TB1175EN00

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General Methodology

The dynamic binding capacity can bedetermined using either partiallypurified monoclonal antibody (MAb),

or feedstock.If partially purified MAb is used,

the breakthrough curve can be seendirectly by monitoring UV absorbance.In most cases, MAb, which hasalready been purified using protein Aaffinity chromatography media, can beused for this with the following notes:1. The pH of the purified MAb must

be adjusted to reflect that of thecrude cell culture supernatant.

2. The partially purified MAb will mostprobably be at a higher concen-tration than that in the cell culturesupernatant. In such cases, it isadvisable to dilute the MAb tobetter reflect the feedstock concen-tration. To some extent, dynamicbinding capacity will be affectedby the concentration of the MAb,especially at low titers of <100mg/liter. More dilute feedstock willrequire longer loading times. If onlylimited time is available, it is pos-

sible to perform a dynamic binding

capacity using higher concentrationMAb, but this should be noted

when presenting the final data.

3. Some antibodies can experience

partial denaturation on passagethough protein A media. For theseantibodies, a second passagethrough the same media may resultin a lower binding capacity.

4. Using MAb, which has alreadybeen partially purified on aprotein A column, means that theeluted MAb from the dynamicbinding capacity experimentcannot be used to give a reliableestimate of protein A leakage.

5. Using eluted antibody fromdynamic binding experiments

where the column is effectivelyoverloaded for the determination ofpurity will impact recovery andmay impact purity.

If clarified cell culture supernatant isused, the intrinsic UV absorbance ofthe feedstock will mask breakthroughof the MAb. In this case, furtheranalysis of the breakthrough fractions

will be required to determine thebreakthrough characteristics.

Determination of

Dynamic BindingCapacity forProSep-vA MediaIntroduction

The dynamic binding capacity of a chromatography media is the amount oftarget protein the media will bind under actual flow conditions before significant

breakthrough of unbound protein occurs. As this parameter reflects the impact ofmass transfer limitations that may occur as flow rate is increased, it is muchmore useful in predicting real process performance than a simple determination

of saturated or static capacity.

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Method

Pack a suitable column (e.g. Omnifit0.66 cm diameter column or VantageVL11 1.1 cm diameter column) to abed height of at least 10 cm (3.4 mLor 10 mL respectively) with ProSep-vA

Ultra media or ProSep-vA HighCapacity media. Ensure the column is

well packed using the column tappingmethod (as detailed in the ProSep-vAOperating Instructions).a. Use clarified cell culture supernatant

freshly passed through a 0.22 or0.1 µm filter, or

b. Prepare a solution of partiallypurified MAb in a loading buffer ata concentration and pH that reflectsthe feedstock conditions (in mostcases the equilibration buffer isappropriate). This feedstock shouldalso be freshly filtered as above.

1. Equilibrate the ProSep-vA column with equilibration buffer.

2. Isolate the column and pass MAbfeedstock through UV detector, todetermine the UV absorbance for100% breakthrough.

3. Flush the system lines and putprotein A column on line.

4. Start loading the MAb feedstock atthe desired flow rate or residencetime.

5. Monitor the UV absorbance of thecolumn eluate.

For an initial screen, it is recom-mended that a residence time of threeminutes be used. Residence time is thetime a nominal non-binding molecule

would take to pass through thepacked bed.

When using clarified cell culturesupernatant, it is normal to observe asignificant amount of UV absorbingmaterial passing through the columnafter approximately one column

volume.

When using partially purified MAb,a UV plateau may also be observed.This represents some impurities, butmainly non-binding or weakly bindingantibodies or aggregates resultingfrom the previous passage through aprotein A column. The level of thisplateau will depend upon theindividual MAb being used.

Figure 1.

Chromatograph with Clarified MonoclonalAntibody Cell Culture Supernatant

Table 1.

Suggested Fraction Sizes for a Range ofAntibody Feedstock Titers

MAb Titer 3 mL Column 10 mL Column(g/Liter) (Omnifit) (Vantage VL11)

0.10 22 mL 75 mL

0.25 9 mL 30 mL

0.50 4.5 mL 15 mL

0.75 3 mL 10 mL

1.0 2.3 mL 7.5 mL

1.5 1.5 mL 5 mL

2.0 1.1 mL 3.8 mL

3.0 0.8 mL 2.5 mL

4.0 0.6 mL 1.9 mL

320030002800260024002200200018001600140012001000800600400200

0-200 0 200 400 600 800 1000 1200

mL

m A U

Feedstock Load

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Using Clarified CellCulture SupernatantFor cell culture supernatant, the highamount of UV absorbing material willmask MAb breakthrough. It istherefore necessary to collect fractions

throughout the loading phase. Theamount of MAb loaded should becalculated so that it will exceed theanticipated capacity of the media, inthe case of ProSep-vA Ultra media,this should be loaded to a capacity of45 – 50 mg MAb/mL media.

The collected fractions can then beanalyzed for the presence of MAb.The early fractions should not containMAb. Initially, fractions at infrequentintervals (e.g. every 10th fraction) can

be tested to find the approximate volume in which breakthrough occurs.Once this region has been identified,all the surrounding fractions can betested. The smaller the fraction volume,the more fractions there will be andthe greater the resolution of thebreakthrough curve. Typical fractionsizes are given in Table 1.

Secondary analysis of the fractionscan be conducted using protein Achromatography (pH adjustment toneutral required). SDS-PAGE or ELISAcan be run as a semi-quantitative stepto screen many fractions to quicklyidentify the fractions of interest.

Once the titer of MAb in each ofthe relevant fractions has beendetermined, the data can be plotted togive a breakthrough curve similar tothat shown in Figure 2.

Using Partially PurifiedMonoclonal Antibody

Continue to load MAb until break-

through of IgG occurs. Breakthrough will be indicated by an increase in theUV absorbance of the eluate (Figure 2).Loading is stopped once the UVabsorbance of the eluting flow stream

reaches approximately 10% of the UVabsorbance of the pure feedstock (10% breakthrough). From the chro-matogram, the total mass of MAb,

which was bound before break-through occurred, can be calculatedby multiplying the concentration ofMAb in the feedstock by the total

volume loaded up to the desiredbreakthrough point (usually 5 or10%)less the column void volume (usuallythis can be determined by inspectionof the chromatogram – a slight shift inthe UV signal occurs after approxi-mately 0.7 x the bed volume). Fordilute feedstocks, this value can

usually be ignored.

Post Load Wash and Elution

Following each load, any remainingunbound material is washed off the

column with 7 –10 column volumes ofequilibration buffer. The addition of0.5 M NaCl to the wash buffer mayreduce any non-specific binding. Thebound antibody can then be eluted bya step change to low pH buffer(typical buffers are 100 mM Citrate,100 mM glycine, 100 mM acetatepH 3 – 4). After each run, anyremaining tightly bound antibodiescan be removed and the columnregenerated using 5 column volumesof a pH 1.5 solution of phosphoric

acid (150 mM) or HCl (10 mM)before re-equilibration in loadingbuffer.

Figure 2.

Breakthrough Curve with Partially PurifiedMonoclonal Antibody

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50

0

-50

-100

0 20 40 60 80 100 120 140 160 180 200

mL

m A U

Breakthrough of Purified MAb Load

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2400

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1200

600

0

-200

mL

Purified MAb Load

100 200 300 400 500 600

m A U

0

Breakthrough Formula

Mass bound = Co x (VL-Vo)

Where:

VL = volume loaded up to the breakthrough point Vo = void volume of the column

Co = concentration of MAb in the feedstock.Note: this is an approximation assuming complete capture of MAb prior to breakthrough.

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Effect of Residence Time

If time is limited, the above experimentshould be performed at the recom-mended operating flow rate for themedia. In the case of ProSep-vA Ultramedia, this should correspond to a

residence time of 3 minutes, or1.14 mL/min in a 0.66 x 10 cm bed.However, for a better understandingof the process characteristics, thedynamic binding capacity over arange of residence times can beconsidered. The efficient mass transfercharacteristics of ProSep-vA mediaallow faster flow rates to be used,decreasing cycle time and increasingthroughput.

In general, dynamic capacity will

decrease as the residence timedecreases, however the rate at whichthe dynamic (i.e. usable) capacitydecreases can vary greatly frommedia to media. An ideal media

would have efficient mass transferproperties across the range of flowrates being used. In practice, there isan upper limit to the flow rate that isdetermined by the mechanical strengthof the media, which is the case forcompressible media such as agarose,

or by the mechanical capabilities ofthe equipment being used (forexample, the pressure rating of aprocess scale column) or by thedegree of loss of capacity at veryshort residence times. An examplecurve of residence time vs. dynamiccapacity for ProSep-vA High Capacitymedia is given in Figure 3.

Shape of Breakthrough Curve

The relative sharpness of the break-through curve is indicative of the

efficiency of mass transfer within thepores of the chromatography media.The higher the efficiency, the sharperthe breakthrough curve. Ideally, underprocess conditions, the column wouldbe loaded to a point just prior tobreakthrough. In practice, a safetymargin is included and the column istypically loaded to 80 – 90% of the

breakthrough point. The sharper thebreakthrough curve, the nearer to100% utilization of binding capacitycan be achieved. As can be seen inFigure 2, ProSep-vA media has a verysharp breakthrough curve.

RecoveryFrom the above experiments, thedynamic binding capacity can bedetermined for a given residence time.Using this data, a column can beloaded to a working capacity of80 – 90% of the theoretical bindingcapacity. The loaded column can thenbe eluted at low pH (e.g.100 mMacetate buffer pH 3.5). The amount ofIgG eluted is determined andcompared to the amount loaded to

obtain the % recovery. Typically aprotein A column should produce>95% recovery. It is important to notethat the column conditions used todetermine the dynamic bindingcapacity will result in overloading thecolumn; therefore these data cannotbe used directly to determine recoveryof MAb, as they will always be low.

For optimum binding, the followingbuffers are recommended (for bufferrecipes, refer to ProSep-vA OperatingInstructions):1. PBS (phosphate buffered saline)

pH 7.4

2. 50 mM phosphate and 0.15 MNaCl pH 7.5

3. 50 mM phosphate, 0.5 M glycine,0.15 M NaCl pH 7.5

4. 50 mM Tris HCl, 0.15 M NaCl,pH 7.5

5. 1M glycine/NaOH + 0.15 MNaCl pH 8.6

6. 0.1 M borate + 0.15 M NaClpH 8.5

Binding buffers with pH greater than

pH 8.6 should not be used.Recommended elution buffers are0.1 M glycine/HCl, 0.1 M Naacetate buffer, 0.1 M citrate buffer,all pH 3.0 – 3.5.

Figure 3.

Effect of Residence Time and Breakthrough Pointon Dynamic Binding Capacity

ProSep-vA High Capacity Media

Residence Time(s)

0 100 200 300 400 500 600 700 800 900 1000 1100 1200

10% BT

5% BT

1%BT

40

35

30

25

20

15

10

5

0

C a p a c i t y ( m g / m L )

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Glossary

BreakthroughThe point at which some MAb passingthrough a packed bed is no longerable to find an available protein Abinding site. This is indicated by anincrease in the UV absorbance of the

process stream leaving the column.Clarified feedstock/cell culturesupernatantThe fermentation liquor from which thecells and large cell debris have beenremoved. This is normally clarified tothe extent that it can be passedthrough a 0.22 – 0.1 µm filter beforechromatography.

Dynamic binding capacityThe amount of antibody that can bindto a chromatography media under

flow conditions. This value is alwayslower than the static or saturationcapacity

FeedstockAny solution passed through thecolumn that contains the target proteinto be purified.

OverloadedA column which continues to beloaded after product breakthroughoccurs. Overloading the column canstill allow extra binding sites to beutilized and the capacity of an

overloaded column may be higherthan the same column loaded just tothe point of breakthrough. However,the efficiency in capturing the targetprotein is greatly reduced and there

will be loss of product as some passesstraight through the column.

Partially purified MAbMonoclonal antibody which hasalready been partially purified bypassage through another chroma-tography media before use in the

capacity determination. Purification isusually on a protein A media. In thiscase, the pH of the feedstock shouldbe adjusted to ensure it reflects the pHof the cell culture supernatant. Usingpartially purified MAb allows directdetermination of breakthrough andeliminates the need to performsecondary assays on the collectedfractions. However, caution must betaken that prior purification of theantibody does not affect its perform-

ance on the protein A media.

RecoveryThe recovery is the amount ofantibody eluted from the columncompared to the amount of antibodyloaded. Recovery is usually expressedas a percentage with complete

recovery being 100%. In dynamicbinding capacity studies, the column isoverloaded. As a result, recovery willalways be below 100% and shouldnot be used as an indication of thecolumn performance in the finalprocess.

Static binding capacityThe static or saturated capacity of acolumn is the amount of target proteinthe column can bind if every availablebinding site is utilized. This is deter-

mined by loading a large excess oftarget protein either at very slow flowrates or after prolonged incubation ina closed system. In practice, the staticcapacity is never available underprocess conditions using packedcolumns.

ThroughputThe throughput of a process is themass of antibody purified per unit oftime. To optimize a separation, it isimportant to consider the throughput

rather than the binding capacity, sincethe highest capacities will usually beachieved at the lowest flow rates andtherefore the slowest cycle times. It isonly by considering capacity and flowrate (i.e. cycle time) together that theoptimum throughput can bedetermined.

Appendix 1.

Suggested Process StepsFlow Rate (Based on

Column 0.66 cm x 10 cmStep Buffer Volumes (CV) Column)

Equilibration PBS pH 7.4 10 2.0 mL/min

Load Direct clarified Dependent on 1.14 mL/minfeed sample volume (Residence time 3 min)

and concentration

Wash PBS pH 7.4 10 – 15 2.0 mL/min

Elute 0.1 M glycinepH 3.5 7 – 10 2.0 mL/min

Regenerate Phosphoric acid

pH 1.5 5 2.0 mL/minRe-equilibrate PBS pH 7.4 10 2.0 mL/min

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Discover the More in Millipore™

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Omnifit is a registered trademark of Omnifit Limited.

Lit. No. TB1175EN00 Rev. - 03/05 Printed in U.S.A. 05-083© 2005 Millipore Corporation Billerica, MA 01821 U.S.A. All rights reserved.

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