A new, integrated, continuous purification process … new, integrated, continuous purification process template for monoclonal antibodies Alex Xenopoulos* Alison Dupont, Christopher

A new, integrated, continuous purification process template for monoclonal antibodies

Alex Xenopoulos* Alison Dupont, Christopher Gillespie, Ajish Potty, Michael Phillips Processing Technologies Merck Millipore Bedford, MA (USA)

Integrated Continuous Biomanufacturing A new ECI conference

Castelldefels, Spain October 20-24, 2013

CONFIDENTIAL

Highlights

We developed a flow-through purification train that enables an integrated, continuous process

We have novel solutions for continuous clarification and capture

Bench-scale proof of principle for several mAbs shown

Breakthrough improvements not possible unless you look at new technologies

AX | ECI Castelldefels | 21Oct2013 2

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Monoclonal antibody production

A mature, robust industry Templated process Protein A chromatography

Yet, several issues remain Stability Capital and utilities Large footprint Frequent bottlenecks Sterility Cleaning validation


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New alternative template


Clarification Capture Purification/polishing

Bioreactor Centrifuge

2° depth filtration

Protein A b/e chrom

CEX b/e chrom

AEX f/t chrom

Bioreactor w/ precipitation

1° depth filtration

Protein A b/e chrom continuous Carbon f/t

device AEX f/t device

CEX f/t device

Virus filtration UF/DF

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Comparison of templates – icons sized by device volume



3.3 m2

4.4 m2

14.1 L 14.1 L 19.3 L

0.6 L each

5 L 0.4 L 3 L

1,000 L @ 2 g/L

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Comparison of templates – pool tanks



1000 L 500 L

50 L

250 L

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Clarification assisted by precipitation and using novel Clarisolve™ filters results in post-Protein A benefits


0100200300400500600700800900

1000

4 4.5 5 5.5 6 6.5 7

Turb

idity

(NTU

)

pH

Depth Filtered

Smart Polymer

Status Three launched Clarisolve™ filters optimized for particle size Portfolio of flocculants Continuous harvesting and loading of protein A column successful and beneficial

Benefits Elimination of centrifuge up to 6,000 L Increased throughput (<3x membrane area) DNA removal (1-2 LRV) Advantages persist post protein A Reduced turbidity Enhanced HCP clearance Reduced resin cleaning

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Capture with continuous multicolumn chromatography and incompressible Protein A resins offers savings

8 AX | ECI Castelldefels | 21Oct2013

RT (min) Effective DBC (g/L)

Productivity (g/L/hr)

1-column batch 4 39 7 1-column batch 0.22 7 19

3-column continuous 0.22 37 136

Effective DBC (g/L)

RT (min)

Consumed resin (L)

Consumed buffer (L)

Batch 39 4 21 2646

Continuous 45 0.5 2.8 2009

Savings 87% 24%

Status Two incompressible resins available Prosep® Ultra Plus Eshmuno® A

Continuous loading from clarified harvest and continuous loading to purification train successfully shown Benefits Higher productivity, especially at low residence times Resin and buffer savings

0

10

20

30

40

50

60

70

80

0 0.5 1 1.5 2 2.5 3 3.5

DBC

@ 1

% B

T (g

/L)

Residence time (min)

Two-column continuousOne-column batch

time savings

buffer/resinsavings

Presenter

Presentation Notes

1000L feedstock, 1 g/L

CONFIDENTIAL

Protein A capture cannot be beaten as part of a holistic process evaluation Why not CEX chromatography? Cheaper resin Cheaper unit operation

Two dilution steps – volume increase Longer processing time Higher water/buffer use Lower selectivity Less virus removal Lower yield Increased process development Less templatable

More expensive

Why not precipitation? Single-use Buffer consumption Processing time

More materials Additional unit operations Precipitant removal No product concentration Dilution steps No purification Increased process development

More expensive at commercial scale AX | ECI Castelldefels | 21Oct2013 9

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Purification in flow-through mode using novel adsorbers, minimum interventions, fewer pool tanks and one skid

Prop

osed

Pr

oces

s

Low pH VI Pool

VF Pool

Carbon + AEX f/t

CEX f/t + VF

In-line pH

Low pH VI Pool

CEX Pool

AEX Pool

VF Pool

Trad

ition

al

Proc

ess

CEX b/e AEX f/t VF with prefiltration


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Novel flow-through adsorber functionalities work synergistically to remove several classes of impurities

MAb

acidic pI basic

Low

M

W

high

Larger acidic HCP, DNA, viruses

AEX

mAb Aggregates CEX

Low MW impurities

(leached Protein A, HCP, fragments)

Carbon

Cell culture components Insulin, methotrexate, Pluronic

F68®, hygromycin, antifoam C Process-related impurities DNA, HCP, leached Protein A,

viruses Product-related impurities Aggregates, fragments


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Benefits of flow-through purification

Disposable chromatography devices connected without pool tanks No bind/elute chromatographic steps Minimal interventions Orthogonal mechanisms for impurity removal Needed pH adjustments incorporated in skid One skid (protein A elution TFF) is possible Enables integrated, continuous process template


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Internal bench-scale experimental case studies: Robustness of flow-through purification train (3 mAbs)


mAb

Monomer Yield (%)

Aggregates ProtA VF pool

(%)

HCP ProA VF pool

(ppm)

VF Capacity (kg/m2)

mAb04 88 N/A 250 2 > 3.5

mAb05 92 5.0 1.0 591 1 >3.6

mAb07 91 1.4 ~0 82 1 >3.7

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External trials: Robustness of flow-through purification train (7 mAbs)


# Monomer yield (%)

Aggregates (%) Fragments (%) HCP (ppm)

1 91 5.1 0.8 1.2 0.1 688 4 2 83 1.0 <0.1 0.3 0 64 <1 3 87 1.6 0.6 n/a 80 3 4 86 2.0 0.8 0.2 0 350 7 5 84 1.6 0.6 0.13 0 155 <1 6 85 9.2 2.7 n/a 600 6 7 91 3.0 0.8 n/a 1468 7

Loadings of activated carbon and f/t CEX devices were 0.5 – 1.0 kg/L

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Internal case studies: Product quality


Current process Alternative process Yield 92% 87%

Process-related impurities HCP: 11 ppm

Leached ProtA: 10 ppm DNA: < 10 ppb

HCP: 2 ppm Leached ProtA : 4 ppm

DNA: < 10 ppb Product-related impurities

(% HMW/Main/LMW) 1/98/1 0.5/99/0.5

Charge variants (% Acidic/Main/Basic) 15/71/13 13/72/15

Glycan profile (% Gal: 0/1/2) 79/19/2 79/20/2

Higher order structure (CD) No change No change

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Cost of Goods: where is the advantage?


% cost savings for DSP process 5 g/L @ 5,000 L commercial

1 g/L @ 1,000 L clinical

Old batch New continuous 24% 35%

12

5

3

3

15

16

4

2

0

10

20

30

40

Old batch New continuous

DSP

cost

($/g

)

5 kL @ 5 g/L commercial laborconsumablesmaterialsfacility

6537

4142

155

91

62

39

0

100

200

300

400

Old batch New continuous

DSP

cost

($/g

)

1 kL @ 1 g/L clinical laborconsumablesmaterialsfacility

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Process modeling: advantages of proposed template


Parameter for DSP portion Units Current process

Alternative process

% change

Equipment cost $M 6.9 3.1 55%

Footprint m2 87 59 32%

Water use (incl cleaning) L/g of mAb 24.2 1.4 94%

Buffer use (excl WFI) L/g of mAb 2.4 1.0 58%

Processing time hrs 55 30 45%

Cost $/g of mAb 219 109 50%

1,000 L @ 2 g/L | 2 kg batch | ~70% yield

CONFIDENTIAL

Key features of the alternative template

An alternative templated process for downstream purification of mAbs is proposed It matches performance of current templates, provides operational advantages Features:

• Novel downstream purification process for mAbs – from bioreactor through formulation • Connected unit operations – continuous operation, minimal interventions • Novel unit operations developed – leverage continuous nature • Clarification toolbox – novel depth filters, precipitating agents • Product capture with continuous multicolumn protein A affinity chromatography –

efficient use of resin and buffer • Flow-through polishing – no bind/elute steps, improved simplicity and economics • Virus filtration and ultrafiltration/diafiltration – no changes • Proof of concept and feasibility data generated – performance equivalent to current,

advantages in overall operational flexibility


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Acknowledgments

Downstream Technologies, MM • Kevin Galipeau • Meghan Higson • Jad Jaber • Mikhail Kozlov • Matthew Stone • William Cataldo • Romas Skudas • Jeff Caron • Jonathan Steen • Scott Bliss • Dennis Aquino • Wilson Moya

Analytical Technologies, MM • Rong-Rong Zhu • Michael Bruce Team Supply, MM • Michael McGlothlen • Patricia Kumpey • Paul Hatch Business Development, MM • Fred Mann BioPharm Services, Inc • Andrew Brown


CONFIDENTIAL

Documents

A new, integrated, continuous purification process … new, integrated, continuous purification process template for monoclonal antibodies Alex Xenopoulos* Alison Dupont, Christopher