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Optimizing Overall Manufacturing
System Performance through the
Use of Animal-free Cell Culture
Supplements
KiriLynn Svay, Jeff Rosenbloom,
Delyan Rusev, and Matt Croughan

Amgen Bioprocessing Center
Keck Graduate Institute, Claremont, CA

BioManufacturing Summit, January 27, 2010
25 Years of Progress in Clinical Protein Production from Recombinant
                                                                     CHO Cells in Batch and Fedbatch Culture
                                                            Professor Matt Croughan, Keck Graduate Institute, Claremont, CA
                                          100000
                                                                       New to Technology           >2 Years Experience
Product Concentration at Harvest (mg/L)




                                           10000
                                                                          0.205x
                                                             y = 9.941e
                                                               2
                                                              R = 0.7939
                                            1000



                                             100



                                              10
                                                                                                                        0.3477x
                                                                                                           y = 0.082e
                                                                                                              2
                                                                                                            R = 0.9593
                                               1



                                             0.1
                                                   0               5               10          15           20                    25   30
                                                                                        Years Since 1980
Analysis of Variance for Experienced Firms
                                                                Professor M Croughan, Keck Graduate Institute
                                                                           att
                                                  100.0
Percent CV for Product Concentration at Harvest




                                                   90.0

                                                   80.0
                                                                      y = -0.0804x + 50.266
                                                   70.0                      2
                                                                            R = 0.0005
                                                   60.0

                                                   50.0

                                                   40.0

                                                   30.0

                                                   20.0

                                                   10.0

                                                    0.0
                                                          0      5          10           15           20        25   30
                                                                                  Years Since 1980
“..It” flows down hill

                    Consequences of
                      10g/L
   Cell
  Culture
  10g/L
                    • Cell density (volume)
                    • Cell debris
                    • Product Mass
                    • HMW
Purification
                     •Drug
                     Product
                     Development
                                     •From Jon Coffman
Fed-batch cell culture in 1990
Cell culture strategies to optimize
overall system performance
 Consistent and sufficient titers
 ◦ Every process, every time
 Shorter culture duration
 ◦ Higher vol. productivity, lower contamination risk
 Reduced cell death
 ◦ Lower degradative enzyme levels
 ◦ Lower HCP and debris loads downstream
 Improved downstream processing
 ◦ Higher yields or fewer/simpler steps
 Improved product quality and/or stability
Recombinant Human Serum Albumin (rHSA) made in
an animal free production host by InVitria
(www.InVitria.com)
Roles of Albumin in Cell Culture:
◦   Binding and transport mechanism
         Supply of Lipids
         Vitamins
         Hormones
◦   Buffering agent
◦   Detoxifying agent
◦   Protectant from shear
Recombinant Lactoferrin is a naturally occurring iron-
binding protein that was made in an animal free
production system by InVitria (www.InVitria.com)
Advantages of Lactoferrin in Cell Culture
◦ Can be used as a growth factor
◦ Protects against oxidation due to Fe3+ ions
◦ Microbial deterrent
◦ Iron transport to cells
Impact of Cellastim/Lacromin Supplements
  on CHO Cell Culture in CD medium
 % Improvement, Supplemented/Control
  (data from five separate experiments)

 Peak viable cell density (VCD)
 ◦ Average: 28%, Range: 2 - 47%

 Peak titer (product concentration)
 ◦ Average: 40%, Range: 2 – 88%

 Volumetric productivity at peak titer
 ◦ Average: 69%, Range: 3 – 162%
Batch Shake Flask Results
                                                Viable Cell Density
                                 Control                         125:125 mg/L Cellastim:Lacromin
                                 250 mg/L Cellastim              500 mg/L Cellastim
                                                                                                                                   Cells supplemented with 250
                         8                                                                                                         mg/L Cellastim or 500 mg/L
                                                                                                                                   Cellastim reached the greatest
VCD (million cells/mL)




                         7
                         6
                         5
                                                                                                                                   cell density in batch shake
                         4                                                                                                         flasks
                         3
                         2
                         1
                         0
                             0     1       2     3    4   5          6            7              8       9       10       11
                                                              Days
                                                                                                                           % Viability
                                                                                             Control                                       125:125 mg/L Cellastim:Lacromin
                                                                                             250 mg/L Cellastim                            500 mg/L Cellastim
                                                                                       100
                                  This data was very                                    90
                                                                                        80
                                  similar to the InVitria                               70
                                                                         % Viability




                                                                                        60
                                  studies using the same                                50
                                                                                        40
                                  conditions                                            30
                                                                                        20
                                                                                             0       1       2        3        4     5          6   7    8      9    10      11
                                                                                                                                         Days
Bioreactor Density Trends
         Similar trends appeared in the fed batch bioreactors

         •Better growth than un-supplemented cells
         •Slower decline rate at end of culture

                                      Bioreactor Viable Cell Densities and % Viability
                                       125:125 mg/L Cellastim:Lacromin VCD          Control VCD
                                       125:125 mg/L Cellastim:Lacromin %VIA         Control %VIA
                         12                                                                                          100
                                                                                                                     95
                         10
                                                                                                                     90
VCD (million cells/mL)




                                                                                                                     85
                         8
                                                                                                                     80




                                                                                                                           %Viability
                         6                                                                                           75
                                                                                                                     70
                         4
                                                                                                                     65
                                                                                                                     60
                         2
                                                                                                                     55
                         0                                                                                           50
                              0   1        2        3       4        5          6          7       8   9   10   11
                                                                         Days
Specific Glucose and Lactate Trends
                           Specific Glucose Consumption in Fed
                                     Batch Bioreactors                                                                                            In fed batch bioreactors there
                                         Control         125:125 mg/L Cellastim:Lacromin                                                          was decreased sp. glucose
                          0.6                                                                                                                     consumption and decreased
sp. Glucose Consumption




                          0.5                                                                                                                     sp. lactate production from
       (ng/cell*day)




                          0.4
                                                                                                                                                  cells supplemented with
                          0.3
                                                                                                                                                  Cellastim and Lacromin
                          0.2
                          0.1
                           0
                                         0-Prefeed               Post Feed - 7                                               7-10

                                                                                                                                  Specific Lactate Production in Fed
                                                                                                                                           Batch Bioreactors
                                     pH vs.Time
                                                                                                                                      Control      125:125 mg/L Cellastim:Lacromin
                                                                                     sp. Lactate Production (ng/cell*day)


                                    125:125 pH            Control pH
                                                                                                                            1.4
                7.6
                                                                                                                            1.2
                7.4
                7.2                                                                                                          1
pH




                  7                                                                                                         0.8
                6.8                                                                                                         0.6
                6.6
                                                                                                                            0.4
                           0    1    2    3   4      5   6   7   8   9   10 11
                                                                                                                            0.2
                                                     Days
                                                                                                                             0
                                                                                                                                      0-Prefeed           Post Feed - 7              7-10
Glucose Trends EXP4 Bioreactors
                                             Control   250 mg/L Cellastim
                      10.00
                       9.00
                       8.00
Concentration (g/L)




                       7.00
                       6.00
                       5.00
                       4.00
                       3.00
                       2.00
                       1.00
                       0.00
                              0      2   4        6    8         10   12                          14         16       18
                                                           Day

                                                                                                         Lactate Trends EXP4 Bioreactors
                                                                                                                               Control       250 mg/L Cellastim
                                                                                                  4.50
                                                                                                  4.00
                                                                            Concentration (g/L)



                                                                                                  3.50
                                                                                                  3.00
                                                                                                  2.50
                                                                                                  2.00
                                                                                                  1.50
                                                                                                  1.00
                                                                                                  0.50
                                                                                                  0.00
                                                                                                         0        2        4        6    8          10     12     14   16   18
                                                                                                                                              Day
pH Trends EXP4 Bioreactors
                         Control       250 mg/L Cellastim
     7.15
     7.10
     7.05
     7.00
     6.95
pH




     6.90
     6.85
     6.80
     6.75
     6.70
            0    2   4        6    8          10     12         14                  16       18
                                        Day

                                                                                  Osmolality Trends EXP4 Bioreactors
                                                                                                      Control   250 mg/L Cellastim
                                                                                500.00

                                                                                450.00
                                                            Osmolality (mOsm)



                                                                                400.00

                                                                                350.00

                                                                                300.00

                                                                                250.00

                                                                                200.00
                                                                                         0        2   4     6   8         10   12    14   16   18
                                                                                                                    Day
Purification Step Using GE-ÄKTApilot

                                                                         a-IL-8




                                                                         Column
  Column               Sample Loading/                      Eluted
                                             Column Wash                    Re-
Equilibration      Flow-Through Collection                 Fractions
                                                                       equilibration
SDS-PAGE
                               Coomassie Blue Staining
        1        2         3        4        5        6        7        8        9          10
75kD

50kD




25kD




  1    Protein Marker
  2    Supernatant: 125/125 mg/L Cellastim/Lacromin
       Purified a-IL-8 by Protein A Column from supernatant containing 125/125 mg/L
  3
       Cellastim/Lacromin
  4    UF Purified a-IL-8 of #3 product
  5    Supernatant: 250 mg/L Cellastim
  6    Purified a-IL-8 by Protein A Column from supernatant containing 250 mg/L Cellastim
  7    UF Purified a-IL-8 of #6 product
  8    Supernatant: 500 mg/L Cellastim
  9    Purified a-IL-8 by Protein A Column from supernatant containing 500 mg/L Cellastim
 10    UF Purified a-IL-8 of #9 product
SDS-PAGE
                                            Silver Staining
           1        2        3        4        5        6        7        8        9        10

75kD
50kD




25kD




1      Protein Marker
2      Supernatant: 250 mg/L Cellastim
3      Flow – through fraction (time point:10-11min) of #2 run through Protein A Column
4      Purified a-IL-8 by Protein A Column from supernatant containing 250 mg/L Cellastim
5      Waste Fraction (time point: 59-60min) of #2 run through Protein A Column
6      Supernatant from Protein A Column after column disassembling
7      Supernatant: 125/125 mg/L Cellastim/Lacromin
8      Flow – through fraction (time point:10-11min) of # 7 run through Protein A Column
       Purified a-IL-8 by Protein A Column from supernatant containing 125/125 mg/L
9
       Cellastim/Lacromin
10     Waste Fraction (time point: 47-48min) of # 7 run through Protein A Column
ELISA
                                                                    IgG Standard Curve
                                     1200



                                     1000



                                      800




                     Titer (ng/mL)
                                                                      y = 197.13e1.28x
                                                                        R2 = 0.9936
                                      600



                                      400



                                      200



                                        0
                                         0.00       0.20     0.40      0.60       0.80   1.00     1.20   1.40

                                                                         OD 450nm


                                                Amount of IgG loaded                Amount of IgG recovered
     Sample                                                                                                     % Recovered
                                                    (milligrams)                         (milligrams)

   CONTROL
 0 mg/L Cellastim                                          784                                  328                41.8
 0 mg/L Lacromin

125mg/L Cellastim
                                                           619                                  321                51.9
125 mg/L Lacromin


250 mg/L Cellastim                                         537                                  315                58.7

500mg/L Cellastim                                          449                                  313                69.7
Summary
 Optimize overall system performance
 Cellastim/Lacromin supplementation
 ◦   Higher product concentration (titers)
 ◦   Higher specific productivity
 ◦   More efficient glucose metabolism
 ◦   Reduced cell death
 ◦   Higher consistency
 ◦   Equal or improved yields at protein A capture
      Supplements in flow through
      Reduced non-specific losses of MAb

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Bio manufacturing summit croughan et al jan 2010

  • 1. Optimizing Overall Manufacturing System Performance through the Use of Animal-free Cell Culture Supplements KiriLynn Svay, Jeff Rosenbloom, Delyan Rusev, and Matt Croughan Amgen Bioprocessing Center Keck Graduate Institute, Claremont, CA BioManufacturing Summit, January 27, 2010
  • 2. 25 Years of Progress in Clinical Protein Production from Recombinant CHO Cells in Batch and Fedbatch Culture Professor Matt Croughan, Keck Graduate Institute, Claremont, CA 100000 New to Technology >2 Years Experience Product Concentration at Harvest (mg/L) 10000 0.205x y = 9.941e 2 R = 0.7939 1000 100 10 0.3477x y = 0.082e 2 R = 0.9593 1 0.1 0 5 10 15 20 25 30 Years Since 1980
  • 3. Analysis of Variance for Experienced Firms Professor M Croughan, Keck Graduate Institute att 100.0 Percent CV for Product Concentration at Harvest 90.0 80.0 y = -0.0804x + 50.266 70.0 2 R = 0.0005 60.0 50.0 40.0 30.0 20.0 10.0 0.0 0 5 10 15 20 25 30 Years Since 1980
  • 4.
  • 5. “..It” flows down hill Consequences of 10g/L Cell Culture 10g/L • Cell density (volume) • Cell debris • Product Mass • HMW Purification •Drug Product Development •From Jon Coffman
  • 7. Cell culture strategies to optimize overall system performance Consistent and sufficient titers ◦ Every process, every time Shorter culture duration ◦ Higher vol. productivity, lower contamination risk Reduced cell death ◦ Lower degradative enzyme levels ◦ Lower HCP and debris loads downstream Improved downstream processing ◦ Higher yields or fewer/simpler steps Improved product quality and/or stability
  • 8. Recombinant Human Serum Albumin (rHSA) made in an animal free production host by InVitria (www.InVitria.com) Roles of Albumin in Cell Culture: ◦ Binding and transport mechanism Supply of Lipids Vitamins Hormones ◦ Buffering agent ◦ Detoxifying agent ◦ Protectant from shear
  • 9. Recombinant Lactoferrin is a naturally occurring iron- binding protein that was made in an animal free production system by InVitria (www.InVitria.com) Advantages of Lactoferrin in Cell Culture ◦ Can be used as a growth factor ◦ Protects against oxidation due to Fe3+ ions ◦ Microbial deterrent ◦ Iron transport to cells
  • 10.
  • 11. Impact of Cellastim/Lacromin Supplements on CHO Cell Culture in CD medium % Improvement, Supplemented/Control (data from five separate experiments) Peak viable cell density (VCD) ◦ Average: 28%, Range: 2 - 47% Peak titer (product concentration) ◦ Average: 40%, Range: 2 – 88% Volumetric productivity at peak titer ◦ Average: 69%, Range: 3 – 162%
  • 12. Batch Shake Flask Results Viable Cell Density Control 125:125 mg/L Cellastim:Lacromin 250 mg/L Cellastim 500 mg/L Cellastim Cells supplemented with 250 8 mg/L Cellastim or 500 mg/L Cellastim reached the greatest VCD (million cells/mL) 7 6 5 cell density in batch shake 4 flasks 3 2 1 0 0 1 2 3 4 5 6 7 8 9 10 11 Days % Viability Control 125:125 mg/L Cellastim:Lacromin 250 mg/L Cellastim 500 mg/L Cellastim 100 This data was very 90 80 similar to the InVitria 70 % Viability 60 studies using the same 50 40 conditions 30 20 0 1 2 3 4 5 6 7 8 9 10 11 Days
  • 13. Bioreactor Density Trends Similar trends appeared in the fed batch bioreactors •Better growth than un-supplemented cells •Slower decline rate at end of culture Bioreactor Viable Cell Densities and % Viability 125:125 mg/L Cellastim:Lacromin VCD Control VCD 125:125 mg/L Cellastim:Lacromin %VIA Control %VIA 12 100 95 10 90 VCD (million cells/mL) 85 8 80 %Viability 6 75 70 4 65 60 2 55 0 50 0 1 2 3 4 5 6 7 8 9 10 11 Days
  • 14. Specific Glucose and Lactate Trends Specific Glucose Consumption in Fed Batch Bioreactors In fed batch bioreactors there Control 125:125 mg/L Cellastim:Lacromin was decreased sp. glucose 0.6 consumption and decreased sp. Glucose Consumption 0.5 sp. lactate production from (ng/cell*day) 0.4 cells supplemented with 0.3 Cellastim and Lacromin 0.2 0.1 0 0-Prefeed Post Feed - 7 7-10 Specific Lactate Production in Fed Batch Bioreactors pH vs.Time Control 125:125 mg/L Cellastim:Lacromin sp. Lactate Production (ng/cell*day) 125:125 pH Control pH 1.4 7.6 1.2 7.4 7.2 1 pH 7 0.8 6.8 0.6 6.6 0.4 0 1 2 3 4 5 6 7 8 9 10 11 0.2 Days 0 0-Prefeed Post Feed - 7 7-10
  • 15. Glucose Trends EXP4 Bioreactors Control 250 mg/L Cellastim 10.00 9.00 8.00 Concentration (g/L) 7.00 6.00 5.00 4.00 3.00 2.00 1.00 0.00 0 2 4 6 8 10 12 14 16 18 Day Lactate Trends EXP4 Bioreactors Control 250 mg/L Cellastim 4.50 4.00 Concentration (g/L) 3.50 3.00 2.50 2.00 1.50 1.00 0.50 0.00 0 2 4 6 8 10 12 14 16 18 Day
  • 16. pH Trends EXP4 Bioreactors Control 250 mg/L Cellastim 7.15 7.10 7.05 7.00 6.95 pH 6.90 6.85 6.80 6.75 6.70 0 2 4 6 8 10 12 14 16 18 Day Osmolality Trends EXP4 Bioreactors Control 250 mg/L Cellastim 500.00 450.00 Osmolality (mOsm) 400.00 350.00 300.00 250.00 200.00 0 2 4 6 8 10 12 14 16 18 Day
  • 17.
  • 18. Purification Step Using GE-ÄKTApilot a-IL-8 Column Column Sample Loading/ Eluted Column Wash Re- Equilibration Flow-Through Collection Fractions equilibration
  • 19. SDS-PAGE Coomassie Blue Staining 1 2 3 4 5 6 7 8 9 10 75kD 50kD 25kD 1 Protein Marker 2 Supernatant: 125/125 mg/L Cellastim/Lacromin Purified a-IL-8 by Protein A Column from supernatant containing 125/125 mg/L 3 Cellastim/Lacromin 4 UF Purified a-IL-8 of #3 product 5 Supernatant: 250 mg/L Cellastim 6 Purified a-IL-8 by Protein A Column from supernatant containing 250 mg/L Cellastim 7 UF Purified a-IL-8 of #6 product 8 Supernatant: 500 mg/L Cellastim 9 Purified a-IL-8 by Protein A Column from supernatant containing 500 mg/L Cellastim 10 UF Purified a-IL-8 of #9 product
  • 20. SDS-PAGE Silver Staining 1 2 3 4 5 6 7 8 9 10 75kD 50kD 25kD 1 Protein Marker 2 Supernatant: 250 mg/L Cellastim 3 Flow – through fraction (time point:10-11min) of #2 run through Protein A Column 4 Purified a-IL-8 by Protein A Column from supernatant containing 250 mg/L Cellastim 5 Waste Fraction (time point: 59-60min) of #2 run through Protein A Column 6 Supernatant from Protein A Column after column disassembling 7 Supernatant: 125/125 mg/L Cellastim/Lacromin 8 Flow – through fraction (time point:10-11min) of # 7 run through Protein A Column Purified a-IL-8 by Protein A Column from supernatant containing 125/125 mg/L 9 Cellastim/Lacromin 10 Waste Fraction (time point: 47-48min) of # 7 run through Protein A Column
  • 21. ELISA IgG Standard Curve 1200 1000 800 Titer (ng/mL) y = 197.13e1.28x R2 = 0.9936 600 400 200 0 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 OD 450nm Amount of IgG loaded Amount of IgG recovered Sample % Recovered (milligrams) (milligrams) CONTROL 0 mg/L Cellastim 784 328 41.8 0 mg/L Lacromin 125mg/L Cellastim 619 321 51.9 125 mg/L Lacromin 250 mg/L Cellastim 537 315 58.7 500mg/L Cellastim 449 313 69.7
  • 22. Summary Optimize overall system performance Cellastim/Lacromin supplementation ◦ Higher product concentration (titers) ◦ Higher specific productivity ◦ More efficient glucose metabolism ◦ Reduced cell death ◦ Higher consistency ◦ Equal or improved yields at protein A capture Supplements in flow through Reduced non-specific losses of MAb