Optimization of Glycosyation & Charge Distribution Through Culture Parameters & Supplements

a customer and science-focused

contract development & manufacturing organization

OPTIMIZATION OF GLYCOSYLATION AND

CHARGE DISTRIBUTION THROUGH CULTURE

PARAMETERS AND SUPPLEMENTS

Sigma Mostafa, Venkata Tayi, Brian Baker, James Smedley, Nate Oien,

Derek Ryan, Abhinav Shukla

Cell Culture Engineering XV

May 11, 2016

• Introduction – KBI workflow

• Case study 1 – PAT approach to meet charge

species target

• Case study 2 – Product quality toolbox

• Case study 3 – Impact of Cu2+ on product quality

• Conclusions

Outline

2 |

KBI Locations

3 |

KBI RTP

• Cell Culture Process Development

• Purification Process Development

• Microbial Process Development

• Analytical Development

KBI Boulder

• Microbial Process Development

• Microbial cGMP Manufacturing

• Analytical Development

KBI Durham

• Analytical and Formulation Dev.

• Cell Line Development

• Cell Culture cGMP Manufacturing

CLD 2%

Dev / Mfg 27%

CLD / Dev / Mfg 32%

Process Characterization

14%

Tech. Transfer/MFG

12%

Dev 11%

PD Supply 2%

Project Types

12%

58% 2%

14%

2% 2%

7% 2%

Molecule Types

Bispecific Ab mAb*

Growth factor Vaccine glycoprotein

Enzyme Recombinant Enzyme

PEGylated protein Fc-Fusion Protein

4 |

Distribution of 2015 Completed Projects: 50+ in

Development

*Includes 5 Biosimilars

Background

• Technology Transfer project

• CHOK1 cell line

• Medium & feeds are off-the shelf

• Limited historical data (n = 2 with varied

data)

• Acidic species desired level within 35 – 45%

5 |

Case Study 1: PAT approach to meet PQ target

• Cell growth and viability were comparable

across bench scale cultures


6 |

• Acidic species range was met only in portion of the

bench scale runs

• Other cell culture and PQ ranges were met by all the

runs


7 |

• Impact of culture length on acidic species was

investigated

• Acidic species was observed to decrease with

culture time

8 |


• At the 200L Demo Run stage daily WCX testing

was carried out to determine harvest day

9 |


• This PAT approach was implemented in the cGMP runs

• The three cGMP runs were harvested on three different

days based on acidic species level


10 |

• Acidic species levels were very similar for all 3

cGMP BDS

• Other product quality attributes were also within

acceptable range


11 |

• A PAT approach using a WCX assay led to a

successful cGMP campaign

• Variable harvest day, within a narrow range,

maintained other cell culture and product

quality attributes in range

• Cross functional collaboration was critical

for successful implementation of this

approach

Case Study 1: Results Summary

12 |

• KBI has an array of tools to customize

product quality

13 |

Case Study 2: Product Quality Toolbox

Galactose

Fucose

Mannose

Sialic Acid

Ratio of

main/

acidic/

basic

species

HMW & LMW

• Impact of key supplements on glycosylation


14

0

20

40

60

80

100

G0 FG0 G1 FG1 G2 FG2

2.0

80.4

11.9

0.6

%

No supplements

0

20

40

60

80

100


7.1

44.9

2.6

35.2

5.5

%

Mn2+ + Uridine + Galactose

0

20

40

60

80

100


29.2

55.3

2.3 8.2

0.5

%

Fucosylation inhibitor (2F-PAF)

0

20

40

60

80

100


36.3

20.6 16.3 17.1

2.1 3.1

%

2F-PAF + Mn2+ + Uridine + Galactose

9/21/2016 15

0%

20%

40%

60%

80%

100%

37C 37C --> 32C

25.2 11.4

67.2

54.6

7.6

24.6

% Acidic % Main % Basic

Effect of temperature shift on charge variants distribution

• Temperature shift decreased the

acidic variants possibly due to

reduction of deamidation

• Temperature shift increased basic

variants possibly due to c-terminal

lysine residues

• Temperature shift to an optimized

value could produce the molecule

with desired profile


16 |


• Temperature shift decreased galactosylation

across clones

• A product quality toolbox was established as

a technology development project

• This toolbox has been successfully used in

multiple biosimilar and novel molecule

process development

17 |


Background

• CHO DG44 derived vendor cell line

• Doubling time <20 hours

• Commercial off the shelf medium & feed

• KBI received a Phase I process that had

historically shown high variability

• Scope of project at KBI – Resupply and

further development

18 |

Case Study 3: Impact of Cu+2 on Product Quality

• Replicate cultures in 3L bioreactors showed lack

of reproducibility

• Reactor 2 had a ‘favorable’ pH correction on day 7

(upward); Reactor 1 had ‘unfavorable’ pH

corrections on days 7 and 10 (toward lower end of

pH dead-band).

Case Study 3

19 |

• Feed addition led to immediate lactate production

and associated pH drop (feed pH is neutral)

• Amount of pH drop after a feed varied from run to

run and could be as high as 0.25 pH unit

20 |

Case Study 3

6.60

6.70

6.80

6.90

7.00

7.10

7.20

7.30

7.40

Day 8 Feed Day 10 Feed

Day 12 Feed Day 14

Feed

• Re-supply Strategy

- Expand pH dead-band

- Glucose range

- Initial glutamine

- Feed addition rate

21 |

Case Study 3

• Shake flask was found to be a good model

to study the high lactate & rapid viability

decline

• Addition of Cu2+ was tested in shake flask

and found to be highly successful in

extending viability and increasing

productivity

22 |

Case Study 3: Impact of Cu+2 on Product Quality

Case Study 3: Impact of Feeds and Supplements in SF

23

0

5

10

15

20

25

30

35

40

45

0 2 4 6 8 10 12 14

VC

D (

10

6 c

ells/m

L)

Time (Days)

0

20

40

60

80

100

0 2 4 6 8 10 12 14V

iab

ilit

y (%

)

Time (Days)

Control with current Feed Current Feed + Cu supplementation

New Feed New Feed + Cu supplementation

New Feed + Amino Acid Cocktail + Cu supplementation

Case Study 3: Impact of Feeds and Supplements in SF

24

0

0.5

1

1.5

2

2.5

3

A B C D ETit

er

(g/L)

0

0.5

1

1.5

2

2.5

3

0 2 4 6 8 10 12 14

La

cta

te (

g/L)

Time (Days)

Control with current Feed Current Feed + Cu supplementation

New Feed New Feed + Cu supplementation

New Feed + Amino Acid Cocktail + Cu supplementation

Case Study 3: Impact of Feeds and Supplements in BRX

25

Control with current Feed New Feed + Amino Acid Cocktail + Cu supplementation

• Supplementation of culture with 400 nM

Cu2+ improved viability, titer, and process

reproducibility dramatically


26 |

• High throughput, high sensitivity analytics

are essential for successful and efficient

process development

• Surrogate outputs, in place of complex

analytics such as mass spec, would enable

implementation of PAT approaches

Final Thoughts

27 |

Upstream Process Development

Brian Baker

Venkata Tayi

Niket Bubna

Cameron Phillips

Jingshu Zhu

Acknowledgements

28 |

Analytical Development

James Smedley

Nate Oien

Derek Ryan

Management

Abhinav Shukla

Tim Kelly

Prathima Acharya

Joe McMahon

Optimization of Glycosyation & Charge Distribution Through Culture Parameters & Supplements

Health & Medicine

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