Alliance HPLC with PDA detection (Waters, MA, USA). Mobile phase A consisted of 10mM sodium phosphate (pH 6.0±0.2). Mobile phase B consisted of 10mM sodium phosphate and 500mM sodium chloride (pH 7.5±0.2). Separation and detection were performed at 30 °C and 214nm using the following operating parameters: Time (min)
100
Figure 2. RP-HPLC Chromatogram. Two 280nm traces (red & green) for a wheat gluten hydrolysate fractionation are shown.
9.00E+06
Viable Cells/mL
Assays were counted using a Vi-CELLTM XR (Beckman Coulter, CA, USA). Spent medium samples were collected for the analysis of nutrients/metabolites and IgG concentration. The secreted IgG was measured by an Octet QK (ForteBio, Inc., Menlo Park, CA, USA) using Protein A biosensors.
50
Minutes
Culture media The media used in this study are all proprietary SAFC Biosciences formulations that are chemically defined or prepared without hydrolysates. SAFC Biosciences’ imMEDIAte ADVANTAGE™ Small Volume Media Program prepared all media. Cell Growth and Recombinant Protein Production Assays The cells were routinely cultured in suspension in shaker flasks and were used to seed experiments conducted in duplicate 50mL (30mL working volume) disposable TPP Bioreactor tubes (Techno Plastic Products AG, Switzerland). Initial cell density was 200,000 viable cells/mL. The cells were cultured in a Multitron incubator (Infors HT, Switzerland) at 37 °C, 5% CO2 and 200rpm shaker speed.
0
60.0% 50.0% 40.0%
5
30.0%
7
20.0% 10.0%
1.00E+06 Feed
0.00E+00 0
1
2
0.0% 3
4
5
6
7
Hydrolysate 2g/L
8
Day
Base + Compound
Base Supplement
Hydrolysate Fractionation Figure 5. Growth Curve for Compound Screening. In this example, a single compound identified from a hydrolysate fraction showed a positive response for cell growth when fed with the nutritive “Base” supplement with the CHO-IgG1 cell line.
The primary chromatography method (TFA Separation) consisted of mobile phase A (0.1% trifluoroacetic acid in HPLC grade water) and mobile phase B (0.1% TFA in acetonitrile). Separation of the hydrolysate components was performed using a 300 minute linear gradient from 0 to 30% B with a 30 minute hold at 30% B. The flow rate was 5mL/min and the column temperature was maintained at 25 °C. The injection volume was 5ml of a 200g/L solution of the representative hydrolysate (soy, wheat gluten, yeast extract, and animal tissue).
The fractions were cell culture tested as outlined above (feeding on Day 2). They were fed at an amount that would be equivalent to the representative quantity in 2g/L of the standard hydrolysate.
500
8.0E+06
No Hydrolysate 7.0E+06
Hydrolysate Control 400
6.0E+06
CD Hydrol. Fusion 1x
No Hydrolysate Viable Cells/mL
The hydrolysate fractions were collected at one-minute intervals with a fraction collector and then pooled in five-minute intervals to simplify testing (66 fractions compared to 330). The samples were frozen in a -70 °C freezer and then lyophilized to remove the solvents. After lyophilization the fractions were resuspended in 5mL water so they would be compatible with cell culture testing and mass spectrometry.
Figure 6. Normalized IgG productivity from Compound Screening. In this example, a single compound identified from a hydrolysate fraction showed a positive response for IgG production when fed with the nutritive “Base” supplement with the CHO-IgG1 cell line. Data is normalized to the “Base” supplement.
5.0E+06
Hydrolysate Control
4.0E+06 CD Hydrol. Fusion 1x 3.0E+06
mg/L IgG
The Reverse Phase HPLC system consisted of a LC-6A separation module (Shimadzu, Japan) equipped with a binary gradient with high pressure mixing and a Shimadzu SPD-6AV UV detector. A preparative C18 column (2.5 cm x 22.5 cm) was used for the separation of the hydrolysates. Data collection and processing were performed using the Class-VP Chromatography Data System.
300
200
2.0E+06 1.0E+06
100
0.0E+00 7
8
9
10
11
0 Max IgG
3.0E+06 2.5E+06 2.0E+06 1.5E+06 1.0E+06 5.0E+05 Feed
1
2
3
4
5 6 Day
7
8
9
10
11
Figure 9. Growth Curve for Fed-Batch Use. A fed-batch study was performed with CHO-IgG2 in EX-CELLTM CD CHO Fusion (catalog #14365C). The maximum cell density for the CD Hydrolysate Fusion is very similar to the hydrolysate controls.
• Reverse Phase HPLC fractionation of four hydrolysate types and subsequent screening with CHO cells led to the identification of active fractions. Further analytical analysis of these fractions led to the discovery of important factors that contribute to the effect seen with hydrolysates.
CEX Product Quality Analysis
• A new supplement was created based on the work above, EX-CELLTM CD Hydrolysate Fusion. It is supplied as a 20x liquid (product # 14700C) or powder (product # 24700C). It can be used as an alternative to hydrolysates in any part of a CHO cell culture process. It also has application with other cell lines, such as NS0 and SP2/0 (data not shown).
50 Hydrolysate Process
45
CD Hydrolysate Fusion Process
% of Peak Area
40 35 30 25 20 15 10 5 0 Acidic
Peak 1
Peak 2
Peak 3
Basic
Figure 11. Product Quality Analysis of IgG produced by CHO-IgG1. A CEX-HPLC method was used to look at product quality difference between IgG produced by a process using hydrolysates and the CD Hydrolysate Fusion. Overall, the profiles are very similar.
www.safcbiosciences.com
CD Hydrolysate Fusion 1x
Glucose Only Soy UF 2g/L Soy UF 4g/L Yeast Extract 2g/L Yeast Extract 4g/L CD Hydrol. Fusion 1x
3.5E+06
Yeast Extract 4g/L
4.0E+06
1400 1300 1200 1100 1000 900 800 700 600 500 400 300 200 100 0 Yeast Extract 2g/L
4.5E+06
Conclusions
71818-25465 0059
Figure 8. Maximum IgG Production for Batch Medium Use. In this example with the CHO-IgG1 cell line, an medium that was dependent on hydrolysates was made into a CD formulation with the addition of the EX-CELLTM CD Hydrolysate Fusion. IgG productivity performance was virtually identical to the undefined control.
5.0E+06
0
• EX-CELLTM CD Hydrolysate Fusion is designed to perform equivalently as compared to undefined hydrolysates. In most instances this goal is met or exceeded.
6
Figure 7. Growth Curve for Batch Medium Use. In this example with the CHO-IgG1 cell line, an medium that was dependent on hydrolysates was made into a CD formulation with the addition of the EX-CELLTM CD Hydrolysate Fusion. Growth performance was slightly improved with the use of this product.
0.0E+00
• By analyzing the amino acid, vitamin, and metal composition of several protein hydrolysates and with subsequent optimization, a hydrolysate “Base” supplement was designed to encompass much of the nutritive functions of hydrolysates. The use of this supplement helped to elucidate the unknown positive effectors contained within hydrolysates.
5
Soy UF 2g/L
All identified active compounds and those contained in the basal supplement were further optimized with multiple CHO lines to develop the EX-CELLTM CD Hydrolysate Fusion product. The design of this product allows it to be used in all CHO applications that currently utilize undefined hydrolysates. Example results for batch medium use are shown in Figures 7 & 8. Figures 9 & 10 demonstrate how the EX-CELLTM CD Hydrolysate Fusion can be used in a fed-batch process. Lastly, in Figure 11 a CEX profile shows that only very minor product quality differences are seen when switching from hydrolysates to a chemically defined process with EX-CELLTM CD Hydrolysate Fusion.
4
Glucose Only
Fractions that gave a positive growth and/or productivity response were subjected to further analytical analysis to determine the components contained within. Orthogonal separation techniques and mass spectrometry methods were employed for this analysis (details and data not shown). Chemically defined versions of the identified compounds were subsequently screened for activity using the same CHO cell culture assay outlined above. An example of a compound that was identified to have a positive effect is shown in Figures 5 & 6.
3
mg/L IgG
Fractions with improved growth or productivity responses were identified from each hydrolysate. Examples of four fractions from a Yeast Extract are shown in Figures 3 & 4. Significant stimulatory effects are seen with addition on these fractions.
2
Day
Average Viable Cells/mL
The process developed for this project is outlined in Figure 1. Four different hydrolysate types were selected for analysis (soy, wheat gluten, yeast extract, and meat). The goal was to identify the unknown active components contained within these hydrolysates, and Reverse Phase HPLC fractionation was used to aid with the separation of these compounds. In Figure 2, two overlaid chromatograms (at 280nm) demonstrate the reproducibility of the separation.
1
Soy UF 4g/L
0
Discussion
Figure 10. Maximum IgG Production for Fed-Batch Use. A fed-batch study was performed with CHO-IgG2 in EX-CELLTM CD CHO Fusion (catalog #14365C). The maximum IgG productivity levels for the CD Hydrolysate Fusion conditions are higher than the 2g/L hydrolysate controls and within 10% of the 4g/L hydrolysate controls.
Results
Abstract Protein hydrolysates are commonly utilized in cell culture processes either as a component of a complete medium formulation or as part of a fed-batch bioreactor process. It is well documented that hydrolysates can have a substantial positive impact on cell growth and/or protein production. Given the undefined nature and the lot-to-lot variability associated with hydrolysates, there exists a need to mitigate these risks with a chemically defined (CD) alternative that can maintain the desired performance. In order to create a CD alternative to protein hydrolysates, a two-pronged approach was taken to capture the associated effects. The first experimental path utilized standard analytical techniques to identify the nutritive components supplied by hydrolysates (i.e. amino acids, vitamins). A “basal” supplement was designed to supply these nutrients, as this is a significant portion of the functionality of hydrolysates. The second path was based upon Reverse Phase HPLC fractionation of multiple different hydrolysate types. Cell culture screening with Chinese Hamster Ovary (CHO) cells led to the identification of “bioactive” fractions. Subsequent identification of the components contained within the fractions led a greater understanding of the effects of hydrolysates. The learnings from both approaches were utilized to generate a chemically defined supplement, EX-CELLTM CD Hydrolysate Fusion, which is capable of replacing the functions of hydrolysates in many CHO cell systems.
Overlaid Traces 062804.001, Chan A 062904.002, Chan A
Screen Hydrolysates
750
750
500
500
RP-HPLC Fractionate & Lyophilize
Screen Fractions (+ Base) with CHO-IgG1
m V o l t s
m V o l t s
ID Fraction Components & Source CD Versions 250
250
Screen Components with Multiple CHO Lines
Materials and Methods
0
Sigma-Aldrich Corporation (St. Louis, MO, USA) supplied all chemicals, media, and solutions unless otherwise stated.
Combine and Optimize with Base Supplement for Final Product
Cell Lines Cell lines CHO-IgG1 and CHO-IgG2 are proprietary Chinese Hamster Ovary (CHO) clones expressing recombinant antibodies.
0
Figure 1. Process Flow Chart
Flow Rate (mL/min)
0 2 10 10.5 15.1 15.3
95 95 60 0 0 95
5 5 40 100 100 5
1.2 1.2 1.2 1.2 1.2 1.2
For fed-batch assays the TPP tubes were fed with a one-time bolus on/around Day 2, depending on the cell density (0.8 to 1.2e6 viable cells/mL). The feed included Glucose (6g/L) to prevent carbon source limitation and a “basal” supplement to provide much of the nutritive function of hydrolysates. It was designed to contain only compounds seen in the hydrolysates or in a standard medium formulation (amino acids, trace elements, and vitamins). The basal supplement is added to help elucidate the unknown positive effectors by supplying known nutritive compounds. This supplement continually evolved over the course of the project as more was learned about the action of hydrolysates. As new compounds were identified they were titrated into the “Base” supplement. This process continued until the final product goals were achieved.
300
350
100.0%
80.0%
1
2
3
4
5
6
7
8
Day
-40.0%
Figure 3. Growth Curve for Fraction Screening. Two fractions (of 66) showed a positive response for growth (Fractions A & D) when fed in addition to the nutritive “Base” supplement with the CHO-IgG1 cell line.
Fraction D
0
0.0%
-20.0%
Fraction C
Feed
0.00E+00
7
Fraction B
1.00E+06
4
20.0%
Fraction A
2.00E+06
40.0%
Yeast Extract 2g/L
3.00E+06
Base Supplement
4.00E+06
60.0%
No Feed
5.00E+06
% Change from Base Control
No Feed Base Supplement Yeast Extract 2g/L Base + Fraction A Base + Fraction B Base + Fraction C Base + Fraction D
6.00E+06
Figure 4. Normalized IgG productivity from Fraction Screening. Four fractions (of 66) showed a positive response for IgG production when fed in addition to the nutritive “Base” supplement with the CHO-IgG1 cell line. Data is normalized to the “Base” supplement.
1.00E+07
70.0%
9.00E+06 8.00E+06 7.00E+06 6.00E+06
Hydrolysate 2g/L
5.00E+06
Base Supplement
4.00E+06
Base + Compound
3.00E+06 2.00E+06
% Change from Base Control
%B
250
200
7.00E+06
Viable Cells/mL
%A
150
8.00E+06
Product quality was measured by Cation Exchange HPLC. MAb charge variants were separated by HPLC by injecting 50ug of purified MAb (1mg/mL) into a 4.6mm × 250mm ProPac SCX-10 strong cation-exchange column (Dionex, CA, USA) connected to an Alliance HPLC with PDA detection (Waters, MA, USA). Mobile phase A consisted of 10mM sodium phosphate (pH 6.0±0.2). Mobile phase B consisted of 10mM sodium phosphate and 500mM sodium chloride (pH 7.5±0.2). Separation and detection were performed at 30 °C and 214nm using the following operating parameters: Time (min)
100
Figure 2. RP-HPLC Chromatogram. Two 280nm traces (red & green) for a wheat gluten hydrolysate fractionation are shown.
9.00E+06
Viable Cells/mL
Assays were counted using a Vi-CELLTM XR (Beckman Coulter, CA, USA). Spent medium samples were collected for the analysis of nutrients/metabolites and IgG concentration. The secreted IgG was measured by an Octet QK (ForteBio, Inc., Menlo Park, CA, USA) using Protein A biosensors.
50
Minutes
Culture media The media used in this study are all proprietary SAFC Biosciences formulations that are chemically defined or prepared without hydrolysates. SAFC Biosciences’ imMEDIAte ADVANTAGE™ Small Volume Media Program prepared all media. Cell Growth and Recombinant Protein Production Assays The cells were routinely cultured in suspension in shaker flasks and were used to seed experiments conducted in duplicate 50mL (30mL working volume) disposable TPP Bioreactor tubes (Techno Plastic Products AG, Switzerland). Initial cell density was 200,000 viable cells/mL. The cells were cultured in a Multitron incubator (Infors HT, Switzerland) at 37 °C, 5% CO2 and 200rpm shaker speed.
0
60.0% 50.0% 40.0%
5
30.0%
7
20.0% 10.0%
1.00E+06 Feed
0.00E+00 0
1
2
0.0% 3
4
5
6
7
Hydrolysate 2g/L
8
Day
Base + Compound
Base Supplement
Hydrolysate Fractionation Figure 5. Growth Curve for Compound Screening. In this example, a single compound identified from a hydrolysate fraction showed a positive response for cell growth when fed with the nutritive “Base” supplement with the CHO-IgG1 cell line.
The primary chromatography method (TFA Separation) consisted of mobile phase A (0.1% trifluoroacetic acid in HPLC grade water) and mobile phase B (0.1% TFA in acetonitrile). Separation of the hydrolysate components was performed using a 300 minute linear gradient from 0 to 30% B with a 30 minute hold at 30% B. The flow rate was 5mL/min and the column temperature was maintained at 25 °C. The injection volume was 5ml of a 200g/L solution of the representative hydrolysate (soy, wheat gluten, yeast extract, and animal tissue).
The fractions were cell culture tested as outlined above (feeding on Day 2). They were fed at an amount that would be equivalent to the representative quantity in 2g/L of the standard hydrolysate.
500
8.0E+06
No Hydrolysate 7.0E+06
Hydrolysate Control 400
6.0E+06
CD Hydrol. Fusion 1x
No Hydrolysate Viable Cells/mL
The hydrolysate fractions were collected at one-minute intervals with a fraction collector and then pooled in five-minute intervals to simplify testing (66 fractions compared to 330). The samples were frozen in a -70 °C freezer and then lyophilized to remove the solvents. After lyophilization the fractions were resuspended in 5mL water so they would be compatible with cell culture testing and mass spectrometry.
Figure 6. Normalized IgG productivity from Compound Screening. In this example, a single compound identified from a hydrolysate fraction showed a positive response for IgG production when fed with the nutritive “Base” supplement with the CHO-IgG1 cell line. Data is normalized to the “Base” supplement.
5.0E+06
Hydrolysate Control
4.0E+06 CD Hydrol. Fusion 1x 3.0E+06
mg/L IgG
The Reverse Phase HPLC system consisted of a LC-6A separation module (Shimadzu, Japan) equipped with a binary gradient with high pressure mixing and a Shimadzu SPD-6AV UV detector. A preparative C18 column (2.5 cm x 22.5 cm) was used for the separation of the hydrolysates. Data collection and processing were performed using the Class-VP Chromatography Data System.
300
200
2.0E+06 1.0E+06
100
0.0E+00 7
8
9
10
11
0 Max IgG
3.0E+06 2.5E+06 2.0E+06 1.5E+06 1.0E+06 5.0E+05 Feed
1
2
3
4
5 6 Day
7
8
9
10
11
Figure 9. Growth Curve for Fed-Batch Use. A fed-batch study was performed with CHO-IgG2 in EX-CELLTM CD CHO Fusion (catalog #14365C). The maximum cell density for the CD Hydrolysate Fusion is very similar to the hydrolysate controls.
• Reverse Phase HPLC fractionation of four hydrolysate types and subsequent screening with CHO cells led to the identification of active fractions. Further analytical analysis of these fractions led to the discovery of important factors that contribute to the effect seen with hydrolysates.
CEX Product Quality Analysis
• A new supplement was created based on the work above, EX-CELLTM CD Hydrolysate Fusion. It is supplied as a 20x liquid (product # 14700C) or powder (product # 24700C). It can be used as an alternative to hydrolysates in any part of a CHO cell culture process. It also has application with other cell lines, such as NS0 and SP2/0 (data not shown).
50 Hydrolysate Process
45
CD Hydrolysate Fusion Process
% of Peak Area
40 35 30 25 20 15 10 5 0 Acidic
Peak 1
Peak 2
Peak 3
Basic
Figure 11. Product Quality Analysis of IgG produced by CHO-IgG1. A CEX-HPLC method was used to look at product quality difference between IgG produced by a process using hydrolysates and the CD Hydrolysate Fusion. Overall, the profiles are very similar.
www.safcbiosciences.com
CD Hydrolysate Fusion 1x
Glucose Only Soy UF 2g/L Soy UF 4g/L Yeast Extract 2g/L Yeast Extract 4g/L CD Hydrol. Fusion 1x
3.5E+06
Yeast Extract 4g/L
4.0E+06
1400 1300 1200 1100 1000 900 800 700 600 500 400 300 200 100 0 Yeast Extract 2g/L
4.5E+06
Conclusions
71818-25465 0059
Figure 8. Maximum IgG Production for Batch Medium Use. In this example with the CHO-IgG1 cell line, an medium that was dependent on hydrolysates was made into a CD formulation with the addition of the EX-CELLTM CD Hydrolysate Fusion. IgG productivity performance was virtually identical to the undefined control.
5.0E+06
0
• EX-CELLTM CD Hydrolysate Fusion is designed to perform equivalently as compared to undefined hydrolysates. In most instances this goal is met or exceeded.
6
Figure 7. Growth Curve for Batch Medium Use. In this example with the CHO-IgG1 cell line, an medium that was dependent on hydrolysates was made into a CD formulation with the addition of the EX-CELLTM CD Hydrolysate Fusion. Growth performance was slightly improved with the use of this product.
0.0E+00
• By analyzing the amino acid, vitamin, and metal composition of several protein hydrolysates and with subsequent optimization, a hydrolysate “Base” supplement was designed to encompass much of the nutritive functions of hydrolysates. The use of this supplement helped to elucidate the unknown positive effectors contained within hydrolysates.
5
Soy UF 2g/L
All identified active compounds and those contained in the basal supplement were further optimized with multiple CHO lines to develop the EX-CELLTM CD Hydrolysate Fusion product. The design of this product allows it to be used in all CHO applications that currently utilize undefined hydrolysates. Example results for batch medium use are shown in Figures 7 & 8. Figures 9 & 10 demonstrate how the EX-CELLTM CD Hydrolysate Fusion can be used in a fed-batch process. Lastly, in Figure 11 a CEX profile shows that only very minor product quality differences are seen when switching from hydrolysates to a chemically defined process with EX-CELLTM CD Hydrolysate Fusion.
4
Glucose Only
Fractions that gave a positive growth and/or productivity response were subjected to further analytical analysis to determine the components contained within. Orthogonal separation techniques and mass spectrometry methods were employed for this analysis (details and data not shown). Chemically defined versions of the identified compounds were subsequently screened for activity using the same CHO cell culture assay outlined above. An example of a compound that was identified to have a positive effect is shown in Figures 5 & 6.
3
mg/L IgG
Fractions with improved growth or productivity responses were identified from each hydrolysate. Examples of four fractions from a Yeast Extract are shown in Figures 3 & 4. Significant stimulatory effects are seen with addition on these fractions.
2
Day
Average Viable Cells/mL
The process developed for this project is outlined in Figure 1. Four different hydrolysate types were selected for analysis (soy, wheat gluten, yeast extract, and meat). The goal was to identify the unknown active components contained within these hydrolysates, and Reverse Phase HPLC fractionation was used to aid with the separation of these compounds. In Figure 2, two overlaid chromatograms (at 280nm) demonstrate the reproducibility of the separation.
1
Soy UF 4g/L
0
Discussion
Figure 10. Maximum IgG Production for Fed-Batch Use. A fed-batch study was performed with CHO-IgG2 in EX-CELLTM CD CHO Fusion (catalog #14365C). The maximum IgG productivity levels for the CD Hydrolysate Fusion conditions are higher than the 2g/L hydrolysate controls and within 10% of the 4g/L hydrolysate controls.
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