Integration of Ion Exchange Chromatography in Downstream ...

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Integration of Ion Exchange Chromatography in Downstream

Processing of Proteins

SCI Training Course, Cambridge, September 2012

Sylvio Bengio, Ph.D.Pall Life Sciences

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DisclaimerThis presentation is the Confidential work product of Pall Corporation and no portion of this presentation may be copied, published, performed, or redistributed without the express written authority of a Pall corporate officer.

© 2012 Pall Corporation.

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Pall Chromatography Toolbox

Lab Scale Methods & Screening Scale-Up & ProductionProcess Development

� Chromatography sorbents� Mustang XT140 and XT5000

capsules� Resolute® columns / Packing

support� Packing stations� PKP and PK systems

� Chromatography sorbents� Mustang XT140 and XT5000

capsules� Resolute® columns / Packing

support� Packing stations� PKP and PK systems

� Chromatography sorbents� Mustang XT5 capsules� Mustang XT Acrodisc� 5 mL prepacked PRC

columns� Empty laboratory LRC

glasscolumns

�AcroPrep™ and AcroWell™ filter plates,SELDI Services for sorbent screening

�1 mL prepacked PRC columns�Prepacked AcroSep™ columns�Mustang® XT Acrodisc or XT5

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Defining Ion Exchange Chromatography

Ion exchange chromatography (�-än eks-ch�nj kr�-m�-tä-gr�-f�):�

A separation technique utilizing a stationary phase that contains either acidic groups for exchanging cations or basic groups for exchanging anions present in the mobile phase

under defined conditions of pH and ionic strength

“”

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Proteins have different isoelectric points

5.6

6.99.7

9.287.49

8.309.59

7.14

4.74

4.06 6.71

6.02

5.22 8.30

Isoelectric point (pI)Calculated using pK values of amino acids as described in: Bjellqvist, et al, The focusing positions of polypeptides in immobilized pH gradients canbe predicted from their amino acid sequences.Electrophoresis 1993, 14, 1023-1031

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Proteins have different hydrophobicities

-0.166

-0.586-0.314

-0.422-0.018

-0.261-0.876

-0.476

-0.553

-0.649 -0.197

-0.080

0.193 0.084

GRAVY (Grand Average of Hydrophobicity) scoreCalculated as the sum of hydrophobicity values of all amino acids, divided by the number of residues in the sequence.

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R-NH4+R-COOH

Binds to Anion exchanger

Binds to Cation exchanger[H+] = 10-1 moles/liter

[H+] = 10-14 moles/liter

When the pH is higher than the pI, the net charge on a protein is negative

-

When the pH is lower than the pI, the net charge on a protein is positive

+

pH 1

pIIsoelectric Point (pI) Net charge is zero 00

pH 14

R-COO- R-NH3

pH = -log [H+]

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Ion-Exchange Chromatography

� Routinely used in process-scale protein purification� Large number of products on the market� Anion and Cation exchangers� Applications : Mabs, polyclonal IgG, Recombinant

proteins, plasma derivatives, Vaccines …

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Main Ligands Used for Ion Exchange� Anion exchange

� Strong

� Weak

� Cation exchange

� Strong

� Weak

R-CH2CH2NH+(CH2CH3)2Diethylaminoethyl (DEAE )

R-N+(CH3)3Quaternary ammonium (Q)

R-CH2COO-

Carboxymethyl (CM or C)

R-CH2SO3-

Sulfonic acid (S)

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Formats: Beads or Membrane adsorbers

Sorbents Membranes

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« Salt Tolerant » Ion Exchange Ligands

� Vast majority of proteins acidic. Will be negatively charged around neutral pH or higher, anion exchange (Q, Deae) is more commonly used than cation (S, CM) for capture steps.

� Novel sorbents and membranes allow protein capture at moderate to high conductivities (e.g. >10 mS/cm) with high binding capacities.� For capture : direct load of feedstock� For polishing (flowthrough mode)

� Usually based on primary amine ligands

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Typical Steps in Ion Exchange

Equilibration Load Feedstock Wash Column

Re-equilibration Clean in Place Elution

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Ion Exchange Typical ChromatogramProteins are monitored by UV absorbance at 280 nm

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UF/DF

BioreactorBioreactorMedia Filtration

UF/DF Virus

Filtration

Membrane Chrom: DNA PolishingFinal Filtration

Media

Cell Separation & Clarification

Chromatography in Process

ChromatographyCapture

ChromatographyIntermediate

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Microfiltration (MF)

BioreactorBioreactor

Protein A

Ultrafiltration/Diafiltration (UF/DF) Virus

Filtration

Media

Example: Typical Mab Platform Process

Cation-exchange (CEX)

Anion-exchange (AEX)

Centrifugation Depth Filtration

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Bind/Elute and Flowthrough Modes

Bind and EluteBind and EluteTarget bindsTarget binds

Contaminants passContaminants pass

Target Target & &

ContaminantsContaminants

FlowthroughFlowthrough ModeModeTarget passesTarget passes

Contaminants bindContaminants bind

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Bind/Elute or Flowthrough mode ?

� Relative concentration of target� Relative pI of target vs main impurity� Feedstock volume � Subsequent downstream process� Target stability to process conditions� Production facilities and equipment

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Selectivity (�) : ��

�� Expressed as : V2 – V0 with V0 : void volume of the column

� = ------------- V1 : elution volume of peak 1 V1 – V0 V2 : elution volume of peak 2

W 1 W 2

V oV o lum e

V1 V 2 V t

Resolution : �� .

Expressed as : 2(V2 – V1) with W1 : peak 1 width at half peak height Rs = -------------- W2 : peak 2 width at half peak height W1 + W2

Selectivity and Resolution

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Chemical Composition of Sorbents

Composite

Polysaccharides

AgaroseDextranCellulose

Synthetic polymersAcrylic

MethacrylatePolystyrene-DVB

Mineral oxidesSilicaCeramic

Zirconium oxideHydroxyapatite

Sepharose®, Capto™, Ultrogel®

Sephadex®

HyperCel™, Cellufine™

Spherodex®, Spherosil®

Ceramic HyperD®

HyperZ®, ZirChrom®

CHT™, HA Ultrogel®,

Trisacryl®, UNOsphere™

Toyopearl® (GigaCap), Fractogel® EMD, Macro-Prep®

Mono®, Source™, Poros®

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Sorbent Chemistry has an impact on Selectivity

Anion exchangers:� Q rigid cellulose sorbent � Rigid Q agarose sorbent� Q polymeric sorbent

Cation exchangers: � S rigid cellulose sorbent� Rigid S agarose sorbent� S polymeric sorbent

Protein mix eluted by Linear salt gradient

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UF/DF Virus

Filtration


Media


Capture Step Chromatography

Capture


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Key parameters for Capture

� Selectivity for the target protein� Dynamic Binding Capacity for the target� Volume and conductivity of feedstock

� Most ion exchangers loose capacity if crude (non-diluted) feedstock is loaded: need for additional unit operations: UF/DF, dilution

� Final column volume and equipment issues

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Enhancing Target Protein Capture by IEX

� Check selectivity for your target� Check « real-life » Capacity for your target

� Vendor’s specs are only given for guidance, « real-life »capacities tend to be lower !

� Optimize loading, wash/elution pH …� Check elution column volumes� Optimize residence time (start with 4-5 min, decrease to

2 min. if positive)

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Prepacked Columns are convenient tools to check both Selectivity and Capacity parameters

� Packing performances guaranteed by the supplier

� Easy to connect to AKTA™ or other system

� Columns can be connected in series to mimic a bed height typical of a pilot-scale column

� Use typical flow rate of 1 mL/min (300 cm/hr)

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Elution Conditions

� Elution can be achieved as usual by salt gradient. � Elution may also be done by decreasing the pH. In this

case, it allows to recover the product at lowerconductivity allowing a direct load to an orthogonal ion exchanger or simplifying formulation

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Ion Exchange for Capture : purification of HSA from crude, undiluted plasma

� Direct capture of HSA from undiluted feedstock: Purity > 99%, Yield ~90%� Elution can be prompted by lowering pH only, no need to add NaCl, this

allows a direct orthogonal load on S HyperCel cation exchange sorbent.

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High Throughput Sorbent Screening

� HTS methods are well suited to quickly select the best sorbent for a specific step

� Saves time, sample and manpower� Needed at early stages of process development� Use 96 well plates combined with Design of

Experiments (DoE) software tools

Second international conference: Avignon, June 4-7, 2012

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High Throughput Sorbent Screening Platform (Pall)

TECAN Freedom EVO® workstation allows flexible and precise fullyautomated high throughput screening of different chromatographysorbents in AcroPrep™ 96-well filter plates.

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Capacity of Ion Exchange sorbents� Dynamic Binding Capacity (DBC) is a key parameter in

process chromatography.� To maximize productivity during capture steps, with

feedstream having high expression titers (e.g. Monoclonal antibodies 5-10 g/L)

� DBC varies according to the sorbent type, the linear flow rate or residence time

� pH and ionic strength of the feedstock also impact DBC.� New generation of « salt tolerant » sorbents

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Dynamic Binding Capacity vs. pH and Conductivity(BSA model protein)

High dynamic binding capacity (DBC) over a wide range of pHs and conductivities at short residence time leads to flexibility and better productivity.

Sorbent: HyperCel STAR AX (Pall) Column: 0.5 cm I.D. x 5 cm bed height (~1 mL); Sample: 5 mg/mL BSA in equilibration buffer; Equilibration buffer: 25 mM Tris-HCl, pH 7.0 – 8.5; Conductivity3 – 20 mS/cm; Residence time: 1 – 4 min (0.25 – 1 mL/min).

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HyperCel STAR AX89% Yield

DBC 30 mg/mLElution in 50 mM Na Acetate, pH 4.0

CEX95% Yield

DBC 60 mg/mLElution in 50 mM Na Phosphate,

0.15 M NaCl, pH 7.0

Cryo Poor PlasmapH 7.6, Conductivity 11 mS/cm

Rigid DEAE Agarose73% Yield

DBC 11 mg/mLElution in 50 mM Na Acetate, pH 4.0

CEX95% Yield

DBC 60 mg/mLElution in 50 mM Na Phosphate,

0.15 M NaCl, pH 7.0

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Loading undiluted plasma (11 mS/cm) on a conventional DEAE sorbent results in loss of Dynamic Binding Capacity (would need 3-6 fold dilution)

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Process Economics AnalysisBioSolve software

� Cost of goods (US$/g) reduced by almost 70%

� Significant savings on labour cost (>50%) and water usage (>55%)

� More than 20% higher throughput (kg/year)

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Ion Exchange at Capture Step: Conclusions� Check selectivity for the target first� Choose sorbent that maximizes Dynamic Binding

Capacity with minimal feed pretreatment (UF/DF, dilution)

� Consider re-equilibration duration, column volumes needed for elution and cleaning in place regimes

� Consider column final volume at production scale and equipment costs

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UF/DF


UF/DF Virus

Filtration


Media


Intermediate Step

ChromatographyCapture


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Key parameters for Intermediate step

� Selectivity for the target protein vs main contaminants� e.g. Host Cell Proteins, aggregates, misfolds….

� Consider Flowthrough mode (contaminants bind)� High resolution steps may help in some cases

� Smaller beads may also increase process costs

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Ion Exchange in Flowthrough mode : Removal of contaminants in rec. Protein (CHO)

� Target in Flow through, contaminants bind with higher capacity vs conventional sorbent

� Capacity at 15 mS/cm for contaminants >2-fold higher than Q rigid agarose

� Other data (not shown) also demonstrates:Excellent target recovery (no protein loss)Good removal of protein contaminants and pigmentsEfficient regeneration with 0.1 M HClfollowed by 1 M NaOH

CHO Cell Culture Supernatant Courtesy of Cytheris, France

0

1

2

3

4

5

6

7

8

HyperCel STAR AX Q Rigid agarose

DB

C f

or c

onta

min

ant p

rote

ins

mg/

mL

15 mS/cm

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Key parameters for Final Polishing Step

� High Resolution sometimes needed� Smaller beads may also increase process costs

� Consider Membrane adsorbers instead of packed columns � DNA and HCP removal� Disposale chromatography

Resolution in IEX is influenced by

� Buffer pH/Protein pI� Particle Size� Load� Gradient Slope� Flow Rate

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Hepatitis B Virus Particle

(0.05um)

B. diminutaBacterium (0.2um)

Yeast Cell (3um)

E. coli Bacterium (0.5 X 1.5um)

Human Hair

(75um)

IgG (0.03um, 160KD)

•Albumin0.01um,

67KD

Drawn to show relative size comparison.Shapes are not accurate representations

Particle Sizes

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50 µm100 µm

30 µm 20 µm

10 µm 5 µm Analytical:HPLC (>500 psi)

Semi-Preparative:MPLC, FPLC , Medium PressureLiquid chromatography(50 - 150 psi)

Preparative:Low Pressure Liquid chromatography (5 - 50 psi)

Bead Size and the Different Types of Chromatography

Small beads give high back pressure but great resolution, good for analytical work and demanding small scale preps

Big beads give low back pressure but less resolution, good for preparative work in large columns

Note that the selectivity is the same. All peaks elute in the same position, they only get broader as the particle size gets bigger.

Resolution and Particle size

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Membrane versus Resin Chromatography

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Benefits of Membrane Adsorbers vs Column Chromatography

� Disposable, plug and play, no time consuming equipment prep

� Less surface area but all of it is accessible to the product� Orders of magnitude faster flowrates� Use of a membrane adsorber will allow the polishing step

to be accomplished using a device of much reduced volume.

� Ideally suited for capture of large proteins, or polishing

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Ion Exchange Mustang� Membranes

� Polyethersulphone (PES) membrane substrate

� Cross-linked polymers containing functional groups – Q, S, E

� Nodular surface - high surface area

� High voids - 0.8 µm pores

� Multiple layers (16) - controlled bed depth

Mustang Q membrane

Mustang Q XT Capsules

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Mustang® Membrane Chromatography

5000 mL140 mL5 mL0.86 mL

XT5000XT140XT5XT Acrodisc®

10 mL

CLM05

60 mL

CL3

780 mL520 mL260 mL0.86 mL

NP8NP7NP6XT Acrodisc®

Single-Use Mustang Capsules : Q, S

Re-usable Mustang XT for Q chemistry

(standard) Acrodisc® Q & S : 0.18 mL not for scale-up Mustang E has different volumes : 0.12mL; 10; 40; 160; 320; 480 mL

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