HCIC IEX Scale Up

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42 BioProcess International February 2009

B i o P r o c e s s Technical

Purifying a Recalcitrant Therapeutic

Recombinant Protein with a Mixed-

Mode Chromatography Sorbent

Andrew Lees, Andrew Topping, Azzam Razzaq, Karine Reiter, and Alberto-Lopez Acosta

Product Focus: Proteins

Process Focus: Downstream

Processing (Purification)

Who should read: Process

DeveloPment anD manufacturing

KeyWords: chromatograPhy,

sorbents, mixeD moDe, hcic, hic,

imac, iex, scale-uP

level: intermeDiate

Mixed-mode chromatography sorbents can save time andmoney by reducing thenumber of steps required to

purify recombinant proteins. They also have the potential to purify proteins that single-mode sorbentscannot. As the term mixed mode suggests, these sorbents containligands that offer multiple modes of interaction. Although mixed-modesorbents are used extensively in solid-phase extraction for high-pressureliquid chromatography (HPLC)sample preparation — and to a morelimited extent in analytical HPLC —

these resins are generally unsuitablefor protein purification because theirpores are too restrictive and they areoften too hydrophobic for goodprotein recovery. Apart fromhydroxyapatite, such sorbents arerelatively new to the market.Currently they are not widely used inprocess-scale purification of recombinant proteins. This situationmay stem, in part, from the widespread assumption that mixed-

mode sorbents are poorly understood,mechanistically, so they would requireextensive studies to achievechromatographic optimization. Asdescribed here, that potentialimpediment does not apply to allmixed-mode sorbents. Based onstudies described here, our view is thatmixed-mode sorbents generally deserve much more seriousconsideration in designing process-scale purification schemes than they have received thus far.

Mixed-mode sorbents areinnovative for three reasons. First,they can facilitate efficient captureand significant purification in a singlechromatographic step because of simultaneous operation of orthogonalmodes of interaction. In our study, themixed-mode sorbent operates by acombination of hydrophobic andelectrostatic interaction. Simultaneousoperation of multiple interaction

modes in a single step may facilitatereduction of a conventional three-steppurification scheme to two steps.Second, mixed-mode sorbents provideunique selectivities that are notachievable by single-mode sorbentsused sequentially, so they enable someproteins to be purified when single-mode sorbent combinations fail. Third, certain mixed-mode sorbentsare capable of reducing endotoxins toclinically acceptable levels.

case history

Here we describe successful use of amixed-mode sorbent to purify arecombinant protein from Escherichiacoli needed for phase 1 and 2 clinicaltrials. We developed our purificationprocess after several other methodsperformed poorly in the capture step.Use of a mixed-mode sorbentproduced clinical-grade material afteronly two chromatographic steps.

avecia biologics ltd (www.avecia.com)




The target protein, a recombinant26-kDa zinc metalloenzyme (see the“Properties” box), was expressed in

E. coli at high yield (~4 g/L) as asoluble, intracellular protein. Proteinquantity and purity were determinedusing sodium-dodecyl sulfatepolyacrylamide gel electrophoresis(SDS-PAGE). Cells were harvested by centrifugation, then resuspended andhomogenized by three passes in ahigh-pressure homogenizer at 4 °C. The extract was clarif ied withpolyethyleneimine (PEI) and

centrifuged again.

initial caPture strategies

Considering the very high isoelectricpoint predicted from the targetprotein’s amino acid sequence (pI~10.5), our obvious choice for thecapture step was cation-exchangechromatography. However, the protein was not soluble in the crude extract atthe low ionic strength needed for IEX binding. Although the presence of

150-mM NaCl in the solutionprevented precipitation of the targetprotein, capture by ion-exchangechromatography at this ionic strength was poor (~50%) at pH 5–7 on severalcation exchangers. We then attemptedto recapture the target protein from

the flow-through fractions. Although yields were considered acceptable forpreliminary studies, for large-scalemanufacture we considered thisrecapture step to be undesirable.

We next evaluated capture f romcrude feedstock using immobilizedmetal-affinity chromatography

(IMAC). IMAC sorbents charged with zinc, nickel , copper, and coba lt were evaluated, with the highestlevel of capture observed used acopper-charged IMAC column.However, enzymatic activity of theeluted target protein wassignificantly reduced by thisprocedure. Furthermore, we foundthe concentrated solutions to beblue, which suggests incorporationof copper into the protein, possibly

through exchange of its zinc ion. Therefore, IMAC was ru led out. We also evaluated hydrophobic-

interaction chromatography (HIC)for the capture step. Screeningstudies using partially purifiedprotein were performed using an

HIC test kit from GE Healthcare,

with the best capture obta ined on aPhenyl Sepharose Hi Sub sorbent at0.5-M (NH4)2SO4, pH 6.9.However, we observed that thetarget protein was not tightly boundbecause it slowly migrated down thecolumn and began eluting duringthe loading process. In consequence, we increased the concentration of (NH4)2SO4 to 0.8 M. In trials withpartially purif ied protein, thisconcentration led to tight binding to

the Phenyl Sepharose, but additionof 0.8-M (NH4)

2SO

4to the clarified

crude feedstock led to precipitation. The prec ipitate conta ined asignificant proportion of targetprotein, which representedunacceptable product loss.

some Brands oF mixed-mode

sorBents For BioProcessing

Capto Adhere, Capto MMC (GE

Healthcare)

Hydroxyapatite (e.g., CHT ceramic from

Bio-Rad Laboratories, Fast Flow

crystalline from Calbiochem, and

HA-Ultrogel crystalline from Pall Life

Sciences)

MEP HyperCel, HEA HyperCel, and PPAHyperCel (Pall Life Sciences)

PolyABX and PolyCSX (JT Baker)

ProPerties oF target Protein

The target protein is a soluble

intracellular zinc metalloenzyme with

no cysteines. Its molecular weight is 26

kDa, and its isoelectric point (pI) is 10.5.

The protein loses activity at >~pH 7.2.

Figure 1: Rpr meP Hyprc

(Pall life sciences)

`

S

N

Derived from 4-Mercaptoethylpyridine

pKa = 4.8

Figure 2: mh

rp meP Hyprc r (Pall life

sciences)

HICAdsorption(hydrophobicassociation)

Desorption(ionic repulsion)

Figure 3: t r h r pr’ y py meP Hyprc r,

r k 2.9 /l 1.6 × 22.5 u pk h meP Hyprc

r 9 l/, pr -u r . r rkhruh urr

r e3, 1.5 l , hh py u 98 r pr

u pr r meP Hyprc r.

200

116976655

37

31

22

14

A 2

B 4

A 4

B 2

m w

KDa B 6

C 1

A 6

C 5

D 2

C 7

E 1

C 3

D 4

E 3

D 6

E 7 G

2 F 6

G 4

E 5

F 2

G 6

E 4

M E P L o a d

MEP Load Fractions

Figure 4: opz h

u pH ur hrrphy meP

Hyprc r (Pall life sciences.)

pH 5.5

pH 5.2

pH 4.9

pH 4.6

pH 4.3

pH 4.0

pH 3.0

Elution of relatively

hydrophilic proteins .


hydrophobic proteins .


basic proteins .


acidic proteins .



What WorKed — and Why

Studies using conventional HICsorbents suggested that a morehydrophobic sorbent might beeffective. Our search led us toevaluate the MEP HyperCel mixed-mode sorbent from Pall LifeSciences (www.pall.com). Its ligandincludes a thioether-linkedheterocyclic ring,4-mercaptoethylpyridine (MEP,

Figure 1). According to themanufacturer’s product literature,the ligand density specification is70–125 µM/mL of sorbent, asignificantly higher degree of substitution than for most HICsorbents and substantially higherthan even Phenyl Sepharose Hi Sub(40 µM/mL sorbent).

Figure 2 illustrates the mechanismof protein binding and elution onMEP HyperCel sorbent. Under near-neutral conditions, the pyridine ringis uncharged and hence hydrophobic.As the pH of the mobile phase isreduced and approaches the ligand’spKa (4.8), the net positive chargeincreases on the ligand, as does thenet positive charge on the boundprotein. Ultimately, desorption isprompted by electrostatic chargerepulsion, so high recovery of boundprotein is achieved despite the highdensity of hydrophobic ligand on thesorbent. Based on hydrophobic-

interaction binding and thepH-induced transition of the l igandfrom an uncharged to a chargedform, this form of mixed-modechromatography is referred to ashydrophobic charge-inductionchromatography or HCIC (1).

Although the target protein inour study is a nonantibody, it isnoteworthy that immunoglobulinsbind to the MEP HyperCel sorbent

through a combination of hydrophobic and af finity interaction.Specifically, antibodies have beenshown to have an affinity for ligandsbearing nitrogen heterocycles andthioether structures (2). Accordingly,this mixed-mode sorbent has beenused as a substitute for protein A inantibody capture (3, 4). It has alsobeen used effectively as a polishingstep in Protein A–based schemes (5).Because of the combinedhydrophobic and af finity interaction,antibodies typically bind to thesorbent without addition of binding-promoting salts (6, 7).

By contrast, nonantibody proteinstypically bind solely throughhydrophobic interaction, promoted by the addition of a lyotropic salt such as(NH

4)2SO

4or in some cases NaCl.

However, because of the high liganddensity of this sorbent, proteinbinding is typically accomplished at alower salt concentration than what is

required with conventional HICsorbents. Indeed, binding of ourtarget protein on MEP HyperCelmedia required addition of 0.5-M(NH4)2SO4, which was less than the0.8 M (NH4)2SO4 concentrationrequired for tight binding on PhenylSepharose Hi Sub media. Moreover,binding was maintained even when,following loading, the column was washed with (NH

4)2SO

4-free

equilibration buffer. This is inmarked contrast to typical behaviorusing conventional hydrophobic-interaction sorbents. Indeed, asdescribed more fully below, apreelution wash with an (NH

4)2SO

4-

free buffer at incrementally reducedpH values prompted desorption of amixture of impurity components butlittle or no desorption of the target

protein (Figures 5 and 6).Before proceeding to our findings,

consider two related mixed-modesorbents in relation to the one wechose (see the “Some Brands” box):HEA HyperCel and PPA HyperCelmedia. These two operate by mixed-mode hydrophobic-interactionchromatography rather than by HCIC. The distinction is a matter of chromatographic mechanism. ForHEA and PPA HyperCel brands, the

ligand does not typically undergotransition from an uncharged to acharged form during binding andelution.

The PPA ligand includes aphenylpropyl amine structure, whereas the HEA l igand includes ahexyl amine structure. The pKa is ~8for both. Thus, under the near-neutral to mildly alkaline conditionstypically used during binding (e.g.,pH 6.5–8.5), these ligands carry asignificant net positive charge. Forboth sorbents, elution is promptedthrough electrostatic charge repulsionby reducing the pH into the range of 3–5 (8), which confers a net positivecharge on the protein and causesdesorption.

If suppression or prevention of ion-exchange binding contributions isdesired during loading, 150-mMNaCl can be included in the buffer. To accomplish binding of very basicproteins, charge-repulsion effects

Figure 5: Rpr hrr , h, u r pr meP

Hyprc

sample load Post load washacetate

pH 6.4 wash

UV

0.0

0.5

1.0

1.5

2.0

2.5

3.0

AU

4.0

6.0

8.0

10.0

12.0

pH

150 200 250 300 350 400 450 500 550

acetate

pH 5.2 elution

citrate

pH 3.0 stripSample Load Post Load WashAcetatepH 6.4 Wash

UV

AcetatepH 5.2 Elute

CitratepH 3.0 Strip

pHpH



generally can be overcome by theaddition of a lyotropic salt and/oroperation at increased pH (e.g., pH ≥9). Except in such cases, binding istypically accomplished without needfor addition of lyotropic salt. As withMEP HyperCel media, binding inthe absence of added salt isaccomplished based on the highligand density of the sorbents.

Taken together, these threemixed-mode sorbents provide a usefulrange of retentivity and selectivity characteristics. As a practical matter,PPA HyperCel sorbent is the mostretentive of the three and MEPHyperCel sorbent is typically theleast retentive.

determination

oF Binding caPacity

To determine the binding capacity of MEP HyperCel sorbent for ourtarget protein, we performed aloading study using the E. coli feedstock. The column wasequilibrated with 25-mM sodiumphosphate buffer and 0.5-M(NH

4)2SO

4at pH 7.2. The PEI-

treated, clarified extract containing2.9 g/L of target protein was made0.5 M in (NH4)2SO4. Afteradjustment to pH 7.2, the feedstock

was fi ltered to remove traceparticulate matter and then loadedonto the column at a flow rate thatprovided a f ive-minute residencetime. Breakthrough occurred after~98 g/L had bound (Figure 3).

oPtimization oF

Washing and elution stePs

During our initial studies, the targetprotein was eluted from MEPHyperCel sorbent at pH 4.0. Theeluent was cloudy and <50% pure. Weachieved more selective desorption of the target protein and recovery of higher-purity product by optimizingthe elution pH, as described by Schwartz, et al. (9). We also paidcareful attention to the ionic strengthof washing and elution buffers, asrecommended by Weatherly et al. (10).Figure 4 illustrates the principlesunderlying our optimization based onelution pH.

As noted above, the pyridine ring

will take on a net positive charge aspH is reduced and approaches the pKa value of the MEP ligand. Forexample, at pH 5.8, the ligand willcarry a 10% net positive charge. Asillustrated in the Figure 4, basicproteins will tend to elute at higherpH values than will acidic proteins.Similarly, hydrophilic proteins willtend to elute at higher pH values than will hydrophobic proteins. Because ourtarget protein was both basic andrelatively hydrophilic, we expected itto elute at a higher pH than many of the host cell proteins, which tend tobe acidic or hydrophobic.

By sequentially washing the MEPHyperCel column with buffers of progressively lower pH, we determinedthe lowest value at which our targetprotein remained bound and the

highest pH at which it eluted. Washing at the lowest feasible pHremoved weakly binding impurities(Figures 5 and 6), with a preelution wash at pH 6.4. Eluting the targetprotein at the highest feasible pHfacilitated retention of more strongly binding impurities (Figures 5 and 6), with elution of the target fraction atpH 5.2. A mixture of strongly bindingimpurities is desorbed at pH 3.0.

Mixed-mode chromatography on

MEP HyperCel media thus achievedboth capture and a significant degreeof purification in one step. Moreover,binding was achieved at a saltconcentration lower than that requiredfor conventional HIC and within aconcentration range that preventedprotein precipitation. Equally important, the target fraction wasrecovered in dilute buffer at a pH value optimized for selectivedesorption of the target protein. Usingconventional HIC, the target fractionis frequently collected in a mobilephase that contains a significantconcentration of lyotropic salt. In suchcases, extensive diafiltration may berequired in advance of the next unitoperation.

Scale-Up: We initially developedour process using a 1-mL column. When scaled to 15 L, the only adjustment needed to widen the wash/elution window (increasing the wash pH and decreasing the elution

pH by 0.2 pH units) to make processspecifications less stringent.

Five 100-L fermentations producedabout 4 g/L of our target protein forpurification. The PEI-clarified extract was made 0.5 M in (NH

4)2SO

4and

adjusted to pH 7.2. It was then loadedonto a 15-L MEP HyperCel columnequilibrated with 25-mM sodiumphosphate and 0.5 M (NH4)2SO4 atpH 7.2. Figure 5 shows a

representative chromatogram, andFigure 6 shows a representative SDS-PAGE analysis of the feedstock andchromatographic fractions. We saw little or no target protein in the flow-through or wash fractions. Virtually all our target protein eluted at pH 5.2.Step yields of 75–95% were obtainedin f ive separate runs, with increasing yields obtained as we gainedexperience during successive runs. Inall cases, purity was >95%.

Finally, use of a preelution washstep at pH 6.4 is noteworthy becausethe MEP ligand would be expectedto carry little net positive charge atthis pH value, given that the pKa of the ligand is 4.8. Desorption of impurities thus would not beprompted by charge repulsion. Wespeculate that the impurity components desorbed at pH 6.4 may be rich in histidine. The histidineimidazole group has a pKa of ~6. AtpH 6.4, histidine imidazole groups

Figure 6: sds-Page y k

hrrph r r

hrrphy meP Hyprc r

FlowTh

rough

pH6

.4Wash

p H

5 . 2

E l u

t i o

n

pH3

Strip

Load




would be protonated to a significantextent. In protonated form, they would display reduced hydrophobiccharacteristics and might be

expected to facilitate desorption of histidine-rich impurities. So ourfindings suggest that a preelution wash step in this pH range may begenerally useful.

meP hyPercel and endotoxin

The capture chromatography step withMEP HyperCel reduced endotoxinlevels by more than 1,000-fold down to~0.3 endotoxin units/mg protein, which was within the tolerance set for the

final product. Endotoxins are bothhydrophobic and acidic, so they wouldbe expected to remain bound to thesorbent, which is positively chargedand hydrophobic at the elution pH.Indeed, buffer conditions that promotedesorption of the recombinant proteinthrough charge repulsion will causeLPS to bind more tightly to thepositively charged ligand. This suggeststhat MEP HyperCel sorbent generally should be effective for endotoxinclearance.

Nucleic acids should also besignificantly reduced by mixed-modesorbents containing a positive charge.Any DNA that does not flow through would be retained on the sorbentunder conditions used to promptelution of the target protein. In thecase of MEP media, lowering the pHto promote elution establishes a netpositive charge on the ligand,increasing binding of the nucleic acid.Very large reductions in nucleic acid

levels have been reported by Ferrieraet al. using this sorbent ( 4).

a successFul case

MEP HyperCel mixed-mode sorbentprovided effective purification of ourrecombinant therapeutic protein, for which other sorbents were unsuccessful. We achieved >95% purity, with 75–95%recovery during the capture step alone.Furthermore, this capture step reducedendotoxin to clinically acceptable levels,even though the feedstock waschallenging (a clarified E. coli extract).Significantly, binding was achieved withlower (NH4)2SO4 concentration than what was required using conventionalHIC. At this lower salt concentration,product precipitation and/or loss did notoccur, rendering mixed-modechromatography practical where

conventional HIC was not. Moreover,the target fraction was recovered in dilutebuffer, reducing or eliminating the needfor diafiltration following thischromatographic step. Given theiradvantages (as described in the“Advantages” box), we predict thatmixed-mode sorbents such as this willfind broad application in the quest torapidly develop cost-effective purificationschemes for therapeutic proteins.

reFerences

1 Burton S, Harding D. HydrophobicCharge Induction Chromatography: SaltIndependent Protein Adsorption and FacileElution with Aqueous Buffers. J. Chromatogr. A 814, 1995: 71–81.

2 Scholz GH, et al. Salt-IndependentBinding of Antibodies from Human Serum to

Thiophi lic Heterocyclic Ligands. J. Chromatogr.B Biomed. Sci. Appl. 709(2) 1998: 189–196.

3 Ghose S, et al. Evaluation and Comparisonof Alternatives to Protein A Chromatography Mimetic and Hydrophobic Charge InductionChromatographic Stationary Phases. J Chromatogr. A 1,122(1–2) 2006: 144–152.

4 Ferreira G, et al. A Two-ColumnProcess to Purify Antibodies WithoutProtein-A. BioPharm Int. May 1, 2007.

5 Chen J, Tetrault J, Ley A. Comparisonof Standard and New Generation HydrophobicInteraction Chromatography Resins in theMonoclonal Antibody Puri fication Process. J.Chromatogr. A 1177, 2008: 272–281.

6 Schwartz W, et al. Comparison of Hydrophobic Charge InductionChromatography with Aff inity Chromatography on Protein A for Harvest andPurif ication of Antibodies. J. Chromatogr. A 908, 2001: 251–263.

7 Guerrier L, et al. New Method forSelective Capture of Antibodies UnderPhysiological Conditions. Bioseparation 9,2000: 212–221.

8 Brochier VB, et al. Fast PurificationProcess Optimization Using Mixed-ModeChromatography Sorbents in Prepacked Mini-Columns. J. Chromatogr. A 1177, 2008: 226–233.

9 Schwartz W, et al. Application of Chemically Stable Immunoglobulin-Selective

Sorbents: Capture and Purification of Antibodies with Resolution of Aggregate.Bioprocessing J. (Sept/Oct) 2004: 53–62.

10 Weatherly G, et al. Initial Puri ficat ionof Recombinant Botulinium NeurotoxinFragments for Pharmaceutical ProductionUsing Hydrophobic Charge InductionChromatography. J. Chromatogr. A 952, 2002:99–110.

For Further reading

Arakawa T, et al. MEP Chromatography of Antibody and Fc-Fusion Protein UsingAqueous Arginine Solution. Prot. Express.Purif. 63(2) 2009: 158–163.

Zhao G, Sun Y. DisplacementChromatography of Proteins on HydrophobicCharge Induction Adsorbent Column. J.Chromatogr. A, 1165(1–2) 2007: 109–115.

Bak H, Thomas ORT. Evaluation of Commercial Chromatographic Adsorbents forthe Direct Capture of Polyclonal RabbitAntibodies from Clarified Antiserum. J.Chromatogr. B 848(1) 2007: 116–130

Dux MP, et al. Purification and Scale-Upof a Recombinant Heavy Chain Fragment Cof Botulinum Neurotoxin Serotype E in Pichia pastoris GS115. Prot. Express. Purif. 45(2)

2006: 359–367.

Mowry MC, et al. Production andPurif ication of a Chimeric MonoclonalAntibody Against Botulinum NeurotoxinSerotype A. Prot. Express. Purif. 37(2) 2004:399–408.

Coulon D, et al. Penicillin AcylasePurif ication with the Aid of HydrophobicCharge Induction Chromatography. J.Chromatogr. B 808(1) 2004: 111–115.

Boschetti E. The Use of ThiophilicChromatography for Antibody Puri fication: AReview. J. Biochem. Biophys.Meth. 49(1–3) 2001:361–389.c

corrspodg utor Andrew Lees s

stf drtor for F Bosoutos llc,

9610 Md ctr Drv Sut 200,

Rokv, MD 20850, 1-301-633-0464;

[email protected]; www.fbo.om.

Andrew Toppi ng s drtor of ry ps

dvopmt, d Azzam Razzaq s sor

t projt dr t av Boogs

ltd. Bgm, UK. Karine Reiter d

Alber to-Lopez Acosta r stff ststs

t Bosyxus, i. of Gtrsburg, MD.

advantages oF mixed-mode

chromatograPhy

Broadly, by combining multiple modes

of interaction in a single sorbent, mixed-

mode chromatography offers unique

selectivities and the potential for

reducing the number of

chromatographic steps in scheme. A

sorbent ligand may combine, for

example, hydrophobic, ion-exchange,

and pi–pi interactions. Specifically, in the

case study presented here, binding was

achieved at reduced salt concentrations

compared with conventional HIC and

allowed for product recovery in dilute

buffer, reducing the need for

intermediate diafiltration.

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