ROF SEIGETARTS EVITAVONNI :ERUPXBAM PESIAD NOITACIFIRALC NI ECNARAELC PCH GNICNAHNE

AND DOWNSTREAM PROCESSING OF MABS

INTRODUCTION

In recent years, biopharmaceutical manufacturing has shown

titers generally associated with higher cell densities. These increases in productivity induces higher levels of process-related impurities like host cell proteins (HCP), DNA, HMW fragments or lipids and consequently shifts the production bottleneck to the downstream

In such context,

limit and will not be able to provide enough protection to the .

TECHNOLOGY

DAISEP MabXpure™

(SUT) combines multimodal depletion mechanisms (Size Exclusion and Anion Exchange) to improve bioburden depletion performances. MabXpure can be used as a single device technology or within a

the resin with very low ratio feed/media, or as a polishing cartridge, when prepacked, without compromising the antibody yield (>90%).

DNA and 1 LRV for HCP under static or dynamic conditions.

as a prepacked cartridge, it is used as a Protein A guard column or as ion exchange DSP step combination. MabXpure improves overall

formulation.

CONCLUSION

HCPs and DNA depletion technology. The use of MabXpure as a

of use are optimal with high mix ratios and short contact times for sample preparation, or lower mix ratios and higher contact times for

MabXpure has a very high capability for depleting host cell proteins and DNA, with extremely high mAb recovery (>95%). Less intrusive

to the product, it allows the removal of up to 1 LRV of HCPs, 2 LRV of DNA depending on the mode of action, and reduces co-eluted HCPs species. MabXpure can supplement, complement, or replace the pre-existing DSP unit operations.

Contact us at cte@daicelbioseparations.comwww.daicelbioseparations.com

MECHANISM OF INTERACTION &

DEPLETION

Small moleculesnucleic acids

HCDNA

HCP

SEC

AEX mAb

SEC

mAb

Small moleculesnucleic acids

HCDNA

HCPs

AEX

DAISEP MabXpure

HCP 27kDa pI 5.8

AEX

DNA 100-300kDa pI 5.5

AEX-SEC

IgG 150kDa pI 8.3

SEC

• SEC prevents antibodies from accessing the interiorof the MabXpure bead. The antibodies remain in the

• HCPs, DNA fragments, and small molecules arecaptured by AEX.

• The highly porous structure brings high-bindingcapabilities with multimodal adsorption features.

SEC (Size Exclusion) and AEX ( Anion Exchange)

$0

$100,000

$200,000

$300,000

$400,000

$500,000

$600,000

$700,000

$800,000

$900,000

$1,000,000

initial capex/10 Total cost/batch Total cost/year/10 Cost/kg mAb

MabXpure as Filter Aid

99 70 79 63

0

50

100

150

Time/batch (hrs)

$3M

$.2M

$30%

$1M

2+ day

COGS

• Various scenarios of MabXpure implementation are possible but the economical savings evaluation ishighly linked to the process steps in place.

• Economic improvements are possible for capital expenditure and total cost per batch and gram of mAb.• The use of MabXpure FT can speed-up the production time and save labor.

–$30%

Feed 1: HCP: 800 mg/mLHMW: 6%

Feed 2: HCP: 200 mg/mLHMW: 8.8%

Feed 1: HCP: 17 mg/mLHMW: 0.6%

Feed 2: HCP: <1.6 mg/mLHMW: 1%

Post ProA step HCP: <50 ppm HMW: 0.5%

Feed 1: HCP: 800 mg/mLHMW: 6%

Feed 2: HCP: 200 mg/mLHMW: 8.8%

Feed 1: HCP: 17 mg/mLHMW: 0.6%

Feed 2:

HCP: <1.6 mg/mLHMW: 1%

Post ProA stepHCP: <50 ppm HMW: 0.5%

Feed 1: HCP: 800 mg/mLHMW: 6%

Feed 2: HCP: 200 mg/mLHMW: 8.8%

Feed 1: HCP: 17 mg/mLHMW: 0.6%

Feed 2 : HCP: <1.6 mg/mLHMW: 1%

Post ProA stepHCP: <50 ppm HMW: 0.5%

OPTIMIZED CLARIFICATION PLATFORM

• rth,

• Orthogonal depletion mechanisms are simultaneously taking place during the alluvial

• After proteinA, the platform is reaching the drug substance criteria for HCP, DNA, HMW…•

100,000

1,000,000

10,000,000

100,000,000

total HCP HCP1 HCP2 HCP3 HCP4 HCP5 HCP6 HCP7 HCP8

Uni

t of

abu

ndan

ce

Post ProteinA

Post Mixed-Mode

Post ProteinA post MabXpure

Post Mixed-mode post MabXpure

Limit of identification

CO-ELUTED HCP DEPLETION PERFORMANCES

• MabXpure FT removes co-eluted HCPs that are not removed by classic technologies.•• MabXpure FT induces purer and safer formulations for the mAbs originally containing co-eluted HCPs.

43

•

• MabXpure depletes HCP and DNA, under staticconditions, by mixing the mAb-containing feedwith the resin.

• ed to recover a

•for impurity capture and polishing purposes.

• MabXpure FT depletes HCP and DNA under dynamicconditions from a mAb-containing feed.

• Traces of co-eluted HCPs and remaining DNA can ber

implementation

UPSTREAM DOWNSTREAM HARVEST FILL & FINISH

BIOREACTOR

CENTRIFUGE

ADF/CF

DFF PA CEX AEX

TFF

UPSTREAM HARVEST DOWNSTREAM FILL & FINISH

Objectives:•• n platform.• Target the capture of co-eluted HCPs all along the process that jeopardize the stability of

the formulation.

PROCESS SCHEME OF MABXPURE IMPLEMENTATION

43

2

1

5

MABXPURE AS FILTER CARTRIDGE (DYNAMIC PERFORMANCE)

• Dynamic conditions expose allspecies to MabXpure’s por ousstructure improving the depletion ofimpurities.

• >75% HCP depletion can beachieved with 90–100% mAbrecovery.

• MabXpure is designed to work with

improve HCP removal.

12%

20% 22%

36%

23%

100%

11% 18%

25%

52%

26%

10%

21%

39%

75%

35%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Feed 1-5cvs 5-10cvs 10-15cvs 15-20cvs Pool

1' 2' 5'

90% 100% 100% 100% 98% 96%

76%

93%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Feed 1-5cvs 5-10cvs 10-15cvs 15-20cvs Pool

1' 2' 5'

HCP Depletion Performances vs residence time mAb Recovery Performances vs residence time

CHO-K1, DNA 1.000 ng/mL, HCP 120 µg/mL, IgG 2 mg/mL

•

Phosphate ions do not impact the depletion.• Thanks to the AEX mechanism, virus clearance can

be claimed and up to 5 LRV for MVM and MuLV canbe reached.

• HCP depletion is observed after classic chromatographic steps of a

• With 3 chromo steps, MabXpure FT helps to streamline the process

• For a two-step process MabXpure can allow the removal of a bindand elute step.

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Feed No DF pH 7.2

50mM Na Acetate pH 5.6

50mM NH4OAc

pH 6.5

50mM Tris HCl pH 7.5

HCP Depletion Performances vs Diafiltration Buffer

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

PostProA

PostMM

PostAEX FT

HC

P L

RV

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

PostProA

PostMM

HC

P L

RV

HCP Depletion Performances vs 2 & 3 Steps DSP platform

0

1

2

3

4

5

6

1:10 1:25 1:50

LRV

Viru

s

MVM MuLV

Virus Clearance with MabXpure FT

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Feed No DF pH 7.2

50mM Na Acetate pH 5.6

50mM NH4OAc

pH 6.5

50mM Tris HCl pH 7.5

HCP Depletion Performances vs Diafiltration Buffer

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Post ProA

Post MM

Post AEX FT

HC

P L

RV

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Post ProA

Post MM

HC

P L

RV

HCP Depletion Performances vs 2 & 3 Steps DSP platform

0

1

2

3

4

5

6

1:10 1:25 1:50

LRV

Viru

s

MVM MuLV

Virus Clearance with MabXpure FT

CHO-K1, DNA 1.200 ng/mL, HCP 460–680 µg/mL, IgG 2.7–2.8 mg/mL

44

3

3

2

5

7 7 MabXpure as Filter Aid (Staticperformance)

100%

9%

60%

7% 9% 12%

37% 35%

69%

2% 5%

9%

23% 26%

88% 83%

97% 100% 98%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Feed 1:5 Silica

1:5 DE

1:5 1:10 1:20 1:50

HCPs DNA IgG

Depletion Performances vs mix ratio (resin:sample)

37%

27% 24%

100%

23%

3% 1%

98% 99% 99%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Feed 1:50 5'

1:50 30'

1:50120'

HCPs DNA IgG

Depletion Performances vs contact times

100%

45% 42% 44% 41%

97% 96% 97% 94%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Feed 6,5 7 7,4 8

HCPs IgG

100%

39% 35%

41%

93% 96% 98%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Feed 0,5 2 4

HCPs IgG

100%

16% 17% 24%

20%

99% 97%

77%

95%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Feed IgG1 IgG2 IgG3 IgG4

HCP IgG

100%

15% 15% 15% 23%

95%

69% 68%

78%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Feed Goat pH5 Rabbit pH5

Mouse pH5

Bovine pH6.7

HCP IgG

Depletion Performances vs Feed pH Depletion Performances vs IgG subclass

Depletion Performances vs mAb titer (g/L) Depletion Performances vs IgG origin

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Feed IgA 5 IgM 7.4 IgM 5

HCP IgG DNA

Depletion Performances vs IgG types

100%

45% 42% 44% 41%

97% 96% 97% 94%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Feed 6,5 7 7,4 8

HCPs IgG

100%

39% 35%

41%

93% 96% 98%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Feed 0,5 2 4

HCPs IgG

100%

16% 17% 24%

20%

99% 97%

77%

95%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Feed IgG1 IgG2 IgG3 IgG4

HCP IgG

100%

15% 15% 15% 23%

95%

69% 68%

78%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Feed Goat pH5 Rabbit pH5

Mouse pH5

Bovine pH6.7

HCP IgG

Depletion Performances vs Feed pH Depletion Performances vs IgG subclass

Depletion Performances vs mAb titer (g/L) Depletion Performances vs IgG origin

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Feed IgA 5 IgM 7.4 IgM 5

HCP IgG DNA

Depletion Performances vs IgG types

• Various process conditions (e.g., titer,pH) can be used without impactingbioburden depletion and mAb recovery.

• MabXpure insures a robust HCPs/DNAdepletion platform.

• All antibodies subclasses, types andorigins can be used with MabXpure.

• Adaptation of pH is required for low IgG’s isoelectric point and balance between HCP depletion and IgG recovery must be assessed.

•bioburden depletion (HCPs and DNA) and mAbrecovery.

• MabXpure guarantees combined performance for

• Flexibility of MabXpure resides in the mix ratio to beused and the time of contact.

• Increasing contact time improves bioburdendepletion.

• MabXpure adapts to your needs with contact timeand mix ratio.

• >1 LRV for HCPs and 2 LRV for DNA can beobtained.

MABXPURE AS FILTER AID (STATIC PERFORMANCE)

CHO-K1, DNA 1.000 ng/mL, HCP 180 µg/mL, IgG 2 mg/mL

SANOFI CASE STUDY

SANOFI CASE STUDY