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

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

Alex Xenopoulos*Alison Dupont, Christopher Gillespie, Ajish Potty, Michael PhillipsProcessing TechnologiesMerck MilliporeBedford, MA (USA)

Integrated Continuous BiomanufacturingA new ECI conference

Castelldefels, SpainOctober 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 | 21Oct20132

CONFIDENTIAL

Monoclonal antibody production

A mature, robust industry

Templated processProtein A chromatography

Yet, several issues remain

StabilityCapital and utilitiesLarge footprintFrequent bottlenecksSterilityCleaning validation


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New alternative template


Clarification Capture Purification/polishing

Current template

Alternative template

Bioreactor Centrifuge

2° depth filtration

Protein Ab/e chrom

CEX b/e chrom

AEX f/t chrom

Bioreactorw/ precipitation

1° depth filtration

Protein Ab/e chromcontinuous Carbon f/t

deviceAEX f/t device

CEX f/t device

Virus filtration UF/DF

CONFIDENTIAL

Comparison of templates – icons sized by device volume



Current template


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



Current template


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


0

100

200

300

400

500

600

700

800

900

1000

4 4.5 5 5.5 6 6.5 7

Turb

idity

(N

TU)

pH

Depth Filtered

Smart Polymer

StatusThree launched Clarisolve™ filters optimized for particle sizePortfolio of flocculantsContinuous harvesting and loading of protein A column successful and beneficial

BenefitsElimination of centrifuge up to 6,000 LIncreased 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 71-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%

StatusTwo incompressible resins available

Prosep® Ultra Plus Eshmuno® A

Continuous loading from clarified harvest and continuous loading to purification train successfully shown

BenefitsHigher productivity, especially at low residence timesResin 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

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

Two dilution steps – volume increaseLonger processing timeHigher water/buffer useLower selectivityLess virus removalLower yieldIncreased process developmentLess templatable

More expensive

Why not precipitation? Single-use Buffer consumption Processing time

More materialsAdditional unit operationsPrecipitant removalNo product concentrationDilution stepsNo purificationIncreased process development

More expensive at commercial scale


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

Prop

osed

Proc

ess

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

alPr

oces

s

CEX b/e AEX f/t VF withprefiltration


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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 AggregatesCEX

Low MW impurities(leached Protein A, HCP, fragments)

Carbon

Cell culture components Insulin, methotrexate, Pluronic

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

virusesProduct-related impurities Aggregates, fragments


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Benefits of flow-through purification

Disposable chromatography devices connected without pool tanksNo bind/elute chromatographic stepsMinimal interventionsOrthogonal mechanisms for impurity removalNeeded pH adjustments incorporated in skidOne skid (protein A elution TFF) is possibleEnables integrated, continuous process template


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


mAbMonomer

Yield(%)

AggregatesProtA VF pool

(%)

HCPProA 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 à 0.1 688 à 42 83 1.0 à <0.1 0.3 à 0 64 à <13 87 1.6 à 0.6 n/a 80 à 34 86 2.0 à 0.8 0.2 à 0 350 à 75 84 1.6 à 0.6 0.13 à 0 155 à <16 85 9.2 à 2.7 n/a 600 à 67 91 3.0 à 0.8 n/a 1468 à 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 processYield 92% 87%

Process-related impuritiesHCP: 11 ppm

Leached ProtA: 10 ppmDNA: < 10 ppb

HCP: 2 ppmLeached ProtA : 4 ppm

DNA: < 10 ppbProduct-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 Lcommercial

1 g/L @ 1,000 Lclinical

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

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Key features of the alternative templateAn alternative templated process for downstream purification of mAbs is proposedIt 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 template for monoclonal antibodies