AbstractThe design of serum-free media for suspension culture of geneticallyengineered Chinese hamster ovary (CHO) cells using classical media asbasis of investigation. An initial control basal medium was prepared,which was similar in composition to HAM’s F12: DMEM (1:1). Plackett–Burman Statistical Design (PBD) was used to screen the componentshaving positive and negative effects on Titer and growth of cells. Wehave investigated 26 media components and result were analyzedusing JMP® software. BME vitamin, Glutathione reduced, ITS, Lipidsupplement were found to have a positive impact on Titer whileUridine and L-serine shown to have positive impact on growth of cells.

IntroductionChinese hamster ovary (CHO) cells are the most widely usedmammalian host for the production of recombinant proteins becauseof compatibility of posttranslational modifications to that of humans.Regulatory constrains abstain the use of serum containing media forproduction of therapeutic proteins, hence development of serum freemedia is essential for cultivation of CHO cells and production oftherapeutic proteins.

References

1. Hideo Miki, Mutsumi Takagi (2014). Design of serum-free medium forsuspension culture of CHO cells on the basis of general commercial media.Cytotechnology. DOI 10.1007/s10616-014-9778-0

2. J. Gonza´lez-Leal, L. M. Carrillo-Cocom, A. Ramı´rez-Medrano, F. Lo´pez-Pacheco, D. Bulnes-Abundis, Y. Webb-Vargas, and M. M. Alvarez (2011). Use of aPlackett–Burman Statistical Design to Determine the Effect of Selected AminoAcids on Monoclonal Antibody Production in CHO Cells. Biotechnol. Prog., 2011,Vol. 27, No. 6

3. Ananth Parampalli Kent Eskridge, Leonard Smith, Michael M. Meagher, MarkC. Mowry, Anuradha Subramanian (2007). Developement of serum-free mediain CHO-DG44 cells using a central composite statistical design. Cytotechnology54:57–68

AcknowledgementThis work was funded by Centre of Excellence for BiopharmacheuticalTechnology grant from Department of Biotechnology, Government of India(number BT/COE/34/SP15097/2015)

ConclusionsPBD design was successfully implemented to screen the mediacomponents for titer and growth. BME vitamin, Glutathione reduced,ITS, Lipid supplement were found to have a positive impact on Titerwhile in case of cell count L-serine, and uridine have shown positiveimpact.

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Industrial SignificanceSerum free media reduce the cost of production of protein therapeuticdrugs, it also decreases the lot to lot variability. Importantly it is as perthe norms of regulatory authorities.

Technology Readiness Level: …

Media development and optimization for CHO cell culture

Anurag S rathore*, Neelesh Gangwar, Rajinder Kaur, Geetanjali Hubli

ResultsData analysis was performed using the JMP® software (SAS Institute,Cary, NC) and the results are illustrated in Fig. 3. It is seen that themodels that are formed are statistically significant (R2 ~ 0.9 in Figs. Aand B) both for the cases of titer and cell count.

In case of titer Out of the 26 input variables 7 i.e. L-leucine, L-Tyrosine,L-Tryptophan, L-cysteine, L-Glutamic acid, Pluronic, L-aspartic acidwere found to have a negative impact on titer while 4 i.e. BME vitamin,Glutathione reduced, ITS, Lipid supplement were found to have apositive impact.

In case of cell count out of the 26 input variables, 2 (i.e L-serine,uridine) found to have positive effect on cell count. While L-Tyrosine,Lipid supplements, L-Tryptophan and L- Glutamic acid have shownnegative effect.

Cell count and Titer was be taken on Day5 show in bar graphs below.

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Material and MethodsCHO cells genetically engineered to express genes encoding the humanimmunoglobulin G (IgG). Commercially available basal media HAM’sF12: DMEM (1:1) purchased from thermo scientific. All 20 amino acids,Pluroninc, ITS, BME vitamins, sodium pyruvate, lipid, Uridine,Glutathione in total 26 components were tested for screeningexperiment. Total 28 formulations were developed using PBD.Experiment was performed in 24 well plate with shaking at 140rpm and5% CO2 in a humidified incubator shaker. Plates were incubated for 5days, after which cell count and titer was taken. Cell count was done byTrypan blue dye exclusion method while titer was taken using protein Achromatography by HPLC

Formulati

onsPattern L-Alanine

L-

Arginine

HCI

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ne.H1O

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Aspartic

Acid

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Cysteine.

HCI.H1-1

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.HCI. H1-

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e

L-Leucine

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Lysine.H

CI

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L-

Phenylal

anine

L-Proline L-Serine

L-

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e

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an

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Tyrosine.

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L-Valine Pluronic ITS BMENa

PyruvateLipid Uridine

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1 −−−−−−−−−−−−−−−−−−−−−−−−−− -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1

2 −−+−++−−+++−++−−+−++−+−+−+ -1 -1 1 -1 1 1 -1 -1 1 1 1 -1 1 1 -1 -1 1 -1 1 1 -1 1 -1 1 -1 1

3 −−+−++−+−+−+−−+−++−−+++−++ -1 -1 1 -1 1 1 -1 1 -1 1 -1 1 -1 -1 1 -1 1 1 -1 -1 1 1 1 -1 1 1

4 −−++−−+−++−+−+−+−−+−++−−++ -1 -1 1 1 -1 -1 1 -1 1 1 -1 1 -1 1 -1 1 -1 -1 1 -1 1 1 -1 -1 1 1

5 −−++−−+++−++−−+−++−+−+−+−− -1 -1 1 1 -1 -1 1 1 1 -1 1 1 -1 -1 1 -1 1 1 -1 1 -1 1 -1 1 -1 -1

6 −−++−+−+−+−−+−++−−+++−++−− -1 -1 1 1 -1 1 -1 1 -1 1 -1 -1 1 -1 1 1 -1 -1 1 1 1 -1 1 1 -1 -1

7 −−+++−++−−+−++−+−+−+−−+−++ -1 -1 1 1 1 -1 1 1 -1 -1 1 -1 1 1 -1 1 -1 1 -1 1 -1 -1 1 -1 1 1

8 −+−−+−++−−+++−++−−+−++−+−+ -1 1 -1 -1 1 -1 1 1 -1 -1 1 1 1 -1 1 1 -1 -1 1 -1 1 1 -1 1 -1 1

9 −+−−+−++−+−+−+−−+−++−−+++− -1 1 -1 -1 1 -1 1 1 -1 1 -1 1 -1 1 -1 -1 1 -1 1 1 -1 -1 1 1 1 -1

10 −+−−+++−++−−+−++−+−+−+−−+− -1 1 -1 -1 1 1 1 -1 1 1 -1 -1 1 -1 1 1 -1 1 -1 1 -1 1 -1 -1 1 -1

11 −+−+−−+−++−−+++−++−−+−++−+ -1 1 -1 1 -1 -1 1 -1 1 1 -1 -1 1 1 1 -1 1 1 -1 -1 1 -1 1 1 -1 1

12 −+−+−+−−+−++−−+++−++−−+−++ -1 1 -1 1 -1 1 -1 -1 1 -1 1 1 -1 -1 1 1 1 -1 1 1 -1 -1 1 -1 1 1

13 −+−+−+−+−−+−++−−+++−++−−+− -1 1 -1 1 -1 1 -1 1 -1 -1 1 -1 1 1 -1 -1 1 1 1 -1 1 1 -1 -1 1 -1

14 −++−++−−+−++−+−+−+−−+−++−− -1 1 1 -1 1 1 -1 -1 1 -1 1 1 -1 1 -1 1 -1 1 -1 -1 1 -1 1 1 -1 -1

15 +−−−−++−−−+−−++−−−−+++++++ 1 -1 -1 -1 -1 1 1 -1 -1 -1 1 -1 -1 1 1 -1 -1 -1 -1 1 1 1 1 1 1 1

16 +−−−−++++++++−−−−++−−−+−−+ 1 -1 -1 -1 -1 1 1 1 1 1 1 1 1 -1 -1 -1 -1 1 1 -1 -1 -1 1 -1 -1 1

17 +−−−+−−++−−−−++++++++−−−−+ 1 -1 -1 -1 1 -1 -1 1 1 -1 -1 -1 -1 1 1 1 1 1 1 1 1 -1 -1 -1 -1 1

18 +−−++−−−−++++++++−−−−++−−− 1 -1 -1 1 1 -1 -1 -1 -1 1 1 1 1 1 1 1 1 -1 -1 -1 -1 1 1 -1 -1 -1

19 +−−++−−−+−−++−−−−++++++++− 1 -1 -1 1 1 -1 -1 -1 1 -1 -1 1 1 -1 -1 -1 -1 1 1 1 1 1 1 1 1 -1

20 +−−++++++++−−−−++−−−+−−++− 1 -1 -1 1 1 1 1 1 1 1 1 -1 -1 -1 -1 1 1 -1 -1 -1 1 -1 -1 1 1 -1

21 +−+−−++−−−−++++++++−−−−++− 1 -1 1 -1 -1 1 1 -1 -1 -1 -1 1 1 1 1 1 1 1 1 -1 -1 -1 -1 1 1 -1

22 ++−+−+−+−+−+−+−+−+−+−+−+−+ 1 1 -1 1 -1 1 -1 1 -1 1 -1 1 -1 1 -1 1 -1 1 -1 1 -1 1 -1 1 -1 1

23 +++−−−−++−−−+−−++−−−−+++++ 1 1 1 -1 -1 -1 -1 1 1 -1 -1 -1 1 -1 -1 1 1 -1 -1 -1 -1 1 1 1 1 1

24 +++−−−−++++++++−−−−++−−−+− 1 1 1 -1 -1 -1 -1 1 1 1 1 1 1 1 1 -1 -1 -1 -1 1 1 -1 -1 -1 1 -1

25 +++−−−+−−++−−−−++++++++−−− 1 1 1 -1 -1 -1 1 -1 -1 1 1 -1 -1 -1 -1 1 1 1 1 1 1 1 1 -1 -1 -1

26 +++++−−−−++−−−+−−++−−−−+++ 1 1 1 1 1 -1 -1 -1 -1 1 1 -1 -1 -1 1 -1 -1 1 1 -1 -1 -1 -1 1 1 1

27 +++++++−−−−++−−−+−−++−−−−+ 1 1 1 1 1 1 1 -1 -1 -1 -1 1 1 -1 -1 -1 1 -1 -1 1 1 -1 -1 -1 -1 1

28 +++++++++−−−−++−−−+−−++−−− 1 1 1 1 1 1 1 1 1 -1 -1 -1 -1 1 1 -1 -1 -1 1 -1 -1 1 1 -1 -1 -1

Figure 1(A) Predictive model for Titer and 3(B) Predictive model for Cell count

Table 1 Media components for screening Table 2 Plackett-Burman design Table

Figure 2 Predictive profile of media components on Titer

Figure 3 Predictive profile of media components on cell count

Figure 4 Average Titer and Vaible cell count