qualification of a chromatographic column

30 BioPharm International MARCH 2003

Preparative chromatography is the dominantpurification technique in the production ofbiological compounds, especially forbioseparations. The column used is centralto the performance of the chromatographic

step. To ensure robust and reproducible columnperformance, columns need to be qualified in anacceptable fashion before each use.

Some of the critical elements of column quali-fication include identifying leachables andextractables from resins, product use, and columnparts and accessories (such as residuals frommanufacturing and shipping). Identity tests mustbe developed for chromatography resins, such asbase matrices (FTIR spectroscopy, for example);functional groups and substitution levels (such ascolorimetric titration); and porosity tests, or ifnecessary, function tests (such as dynamic capac-ity, selectivity, or real runs). Column qualifica-tion also involves writing protocols for trainingplant personnel and protocols for column hard-ware, packing, and operation. And qualificationrequires protocols for measuring bed integrity,such as background buffers, tracers, and injection

Qualification of aChromatographic ColumnWhy and How to Do ItAnurag S. Rathore, Robert M. Kennedy, J. Kevin O’Donnell, Ivars Bemberis, and Oliver Kaltenbrunner

volumes (see the “Measuring Column BedIntegrity” sidebar for further information andequations that help confirm the quality of chro-matographic operations).

This BioPharm International series on the “Elements of Biopharmaceutical Production”presents the viewpoints of industry experts onissues commonly encountered in developing andmanufacturing biopharmaceuticals. The serieshas previously covered process validation (1) andthe optimization and scale-up of preparative chro-matography (2). In this article, our series coordi-nator has brought together four other expertsrepresenting major chromatography media andequipment manufacturers and from biopharma-ceutical companies to offer their viewpoints,insights, and experience on various facets ofchromatography column qualification.

ROBERT M. KENNEDYQualifying Protocols and HardwareQualification is thepart of process vali-dation that ensuresthat the equipmentand protocols are ca-pable of fulfilling thespecified require-ments. Column qual-ification is documen-tation of work doneto ensure that the column and the protocols writ-ten to direct operation of the column are appro-priate for the purification step intended. Columnqualification can be divided into two broad sub-

Elements ofBiopharmaceutical Production

FDA has become more knowledgeable about processchromatography — and more demanding about columnqualification. In the latest installment of BioPharmInternational’s “Elements of BioPharmaceuticalProduction,” five industry experts share their insightson how to qualify a chromatographic column. Theiradvice: Write unambiguous SOPs. Focus onreproducibility of column packing. Choose appropriatemetrics. And analyze your testing procedures to reducethe chance of erroneous results.

BioPharm International MARCH 2003 31

A reference to the process workflow diagramshould call out all the details of an individual col-umn. The column can be defined by its serialnumber, its asset property number, or its unitoperation number. The construction materialsshould be verified as part of the overall validationprogram, and records of those materials need tobe kept in the validation record. Leachables andextractables from those materials need to be veri-fied as well.

If the column is mobile; the process work dia-gram should show how the column travels, thatis, where it is stored, where it is packed, how it istransported, where it is tested, and how it isremoved from service and transported out of theoperations area.

ject areas: column hardware qualification andcolumn protocol qualification.

In column hardware qualification, the column sizeand manufacturing materials need to be docu-mented. The intended packing material needs tobe associated with a particular column. Resinqualification and the workflow associated withresin is another topic to be documented. The lo-cation in the plant needs to be established. Aschematic needs to diagram the column and itsassociated peripheral equipment. Peripheralequipment, such as tubing and hoses, pressuregauges, clamps, and gaskets needs to be docu-mented. Spare parts must be associated with aparticular column as well.

After a column is packed, integrity of the column bed needs tobe measured to confirm the quality and consistency of thechromatographic operations. Several measures are commonlyused for this purpose.

Number of Plates (N)

To calculate the number of theoretical plates for a column, use:

where tr is the retention time of the probe molecule, and Wb isthe width of the peak at the baseline.

Alternatively, use

in which W1/2 is the width of the peak at one half the maximumheight (figure a). Both of these equations are used widely forestimating N and yield very similar results.

For comparing two different columns, number of plates permeter can be calculated using:

where L is the total length of the column.

Height Equivalent to the Theoretical Plate (HETP)

This equation also normalizes column performance to the lengthof the column, so it can be used to compare performance:

Another equation that uses a variant of HETP is reduced plateheight h, which normalizes HETP for particle diameter dp, and isdefined as:

Tailing Factor (T)

The tailing factor of the peak can be defined as:

in which W0.05 is the width of the probe peak at 5% of full height,and f is the distance from the leading edge to the midpoint of thepeak.

Asymmetry (As)

Asymmetry is defined as:

where a is the distance from the leading edge of the peak to themidpoint of the peak, and b is the distance from the midpoint ofthe peak to the trailing edge (figure b). As is routinelydetermined at 5% or 10% of maximum peak height. As valuesbetween 0.8 and 1.4 are typically suitable. As shown in the Tand As equations, As is not the same as peak tailing.

A s = ba

T=W0.052 • f

h = HETPdp

HETP= LN

platesmeters

= NL

N =5.54t r

W1/2

2

N =16t r

Wb

2Injection

tr

W1/2 1/2 h

h

(a)

(b)

Injection

a b 1/10 h

h

Measuring Column Bed Integrity


The schematic diagram shows all the associ-ated lines into and out of the column. The processworkflow diagram indicates the tanks that areconnected to the inlet side and which tanks areconnected to the outlet. Product is collected intoone of the tanks on the outlet. People responsiblefor the process need to know the location and thecondition of the product at all times. Qualifica-tion of these tanks and fluid path transfers isanother part of hardware qualification. Includedin the validation package for the column is theengineering drawing of the column. Spare partsare associated with an individual column, andengineers performing the scheduled maintenanceon the column can be expected to require accessto this part of the validation record to ensure thatthe appropriate spare parts are used in mainte-nance and reordered once they are consumed.Documentation of the gauges used to operate acolumn should include the calibration record ofthe gauge as well as the serial numbers or assetnumber to ensure that the correct gauge is inplace during operation.

In column protocol qualification, process develop-ment work can contribute to the validation of thechromatography step by providing data on thestability of the chromatography resin under equi-libration, operating, regenerating, cleaning andsanitizing, and storage conditions. To operate thecolumn, an associated set of standard operationprotocols (SOPs) needs to be written and tested:SOPs must be unambiguous. Regulatory require-ments exist to ensure that operators are trainedand regularly retrained to maintain their compe-tence in performing the operation.

Each column in the purification process needsto have SOPs for column packing and testing.Once accepted for use, each column needs SOPsfor equilibration, running (that is, applying feedto a collection of product), regenerating, andcleaning. SOPs are needed to direct the collectionof data for use in ascertaining performance qual-ity, to document how preventive maintenanceshould be done, and to instruct on how materialshould be stored.

Assistance with writing many of these SOPs isoften available from the appropriate equipmentand chemical suppliers. The supplier’s technicaldepartment can be a valuable contributor to in-house writers of these protocols. Chromatographysuppliers can contribute to SOPs on columnpacking, performance quality, maintenance, andstorage, for example. Engineering companies can

provide assistance with the process workflow dia-grams and advice on data storage related to thephysical column and the drawings and parts.

Purification by chromatography is at the heartof biopharmaceutical production. Column qualifi-cation is a critical activity in the assurance ofquality in the production area.—Robert M. Kennedy is department director, separation

sciences, Amersham Biosciences Corporation, 800 Cen-

tennial Avenue, Piscataway, NJ 08855, 732.457.8438,

[email protected].

J. KEVIN O’DONNELLThe Science of Column PackingRecent FDA inspections have shown an increasedinterest in the packingcharacteristics ofprocess-scale, liquidc h r o m a t o g r a p h ycolumns. That is man-ifested by theagency’s emphasis onreproducible packingof chromatographycolumns by pharma-ceutical and biotechnology companies. Althoughprocess-scale column packing is still, in manycases, more of an art than a science, FDA — by itsinquiries at inspections — is encouraging compa-nies to move toward a column-packing science. Alarge part of that science is how a column is charac-terized or qualified after it is packed.

Resins. Many variables influence the science ofcolumn packing. The first variable is the chro-matographic resin. A chromatographic resin isselected primarily for its ability to separate themolecule of interest from its impurities, not onhow well it packs into a manufacturer’s column.Many different resins are used in process chro-matography, and all have individual column-packing characteristics.

Some resins are soft and cannot withstandeven moderate pressures. As a result, whencolumns are packed with this group of resins,20–30% more resin often has to be added to getthe desired column volume at standard operatingpressures. Semirigid resins, including those basedon silica, are better able to withstand normaloperating pressures and require only 5–15% extraresin to get to the specified volume. Silica resinsare pressure resistant, so they can be used incolumns with extremely high backpressures.Silica resins require little additional resin for effi-cient column packing.


BioPharm International MARCH 2003 33

Resin slurry concentration is often over-looked when packing process-scale columns. Forunknown reasons, resin can pack differentlywhen charged to the column at different concen-trations. A slurry concentration that works in onecolumn may not work as well in another manu-facturer’s column. Resin manufacturers generallyhave a good idea of what slurry concentrationworks best with different column technologies.

Column packing. Traditional columns withadjustable upper adaptors can be packed repro-ducibly with repeated practice. The caveat here isthat it may take several attempts to get the firstoptimal pack. Once the technique is determined,subsequent packing can proceed more efficiently.Newer process-scale column designs, whichinclude dynamic axial compression and self-packing technologies, are generally more userfriendly and can be packed successfully on thefirst try. However these columns require moreup-front training, and the hardware is usuallymore expensive because it includes additionalequipment specially designed to assist in columnpacking. Expanded-bed columns may be the easi-est to pack; however they also require more su-pervision while running.

Ideally, all resins should be defined beforepacking, eliminating any small fragments that canbe generated by shipping and handling resin.Trace amounts of fines may not have any effecton the actual chromatography, but can eventuallylead to occlusion of the bottom screens or frits.Such occlusion would ultimately create increasedbackpressure, which would require a reducedflow rate. As a result, the column would have areduced throughput.

The mobile phase plays an important role inpacking process columns. Generally the solventthat packs the resin most tightly is preferred.Placing and securing the upper adaptor directlyon the top of the settled bed minimizes swellingwhen the mobile phase is changed. Exceptions tothis method include some polymeric reversed-phase resins that can swell 15–25% in organicsolvents. Under those conditions, a fixed adaptoror column housing could fail, with resulting obvi-ous safety concerns. Allowing a void above theresin bed or using a dynamic axial compressioncolumn that moves with the shrinking andswelling of the resin prevents that.

Test result variations. Once a column is packed,a series of standard tests should be conducted toevaluate performance and qualify the column.The results of these tests do not predict success inthe actual chromatography step; however, theresults are useful for column packing repro-

ducibility. It is this reproducibility that mostinterests FDA.

Usually a small, unretained probe molecule isused for standard column tests. Most chromatog-raphers use a UV-absorbing molecule such as p-aminobenzoic acid (PABA) or acetone.Alternatively, concentrated sodium chloridespikes are monitored using a conductivity detec-tor. Many more companies use sodium chloridebecause it is almost always used in the mobilephase of applications that don’t use reversed-phase chromatography (RPC).

Injection volume becomes more important asparticle size decreases. A 0.1% column volumeinjection is a good starting point. The flow ratefor such a test should be 60–80 cm/hr for best re-sults. Data can be analyzed by computer ifrecorded digitally, or if not, data can be calcu-lated by hand.

The location of the “injection loop” is impor-tant and should be located as close to the columninlet as possible. The detector at the bottom of thecolumn should also be located as close to the col-umn as possible. Sometimes neither suggestion ispossible. In such cases, column performance willbe noticeably less than what is observed at benchscale. That might not be interpreted as a negativeresult as long as the root cause is known, and theresults are consistent. In future column-packingtests, similar results must be observed.

Fronting can occur if sodium chloride isinjected onto an ion-exchange resin with a watermobile phase. Adding salt to the mobile phase

As�0.8• Overpacking the column

• Packing at too high of a pressure

• Column bed cracking

As�1.4• Column not packed “tight” enough

• Clogged screens or frits at top or bottom of the column

• Air pockets in column hardware void spaces

• Poor injection technique

High HETP• Injection sample or detector too far from

column

• Column not packed efficiently

Low HETPProbe molecule retained on column becauseof interaction with functional group orbackbone

Troubleshooting Performance Evaluation


(0.1 M NaCl, for example) usually eliminates thatproblem. With hydrophobic-interaction chro-matography (HIC) or RPC resins, using acetoneor PABA can generate tailing or higher-than-nor-mal height equivalent to a theoretical plate(HETPs) because of interaction with the func-tional group or resin backbone. Adding smallamounts of organic solvent (10–20%) to the mo-bile phase eliminates that interaction (see Table 1for troubleshooting performance evaluations).

Operators on the manufacturing floor shouldknow how column performance affects the sepa-ration of the target molecule. In some cases, less-than-optimal column performance values may betolerated because of the column’s function. Forinstance, higher HETPs may be acceptable whenthe column is used for a simple capture step witha step elution. Tailings may also be acceptable ifno closely eluting impurities are found and theextra volume is handled easily in subsequent unitoperations.

FDA is now more knowledgeable about opera-tions using process-scale column chromatogra-phy. It is now incumbent on pharmaceutical andbiotechnology companies to address FDAinquiries with more appropriate scientificresponses. One of the ways to do this is throughreproducible column packing as indicated bycomparing the performance of one column packto another. That, in turn, helps to predict repeatedsuccess in purifying the target molecule. —J. Kevin O’Donnell is the technical service manager at

Tosoh Biosciences LLC, 156 Keystone Drive, Mont-

gomeryville, PA 18936, [email protected].

IVARS BEMBERIS Choosing the Right MetricTo ensure robustchromatographic per-formance, it is essen-tial to have a properpacking and qualifi-cation protocol. Be-cause chromato-graphic purificationtypically involvesseveral columns,packing and qualifying columns is an importantprocess validation concern. An effective packingand qualification protocol typically consists of aprepacking checklist, a description of the media’sproperties, choosing the right metric for columnqualification, and qualifying the packing protocol.

Prepacking checklist. A column’s mechanicalrobustness must be ensured before packing. That

should include an inspection of all column seals,bed supports, and bolts to ensure they are fit foruse. Hydrophobic bed supports, such as polyeth-ylene, must be prewetted and checked for uni-form wetting. Before packing, the column shouldbe assembled and pressure tested with water-for-injection (WFI) to a pressure in excess of thehighest operating pressure anticipated during theprocess.

Media properties. Understanding the flow andpressure characteristics is essential to reliablepacking. Determining the compressibility of themedia to be packed is most critical to ensuringthat the proper amount of media is packed intothe column. As always, the conditions of packingmust exceed those to be experienced during theprocess to ensure bed stability and an extendedcycle life for the packed column.

Choosing the metric. An appropriate metric mustbe chosen for qualifying the column. Conven-tional practice is to measure the HETP and theasymmetry (As). However, the usefulness of amethod can be realized only if the method can berepeated with sufficient precision to allow it to beused to compare the packing performance over aperiod of time. HETP and As test results can beapplied in many ways to aid in the overall controlof the chromatographic process, from initialpacking qualification to use as a periodic in-process check of bed stability. HETP and As testsare, with increasing frequency, used to verify bedintegrity throughout the service life of a packedcolumn. In the biotechnology industry, whereprocesses are frequently campaigned, the use ofHETP and As testing to requalify a column com-ing out of storage has particular merit.

Choosing the marker. Usually, pulse injectionof a nonreactive marker is used to qualify a col-umn. The marker must be chosen carefully toensure that it is representative of the process andbecause it affects qualification. The marker canbe an “internal” standard, selected to mimic theprotein to be processed. Or the marker can be ageneralized, “external” standard, such as acetone,NaCl, or NaOH.

Pulse injection is the favored packing methodbecause it uses a small injection volume. How-ever, because precise, nondiluted injections areneeded, that small volume places a great burdenon the test. The injection method must be refinedto ensure it is precise, repeatable by many opera-tors, and includes this desideratum: It is stronglysuggested that the qualification test be carriedout using an automated skid.



Pulse injection tests are intended for assessingthe uniformity of the packed bed, not for assess-ing aspects of the hold-up volume of the HETPtest rig, the inlet, or the flow cell. Therefore, theinjection volume should be sufficiently large sothat it remains undiluted by such hold-up vol-umes. Generally, media vendors suggest injectionvolumes of 1–2.5% of the packed-bed volume.For consistency, Millipore suggests that the vol-ume be fixed at 1% of the total column volume.For a column with a tube height of 50 cm, theinjection volume should be equivalent to the vol-ume contained in 0.5 cm or 5 mm of the columnto be tested. (For a column with a 45-cm diameterand a 50-cm height, the injection volume shouldbe 750 mL.)

In its purest sense, the qualification procedureis really an “integrity test” of the packed bed.Postpacking testing is advised as a means to peri-odically validate column integrity. The repeat-ability of HETP and As data over the life of thepacked column gives assurance that the packedbed remains stable and the column continues tobe suitable for service.

An alternate metric for column qualifica-tion. In normal chromatographic operations, thecolumn is exposed to a variety of buffers duringequilibration, loading, washing, eluting, regener-ating, and cleaning. During these steps, thecolumn undergoes a number of buffer exchanges.Data from these steps can also be used for col-umn qualification. The process is similar to thefrontal analysis used for determining mediacapacity. However as a qualification technique,data from buffer exchanges have the advantage ofrequiring no process piping changes to run thetest. So the potential for operator-induced vari-ability is greatly reduced.

Frontal curves and pulse injection peaks don’tlook the same, but they can provide precisely thesame data. A uniform frontal curve indicates thatthe packing is of high quality, the same as a nar-row, symmetrical injection peak. The frontal peakdata can be manipulated to compare it with apulse injection peak. Taking the first derivative ofthe frontal curve produces the same curve as itsanalogous injection peak (see Figure 1).

Qualification of the packing protocol. A packingprotocol must be rigorous to ensure good pack-ing. Figure 2 shows the development of a packingprotocol for Sephacryl S-200 HR resin — a gelfiltration resin, which is a challenge to consis-tently pack well. The acceptance criteria requiredthat Millipore demonstrate five repeat packings(in an IsoPak 630-mm column) meeting the per-formance specification: HETP � 0.02 cm and As � 0.8–1.2. Client operators would then usethe same packing protocol to pack the column towithin the performance specification. The data inFigure 2 confirm that the seven packings all metthe qualification (and process) specifications.

A rigorous packing protocol ensures goodpacking, which in turn ensures that columns canbe well packed by any capable operator. Variabil-ity in packing methods must be understood, andthe test method must be properly set to the quali-fication specifications. Some conservatism is sug-gested in finalizing the specifications becausetesting may be conducted on initial packing aswell as throughout the service lifetime of the col-umn and gel. Robust packing methods makequalification test results more consistent andreproducible. —Ivars Bemberis is technology manager,

chromatography, Millipore Corporation, 80 Ashby

Road, Bedford, MA 01730, 781.533.8452,

fax 781.533.3110, [email protected].

OLIVER KALTENBRUNNER Preventing Erroneous Results

C h r o m a t o g r a p h ycolumn packing in-tegrity is usuallytested before processuse of a column.Typically, a pulse ofa noninteracting sam-ple tracer is appliedwhile flowing a col-umn signal (which

alters the initially rectangular tracer) under de-fined conditions. An analysis of the response sig-nal of the pulse sample can be used to assess

Figure 1. Frontal analysis curve and calculated peak

0

100

50

0

dC/dV

Feed Volume

Pro

duct

Con

cent

ratio

n

Per

cent


packing quality by determining the number oftheoretical plates (N) of the column. N can beinterpreted as the signal-to-noise ratio of theretention time, that is, the ratio between retentiontime and the peak width of the tracer pulse (seeFigure 3). By relating N to column height, theHETP can be calculated and compared to theresin bead diameter to calculate the reduced plateheight h. Chromatographic theory based on thevan Deemter equation (2) provides a clear inter-pretation for the meaning of h as

where h is influenced by the quality of the pack-ing (A), diffusivity of the tracer molecule (B), andthe mass transfer characteristics of the resinbeads (C). For column qualification, test condi-tions have to be chosen to maximize the contribu-tion of the packing quality and minimize the con-tribution of diffusivity and mass transferresistance.

Making diffusivity invariable. To optimize col-umn packing for qualification, the A term needsto be maximized by minimizing the B and Cterms. Whereas in typical protein chromatogra-phy the B term can be ignored because the diffu-sion of macromolecules is insignificantly smallcompared to the other contributions, the same isnot true for small molecule tracers. As demon-strated by Knox, h has its minimum value at Pe � 5. Because h grows quickly at Pe � 5 andincreases gradually at Pe � 5, the flow rate re-gion between 5 � Pe � 10 gives the most stableresults for HETP testing (4). The definition of theflow rate range based on the Péclet number indi-cates that, for reproducible and transferable testresults, the diffusivity of the tracer molecule hasto be invariable.

h = A + BPe

+ C × Pe

Additional information can be gathered whenanalyzing the As of the peak. Typically, afronting peak indicates a compression that is toolow (with an increased risk of column channel-ing), whereas peak tailing indicates excessivepacking compression. However, interpretation ofAs is normally not that simple because most extracolumn equipment introduces exponentialwashout behavior that also manifests as peak tail-ing. Nevertheless, for a given column in a definedtest setup, a change in peak As can typically betraced back to over or under compression of thepacking.

Accounting for broadening. In large-scalechromatography, less-than-ideal theoreticalHETP results are typically accepted because thechromatographic setup in a manufacturing plantis designed to handle larger volumes and flowrates than were used for the column qualificationtest. The broadening introduced by switchingvalves and the monitoring unit can be significantfor an integrity test. To assess a specific equip-ment setup, a UV detector can be put before themonitoring assembly. The alteration of a UVtransition signal on its pass through the assemblycan be used for qualifying the monitoring setup.In our measurements, typically the apparent mix-ing volume of an assembly is between 20% and40% of its actual volume. The volume of a 3/4-inch OD monitoring assembly, including acolumn-switching valve, can easily exceed 200 mL with about 50 mL of apparent mixingvolume. With a simple estimate of retention for anonretained small tracer molecule of about 0.95 column volumes, and an ideal HETP ofabout 5 particle diameters, we can easily estimatethe expected band broadening (�) in the packedbed:

Comparing this column broadening to externalbroadening of the system reveals that, in manycases, the monitoring equipment used forpreparative runs significantly affects the columnqualification result. Ignoring this aspect cancause problems when setting column acceptancecriteria during process transfers. Difficulties aretypically exacerbated when packing with a resinwith a small particle size, for which the packingquality process requirements are higher and, atthe same time, the relative negative effect of the

σColumn2 =

VR2

N=

d2π4

× L × 0.95

2

L5 dp

Figure 2. Summary of Sephacryl S-200 HR packing qualification

0.020

0.015

0.010

0.005

0.000

1.4

1.2

1

0.8

0.6

0.4

0.2

0

HETP (cm)As

Average Results:HETP � 0.014 crAs � 1.082

Pack 1

Pack 2

Pack 3

Pack 4

Pack 5

Avera

ge

Run 1

Run 2

Packing Number

HE

TP

(cm

)


setup is also higher. Obviously the tracer applica-tion technique can have an even bigger effect onthe test result, and a defined and reproduciblesample application method or device needs to beestablished.

Preventing erroneous conclusions. Oncereproducible test methods are in place, HETPresults can be used to accept or reject columns forprocessing. Acceptance criteria should be set pri-marily by considering the best practice for col-umn packing and the available equipment, thenestablishing criteria that are specific to that sys-tem. Consequently, there is not necessarily aclose correlation between a column’s qualifica-tion test result and its process performance.However, as in any acceptance test, there are twopossible erroneous conclusions.

The most likely error is that a column fails thetest criteria but would not fail to meet processrequirements. This is the typical error to expect ifthe testing operation failed to follow the requiredbest practices. It causes an operational problem ofproducing extra work and unnecessary repacking,but it does not set the process at risk. Settingappropriate acceptance criteria, reproducible testmethods, and operator training can minimize therisk.

The second type of erroneous result is toaccept a column that is not fit for the process.This obviously sets the process at risk and needsto be avoided. Reasons for this error can be col-umn failure occurring at a particular flow rate, orviscosity of a process feed while the test condi-tions allow good packing integrity.

Bed stability is not only a function of the bedheight and compression, but also of the height-to-diameter ratio of the packing. Identical linear ve-locities cause increasing bed compression withincreasing column diameter. This leads to de-creasing maximum operating linear velocities,with increasing column diameter (5). If theprocess flow rates are developed using small col-umn diameters without consideration of potentialflow rate limitations at large column diameters,bed instabilities can occur that are not evidentduring typical column qualification conditions.Consequently, test conditions have to be evalu-ated column by column, considering the processconditions at the respective scale.—Oliver Kaltenbrunner is in process engineering,

MS: 30W-2-A, Amgen Inc., One Amgen Center Drive,

Thousands Oaks, CA 91320, 805.447.2641,

fax 805.499.5008, [email protected]

ANURAG RATHORE From the Series CoordinatorBioPharm Interna-tional and I hope thatthe insightful com-ments from these au-thors are useful for bio-chemists and engineerspracticing preparativechromatography in thebiopharmaceutical in-dustry. We concludethis article with a caution about some commonpitfalls found in preparative chromatography:• Packing procedures need to be optimized for

each different type of resin to get reproduciblepacking;

• Measures of bed integrity need to be carefullychosen along with their specifications toguarantee robust column performance;

• The tracer used for estimating HETP or As caninteract with column packing, yieldingerroneous results;

• Flow rate or particle size effects must beeliminated when qualifying column packing toachieve consistent results;

• Function tests might be necessary, and lot-to-lotvariations in chromatography media may beunacceptable for some applications if theprocess is very sensitive; and

• Resin qualification procedures need to becarefully chosen because excessive testing canbe expensive and should be reduced graduallythrough reexamination.

—Anurag Rathore , series coordinator, is at

Pharmacia Corporation, 700 Chesterfield Parkway North,

Chesterfield, MO 63017, 636.737.6790, fax 636.737.7281,

[email protected]. BPI

REFERENCES(1) Rathore, A.S. et al., “Process Validation: How Much

to Do and When to Do It,” BioPharm 15(10), 18–28(October 2002).

(2) Rathore, A.S. and Velayudhan, A., “Guidelines forOptimization and Scale-Up in PreparativeChromatography,” BioPharm International 16(1),34–42 (January 2003).

(3) Van Deemter, J.J., Zuiderweg, F.J., and Klinkenberg,A., “Longitudinal Diffusion and Resistances to MassTransfer as Causes of Nonideality inChromatography,” Chem. Eng. Sci. 5, 271–289(1956).

(4) Kaltenbrunner, O., Watler, P., and Yamamoto, S.,“Column Qualification in Process Ion-ExchangeChromatography,” Progress in Biotechnology:Bioseparation Engineering, Vol. 16, I. Endo et al.Eds. (Elsevier Science, Amsterdam, The Netherlands,2000), pp. 201–206.

(5) Stickel, J.J. and Fotopoulos, A., “Pressure–FlowRelationships for Packed Beds of CompressibleChromatography Media at Laboratory andProduction Scale,” Biotechnol. Prog. 17(4), 744–751(2001).

Corresponding author and seriescoordinator Anurag S. Rathore is atPharmacia Corporation, GG3K, 700Chesterfield Parkway North,Chesterfield, MO 63017,[email protected];Robert M. Kennedy is departmentdirector in separation sciences atAmersham Biosciences Corporation,J. Kevin O’Donnell is technicalservice manager at Tosoh BiosciencesLLC, Ivars Bemberis is technologymanager at Millipore Corporation,and Oliver Kaltenbrunner is inprocess engineering at Amgen Inc.


qualification of a chromatographic column

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