Novel Approach to Chemically Defined Platform Medium and Feed Optimization for CHO Cell Culture

David (Xiaojian) Zhao, Richard Fike, Zhihua Xiao, Scott Jacobia, Robert Kenerson, Brian Horvath, Richard Hassett and Borka Naumovich
Abstract
Developing fed-batch media platforms with chemically defined base medium and feed for bioproduction cell lines is traditionally a difficult and time consuming process. Historically, strategies used have ranged from the simple iterative process of cell based assays and spent media analysis to highly complex DOE designs also requiring multiple iterations of cell based assays followed by data analysis. This methodology typically results in optimization that is highly clone specific. It has been challenging to translate this methodology and its results into a platform optimization system. Using multiple approaches and our proprietary AGTTM format, we have identified a way to develop well balanced chemically defined cell culture platforms consisting of base medium and concentrated feed formulation with sufficient diversity to support fed-batch optimization. This approach results in optimized base medium, feed formulation and fed batch process for both the high density growth and the recombinant protein production for many recombinant CHO cell lines expressing different proteins including IgG from different parental CHO cells.
1Invitrogen

Corporation 3175 Staley Road Grand Island, New York 14072 USA

Fed-batch in shake flasks

CHO CD A and B are based on the chemically defined batch media formulations (not the individual cells requirements), which facilitates their application in multiple cell lines. They were made highly concentrated, which enables small volume delivery. A simple experiment using a Full Factorial Design of Experiment (DOE) of the two components at three levels (9 culture conditions) supplemented on day 0 using shake flasks gave a optimized base medium for a specific cell line. Then the culture was fed with different feed combinations under different feeding strategies. This approach is a paradigm shift for fed-batch development that allows us to get good fed-batch protocols in very short time (less than a month). This simple strategy was successfully used to demonstrate the ability to develop a superior combination of base medium and feed strategy for various recombinant CHO cell lines including several IgG producing cell lines using the two feeds independently or as mixture. Additionally from the same data, the best platform media including base medium and feeds can be developed for multiple objectives such as high peak cell density, high peak titer or higher titer in long culture (Figure 1-3). Cell culture maintenance, scale-up and production media platform can be developed and optimized simultaneously.
Fed-batch culture using CD OptiCHO as base medium Recombinant IgG Clone #1
CD OptiCHO batch Fed with 15% A on day 0 and 10% A on day 3 Fed with 30% A on day 0 Fed with 30% of A/B mixture on day 0 CD OptiCHO batch, IgG Fed with 30% of A/B mixture on day 0, IgG Fed with 15% A on day 0 and 10% A on day 3, IgG Fed with 30% A on day 0, IgG

Chemically defined feeds superior to hydrolysates

Different hydrolysates were also tested as feeds comparing to these two feeds. Using CD OptiCHOTM as base medium, EfficientFeedTM A gave significantly higher protein expression than any of the hydrolysates tested (Figure 4). Addition of hydrolysates to EfficientFeedTM A did not result in any increase in protein expression. In fact, a slight improvement may be indicated with EfficientFeedTM A alone (Figure 5). Further, potential osmolality excesses from both hydrolysates as well as EfficientFeedTM A additions were not indicated as Soy yielded expression levels closest to those of EfficientFeedTM A alone with an osmolality increase of 43 mOsm while the remainder of the hydrolysates averaged only 25 mOsm.
Fed-batch IgG production with 10 g/L hydrolysate feeds using CD OptiCHOTM as base medium
500
1- Pea (10g/L) [Days: 3, 6, 9]

Fed-batch Growth Performance in DASGIP Bioreactors

16 Batch 10% CHO Feed A fed on D3, 6, 8
IgG titer 10% 1:1 CHO Feeds A and B fed on D3, 6, 8 (mg/L)

Day 9 IgG Titer in DASGIP Bioreactors

2000

Viable cell density (106cells/ml)

EfficientFeedTM

14 12 10 8 6 4 2 0

10% 1:1 CHO Feeds A and B fed on D2, 4, 6, 8

IgG Concentration (mg/L)

1500

1000

500

0
0 2 4 6 8 10 12
Batch Fed-batch 1 Fed-batch 2 Fed-batch 3

Time (days)

Figure 6. Cells have been growing in CD OptiCHO Medium for three passages prior to the study. Cultures were seeded at 3e5
viable cells/ml in 0.5 liter bioreactors and in 50ml working volume in 250ml shake flask. Bioreactors were set at 37C, pH 7.15 and DO at 50%. Shake flask cultures were incubated at 37C, 8% CO2, and shaken at 125 rpm. Different feed combinations and feeding strategies were tested. Only some examples of Day 9 IgG levels presented here. Fed-batch 1 (Red) : Fed with 10% CHO feed A on Day 3, 6 and 8; Fed-batch 2 (Dark Green): Fed with 10% of 1:1 mixture of CHO feed A and B on Day 3, 6 and 8; Fedbatch 3 (Dark Blue): Fed with 10% of 1:1 mixture of CHO feed A and B on Day 2, 4, 6 and 8.

450 400 350

2- Wheat (10g/L) [Days: 3, 6, 9] 3- Soy (10g/L) [Days: 3, 6, 9] 4- Rice (10g/L) [Days: 3, 6, 9]

IgG level (mg/L)

Introduction
Fed-batch can be used to improve the yield of recombinant proteins and the rate of cell expansion. However, the process and mechanisms for developing good fed-batch processes with chemically defined feeds are inefficient. Two chemically defined CHO feeds (CHO CD EfficientFeedTM A and B) have been developed to enable media platforms using fed-batch processes. The use of these two feeds with different nutritional structures allows users to accommodate unique cell culture requirements using mixing/combinatorial approaches. These feeds used independently and in various combinations with different feeding strategies have been explored using shaker flasks, bioreactors and microbioreactor system (SimCellTM). A chemically-defined medium as defined by Invitrogen such as CD CHOTM and CD OptiCHOTM, does not contain recombinant protein or hydrolysate. Chemically-defined media developed support high performance seen typically in media containing hydrolysates, peptones, and/or growth factors but without the commonly associated variability. Additionally the development of robust chemically-defined feed/supplements for use with chemically-defined base media enable users to develop chemically defined fed-batch media platforms . Hydrolysates, peptones and growth factors have been demonstrated to improve both peak cell density and recombinant protein production in cell culture medium. However they contribute to increased variability for the fed-batch processes. There are strong interests to remove hydrolysates from bioproduction processes. AGTTM (Advanced Granulation Technology) is a proprietary technology developed by Invitrogen as a dry format having a pre-adjusted pH, osmolality, homogeneous ingredient distribution, very low dust generation, and rapid dissolution vs. standard powder media. CHO CD EfficientFeedTM A and B were developed from AGTTM formulations as chemical stocks. This approach provides a convenient liquid media format for use in small scale development work and compatible with AGTTM format for future scale up.

5- Feed A (10%) [Days: 3, 6, 9]

300 250 200 150

Comparison of fed-batch IgG production from different cell culture systems

Different bioreactors and SimCellTM High-Throughput Automated Cell Culture Microbioreactor System from Bioprocessors have also been used to culture recombinant CHO cell lines and similar IgG titers achieved as in shake flasks (Figure 7). SimCellTM allows hundreds of parallel bioreactors runs under different conditions. Larger experiments can be run in the future in SimCellTM to further optimize performance.

5000

1e+7 Viable Cell Density (cells/ml)

4000

100 50

3000

IgG level (mg/L)

0 day7 day8 day9 day10 day11 day12 day13 day14

5-fold increase

Time
Figure 4. Cells was inoculated at 3e5 viable cells/ml into a 30 ml working volume in 125 ml shake flask (125 rpm) in CD OptiCHOTM medium at 37C in 8% CO2. On days 3, 6 and 9, a 10% volumetric addition of hydrolysate (Kerry Bio-Science) at a stock concentration of 100g/L or CHO CD EfficientFeedTM A was added to the culture. Samples were withdrawn for HPLC determination of IgG concentration on Days 7-14. , but when cell viabilities dropped below 20%, samples were no longer obtained.

2000

1e+6
2-fold increase

IgG Production in Three Systems

1600

1000

Time (days)

Figure 1. Cells have been growing in CD OptiCHO Medium for three passages prior to the study. Cultures were seeded in triplicate at 3e5 viable cells/ml in 50ml working volume in 250ml shake flask. Cultures were incubated at 37C, 8% CO2, and shaken at 125 rpm. Different fed-batch strategies as illustrated in the legends. No further supplementation was performed during culture. Viable Cell densities determined using Coulter ViCELL. IgG level determined by protein affinity HPLC.

IgG mg/L

10

12

14

16

Fed-batch IgG production with 10 g/L hydrolysate feeds in combination with CHO CD EfficientFeedTM A using CD OptiCHOTM as base medium
500
1- Pea (10g/L) + feed A (10%) [Days: 3, 6, 9]

1200

800

450 400

2- Wheat (10g/L) + feed A (10%) [Days: 3, 6, 9] 3- Soy (10g/L) + feed A (10%) [Days: 3, 6, 9] 4- Rice (10g/L) + feed A (10%) [Days: 3, 6, 9]

Fed-batch culture using CD OptiCHO as base medium Recombinant IgG Clone #2

1e+7
CD OptiCHO batch Fed with 15% A on day 0 and 10% A on day 3 Fed with 30% A on day 0 and 10% A on day 3 Fed with 30% of A/B mixture on Day 0 CD OptiCHO batch, IgG Fed with 30% of A/B mixture on Day 0, IgG Fed with 15% A on day 0 and 10% A on day 3, IgG Fed with 30% A on day 0 and 10% A on day 3, IgG

400

IgG level (mg/L)

5000

350 300 250 200 150 100 50 0

5- CD OptiCHO + feed A (10%) [Days: 3, 6, 9]

0 Bioreactor SimCell Shake Flask

4000

Viable Cell Density (cells/ml)

3000

6-fold increase

2000

IgG level (mg/L)

Figure 7. Cells have been growing in CD OptiCHO Medium for three passages prior to the study. Cultures were seeded at 3e5 viable cells/ml in 0.5 liter DASGIP bioreactors, 0.6 ml mini-bioreactor chambers of SimCellTM and in 50ml working volume in 250ml shake flask. Bioreactors and SimCellTM were set at 37C, pH 7.15 and DO at 50%. Shake flask cultures were incubated at 37C, 8% CO2, and shaken at 125 rpm. 10% of initial culture volume was fed using CHO CD feed A on day 3, 6, and 9. IgG level at Day 11 determined by protein affinity HPLC.

1e+6
1000

day7

day8

day9

day10

day11

day12

day13

day14

Materials and methods

Cells, Media and Feeds: A variety of different recombinant CHO cell lines expressing proteins including IgG(s) and EPO were used. The IgG producing CHO cell lines were from our collaborators. EPO DG44 CHO cell lines were developed by Invitrogen. Different hydrolysates were from Kerry BioScience. CD CHOTM and CD OptiCHOTM were used as base media. CHO CD EfficientFeedTM A and B developed by Invitrogen were used for fedbatch development. Universal nutrient requirements for cell growth and recombinant protein expression were considered in feed design. Solubility of medium components and osmolality of the final feeds were also considered. Feeding strategies Different feeding timing, feed concentrations and mixtures of the CHO CD EfficientFeedTM A and B were evaluated in different IgG CHO cell lines and EPO DG44 cell lines to demonstrate improved productivity, fast cell expansion, high viable cell density or long cell culture.. Assays Viable cell density and cell viability were determined using Coulter ViCELL. IgG titers were quantified using protein G affinity HPLC. EPO levels were determined using the Quantikine IVD kits (ELISA) from R&D Systems, Inc.
0 2 4 6 8 10

Time
2.5-fold increase

Summary
Concentrated chemically defined feed formulations for recombinant protein expression in CHO cells (CHO CD EfficientFeedTM A and B) have been developed to enable a media platform using fed-batch processes. Also, the use of these two feed formulations independently and in various combinations demonstrated various outcomes, enhancement of recombinant protein production (2-8 fold improvement) and rapid cell growth accelerating the scaleup (See Figure 1- 3). In addition, we achieved over 1g/liter yield of IgGs with several recombinant CHO lines. Further, high titers and high viable cell densities similar to those observed in shake flasks were achieved in bioreactors and microbioreactor system (SimCellTM) using different feeding strategies without process optimization. With additional process optimizations and feeding strategies, we believe higher viable cell density and titers can be achieved in bioreactors. The observations with multiple cell lines expressing various proteins including IgG demonstrated that chemically defined feed formulations such as CHO CD EfficientFeedTM A and B will allow users to build robust CHO fed-batch processes in different CHO systems.

Figure 5. Cells was inoculated at 3e5 viable cells/ml into a 30 ml working volume in 125 ml shake flask (125 rpm) in CD
0

OptiCHOTM medium at 37C in 8% CO2. On days 3, 6 and 9, a 10% volumetric addition of different hydrolysates and CHO CD EfficientFeedTM A or EfficientFeedTM A alone were added to the cultures. Samples were withdrawn for HPLC determination of IgG concentration on Days 7-14.

10

12

14

16

Time (days)

Figure 2. Cells have been growing in CD OptiCHO Medium for three passages prior to the study. Cultures were seeded in triplicate at 3e5 viable cells/ml in 50ml working volume in 250ml shake flask. Cultures were incubated at 37C, 8% CO2, and shaken at 125 rpm. Different fed-batch strategies as illustrated in the legends. No further supplementation was performed during culture. Viable Cell densities determined using Coulter ViCELL. IgG level determined by protein affinity HPLC.

Fed-batch in bioreactors
Batch and fed-batch experiments using DASGIPTM parallel bioreactor system (8 chambers) with 0.5 L working volume were executed to further evaluate the two chemically defined CHO feed formulations demonstrating applicability of the feeds and feed strategies in bioreactors. CD OptiCHOTM was used as base media. Different chemically defined CHO EfficientFeedTM A and B combinations and feeding strategies were evaluated.

Fed-batch culture using CD OptiCHO as base medium recombinant EPO DG44 CHO
CD OptiCHO batch Fed with 30% of A on day 0 Fed with 30% of A/B mixture on day 0 CD OptiCHO batch, EPO Fed with 30% of A/B mixture on day 0, EPO Fed with 30% of A on day 0, EPO

Viable Cell Density (cells/ml)

1.0e+7

EPO level (mg/L)

8-fold increase

300

200

1.0e+6

4-fold increase

100

With minimal process optimization, similar results were achieved with respect to viable cell density and peak IgG titers as observed in shake flasks. Representative results of day 9 IgG levels were shown (Figure 6). In bioreactors, significant improvements with day 0 supplements (rich base media) were not observed as in shake flasks, that may be caused by different nutrition usage patterns in different systems. Further performance enhancement can be achieved by supplementing some key nutrients such as glucose or glutamine, which were depleted after day 9 based on limited depletion analysis for above experiments. In-depth spent media analysis will also be helpful for designing better feeding strategies.

Acknowledgments
We would like to thank Invitrogens Media Analytical Services laboratory along with Brian Burgin, Lori Burgin, Melanie Kester, Lynne Pajak and Steve Peppers.

12

14

16

Time (days)
Figure 3. Cells have been growing in CD OptiCHO Medium for three passages prior to the study. Cultures were seeded in triplicate at 2e5 viable cells/ml in 50ml working volume in 250ml shake flask. Cultures were incubated at 37C, 8% CO2, and shaken at 125 rpm. Only day 0 feeds with different supplement combinations were used as illustrated in the legends. No further supplementation was performed during culture. EPO determined by ELISA.