Continuous and Semi-continuous Cell Culture for Production of Blood Clotting Factors

Sunil G. Desai, Ph. D. Sr. Manager, Manufacturing Sciences and Technology

2

Sanford , NC

Grange Castle, Ireland Andover, MA

Pearl River, NY

Pfizer Biotech

CMOs

Strangnas, Sweden

Algete, Spain

Biologics Vaccines Biosimilars Robust pipeline

BeneFIX or rFIX

• Recombinant therapy for Factor IX deficiency (hemophilia B)

• 55 kDa zymogen with 12 γ-carboxylation sites

• Several other complex post-translational modifications

• Extensive structure and functionality assays

• First approved recombinant factor IX product (1997)

• CHO-based batch re-feed process • ADRM free culture medium from

the beginning

3

318316

312

3

ReFacto AF/ Xyntha or BDD-rFVIII

• Recombinant therapy for Factor VIII deficiency (hemophilia A)

• FVIII gene sequenced in 1984 • Engineered version of FVIII (B

domain deleted), maintains procoagulant function

• Increased transcription efficiency • ReFacto approved in U.S., EU (1st

licensed in 1999) • ReFacto AF approved in 2008 • CHO-based perfusion process • DS manufactured at an external

partner site for Pfizer

4

Full Length FVIII

NH2 90 B 80 COOH

ReFacto

NH2 COOH 90 80

50 43

73

Me2+

Activated ReFacto

3D picture reference: Orlova et al., 2013, Acta Naturae, 5(2).

Key Process Challenges

• Low level expression (transcription/ translation level) • Growth associated expression • Labile product

– Degradation during processing – Activation during processing

• Complex post-translational modifications – Disulfide bond formation (11 for rFIX) – N-linked and O-linked glycosylation – Tyrosine sulfation (rFVIII, rFIX) – Metal ion binding – Heavy and light chain cleavage – γ-carboxylation sites

• Process changes while maintaining quality/ comparability – Removal of ADRMs (Albumin) from culture medium (BDD-rFVIII) – Changes in production sites/ scales – Use of disposables – Ongoing process updates (PAT, vendor changes, ICH guidelines)

• Supply sensitive customer base

5

Cell culture process - BeneFIX

6

Low cell density

Bio

reac

tor

Har

vest

Re-

feed

Recovery Nutrient media

Seed

Re-feed Process

High cell density

Retained culture

Low cell density

After n days

BeneFIX production data

7

BeneFIX (1997 – present)

Non-validated database of GMP data Bioreactor

Num

ber o

f bat

ches

2.5kL 6kL 12kL

1 2 3 4 5 6 7 8 10 9 11

Benefits/ challenges of continuous process

•Productivity vs Titer – Perfusion/ re-feed processes reduce BRX turn-around time – Lower product quality risk than increasing specific productivity of cells – Easy to model volumetric productivity vs titer benefit analysis

8 •Data from multiple bioreactors •Range of variation in productivity yet consistent product quality

LCL

Bioreactor batch

Cel

l Spe

cific

Pro

duct

ivity

0 Non-validated database of GMP data

Benefits/ challenges of continuous process

•Campaign lengths – Several weeks to months – Multiple bioreactors allow back up inoculation sources – Savings in reduced SIP/ CIP cycles of bioreactors

•Number of cell generations

– Extensive LIVCA studies required • At scale EOP samples tested for genetic stability

– Routine testing for culture purity (mycoplasma and virus) at EOP only

– Additional product characterization to account for cell age (early-mid-late stage campaign effects)

– CHO, in general, stable for extended number of generations

9

Specific growth rate vs generations

10

•Growth rate is independent of cell generation number •Range in growth rate giving consistent product quality

Culture generations

Spe

cific

gro

wth

rate

LCL

0 85

Non-validated database of GMP data

Benefits/ challenges of continuous process

• Maintenance of aseptic BRX conditions for extended time – A major concern with extended period cell culture – SIP / CIP procedures have vastly improved • Years of experience • Multiproduct facilities • Rigorous PM and engineering controls (o-rings!) • Disposable use with minimum open operations

11 1.3% contaminated Two of eleven bioreactors were never contaminated

Campaign

Bat

ch c

ount

Contaminated batches

Non-validated database of GMP data

Period with non contaminations

Cell culture process – ReFacto AF

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Culture medium

Cell separator

Return of CHO cells to the bioreactor

Harvest of product containing broth

Bioreacto

To clarification, primary capture

Perfusion Process

Bioreactor

Harvest tank

ReFacto AF •New MCB for HSA-free ReFacto

– Same cell lineage, BDD-rFVIII genotype unchanged – Albumin-free media for production

•Replace hybridoma-derived MAb column •Analytical Comparability

– Very similar biochemical profile – Relationship to historical ranges well understood

•Non-clinical/Clinical Comparability

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ReFacto 2.0 1.6 1.2 0.8 0.4

0

ReFacto AF

15 20 25 30 35 40 45 50 55

1.6 1.2 0.8 0.4

0

2.0 2.4

Time (minutes)

1 2 3 4

Lane Sample

1 MW Markers 2 Blank 3 ReFacto (Site 1) 4 ReFacto (Site 2) 5 ReFacto AF

5

Kelley et al., 2009, Haemophilia.

80 kDa

170 kDa 90:1 90:2

ReFacto AF production data

14 Consistent product quality data over the years

~5 years of data shown

Non-validated database of GMP data

LCL

LCL

UCL

UCL

% P

rodu

ct

Isof

orm

1

Sp.

Act

ivity

Bioreactor 1 Bioreactor 2

Chromatography 2

Bioreactor 3

Harvest and Cell Separation

Concentration/Diafiltration by Ultrafiltration 1

Chromatography 1

BeneFIX

Purification

Bioreactor

Harvest and Cell Separation

Chromatography 1Frozen eluate

ReFacto AF

Viral inactivation &

Purification

Eluate pool

Benefits/ challenges of continuous process

• Critical for biologics from a compliance perspective

• Each DS lot traces back to a single cell bank thaw event

• Pooling of multiple batch streams during purification

• Batch stream could originate from multiple scales

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Batch definition

Conclusions

• Semi-continuous (re-feed) and continuous (perfusion) processes of complex recombinant proteins commercially successful for over 15 yrs

• Product/ process life-cycle changes successfully implemented • Large data sets demonstrate process robustness and cell line

stability • Increased plant productivity with shorter bioreactor turn-

around times • Manageable contamination rates over extended periods of

operation add substantial operational savings

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Acknowledgements

Almost two decades of contributions towards continuous development and manufacturing of BeneFIX and ReFacto AF/ Xyntha

– Pfizer Global Supply (Tanya Alcorn) Site Operations (Erin Beal, Erik Roos) Quality Manufacturing Sciences and Technology (Dan Lasko,

Enda Moran, Kesav Reddy, Jim Booth) – Pharmaceutical Sciences (Tim Charlebois, Mike Jankowski,

Shamik Sharma) – External supply partners