Separation of Human Serum Albumin Components by
`RP-HPLC and CZE and their Characterization by ESI-MS
`
`M. Girard* IT. Cyr IN. Mousseau I 1.-C. Ethier
`
`Bureau of Biologics and Radiopharmaceuticals, Therapeutic Products Programme, Health Canada, Sir F.G. Banting
`Research Centre, Tunney's Pasture, Ottawa (ON) KlA OL2, Canada
`
`Key Words
`Column liquid chromatography
`Capillary zone electrophoresis
`Electrospray ionization-mass spectrometry
`Human serum albumin
`
`Summary
`Human serum albumin (HSA) is one of the most abun(cid:173)
`dant human proteins and has been shown to be hetero(cid:173)
`geneous. A RP-HPLC method has been developed to
`separate HSA components in commercially available
`preparations. Separations were carried out on Aqua(cid:173)
`pore RP-300, CS columns using gradient elution with a
`combination of acetonitrile/water mobile phases con(cid:173)
`taining 0.05 % trifluoroacetic acid as ion-pairing agent.
`Optimum resolution was attained on narrow-bore col(cid:173)
`umns using a stepwise, linear gradient that incorporated
`a shallow intermediate step of 0.20 %/min in Mobile
`Phase B. Under similar elution conditions, separations
`carried out on standard-size columns showed the ex(cid:173)
`pected decrease in resolution due to increased peak
`widths. A comparative analysis of three commercial
`products highlighted qualitative and quantitative differ(cid:173)
`ences. Capillary zone electrophoresis was used for the
`analysis of collected RP-HPLC fractions. Results indi(cid:173)
`cated that while the HPLC separation was incomplete,
`one of the major HPLC peaks was primarily composed
`of one of the three main components typically separated
`by CZE. ESI-MS was used to characterize the two ma(cid:173)
`jor RP-HPLC fractions and also showed that the HPLC
`separation was incomplete. The MaxEnt transform of
`the HPLC peaks was consistent with components all be(cid:173)
`ing HSA and closely related derivatives.
`
`Presented in parts at the Symposium on High-Performance Li(cid:173)
`quid-Phase Micro Separations, Lund, Sweden, June 7-10, 1998
`
`Introduction
`Methodologies using high performance chroma(cid:173)
`tographic and electrophoretic separation techniques
`have been increasingly used over the last two decades
`for the purification, characterization and quality assess(cid:173)
`ment of biomolecules ~ 2]. These methods have been
`particularly useful for biopharmaceuticals, especially
`for biologically important proteins and glycoproteins
`produced
`through recombinant DNA
`technology.
`Reversed-phase HPLC (RP-HPLC) has been one of the
`more versatile chromatographic modes to study pro(cid:173)
`teins and their impurities, owing in part to advances
`made in microparticulate stationary phases and to the
`development of a wide range of columns of differing se(cid:173)
`lectivities [3, 4]. For instance, several methods have de(cid:173)
`scribed the separation of variants differing by a single
`amino acid from the native protein. Capillary zone elec(cid:173)
`trophoresis (CZE) has rapidly become a valuable ana(cid:173)
`lytical technique by virtue of the exceptional separation
`efficiencies attained and its capability for rapid, auto(cid:173)
`mated and reproducible separations [5]. These consid(cid:173)
`erations make CZE particularly well suited for the sepa(cid:173)
`ration of closely-related protein variants.
`Human serum albumin (HSA) is a single chain, non(cid:173)
`glycosylated protein [6]. However, studies have demon(cid:173)
`strated that it is composed of a complex mixture of vari(cid:173)
`ants such as mercapto and non-mercapto [7], glycated
`[8] and polymerized [9, 10] forms. Conditions of storage
`and manufacture can further increase heterogeneity by
`introducing deamidation, oxidation or polymerization
`[10]. Chromatographic separations of HSA components
`based on size-exclusion [7, 11, 12], ion-exchange [13-16]
`and reversed-phase [17] HPLC have been reported. Re(cid:173)
`cent studies have also demonstrated that HSA hetero(cid:173)
`geneity can be characterized by CZE on bare-silica cap(cid:173)
`illaries under conditions that minimize protein adsorp(cid:173)
`tion on walls [18, 19] or by using coated capillaries [20].
`In this study, we report on the development of a rapid
`RP-HPLC method that provides reproducible separa(cid:173)
`tion of RSA-related components in commercially avail(cid:173)
`able HSA products, with on-line characterization of
`components by ESI-MS.
`
`Original
`
`Chromatographia Supplement I, Vol. 49, 1999
`
`S-21
`
`0009-5893/99
`
`S-21-07 $ 03.00/0
`
`© 1999 Friedr. Vieweg & Sohn Verlagsgesellschaft mbH
`
`Abraxis EX2024
`Actavis LLC v. Abraxis Bioscience, LLC
`IPR2017-001101; IPR2017-01103; IPR2017-01104
`
`

`

`Table Gradient profile 1 for the separation of human serum albu
`min by RPHPLC The flow rate was that used with the narrow bore
`column
`
`Table II Gradient profile 2 for the separation of human serum al
`bumin by RPHPLC The indicated flow rate was for a standard
`size column
`
`A
`
`70
`
`70
`
`65
`
`61
`
`50
`
`70
`70
`
`B
`
`30
`
`30
`35
`
`39
`
`50
`0
`
`0
`30
`30
`
`C
`
`Flow Rate
`
`mL min1
`
`0
`
`0
`
`0
`0
`
`0
`100
`
`100
`
`0
`
`0
`
`070
`070
`070
`070
`070
`200
`200
`200
`070
`
`Time
`
`mM
`
`1 6
`
`25
`
`30
`35
`
`41
`45
`46
`
`B
`
`30
`30
`
`35
`37
`
`50
`
`10
`
`30
`30
`
`C Flow Rate
`in mL min1
`
`015
`015
`015
`015
`015
`05
`05
`05
`015
`015
`
`100
`
`100
`
`Time min A
`
`0
`
`10
`
`15
`30
`
`35
`40
`
`50
`59
`
`60
`70
`
`70
`70
`
`65
`63
`
`50
`
`90
`
`70
`70
`
`Experimental
`
`Materials
`
`Pentex® HSA fraction V was
`from Miles Inc
`II Albuminar®25 HSA preparations
`Kankakee
`lots No M65205 M66206 and M67007 from Armour
`Pharmaceuticals Kankakee IL and crystallized and
`lyophilized HSA lot No 126C8070 from Sigma St
`Louis MO Buffer salts and additives were HPLC or
`Molecular Biologiy grade reagents OmniSolv HPLC
`grade solvents were obtained from EM Science Hen
`egg lysozyme was from Sigma St Louis MO Aqua
`pore RP 300 Brownlee columns 250 x 46 mm id were
`purchased from Chromatographic Specialities Brock
`ville ON
`
`Methods
`
`HPLC
`
`Mobile Phase A consisted of 005 `0 trifluoroacetic acid
`TFA in 10
`water Mobile Phase B
`acetonitrile90
`was 005 `0 TFA in 90
`water and
`acetonitrile10
`TFA in acetonitrile
`Mobile Phase C was 005
`Narrow bore column separations were performed on
`ternary DR 5 pumps
`Hewlett Packard 1090 Series II
`6 mm SST micro 17 iaL high pressure flow cell The col
`umn was an Aquapore RP 300 7 gm 220 x 21 mm id
`and was equilibrated with a mixture of Mobile phases A
`and B 7030 until a stable baseline was obtained Elu
`tions were carried out using multistep gradient profiles
`1 and 2 as described in Table I and II respectively The
`flow rate was that presented in Table I The column was
`maintained at a temperature of 50 °C The effluent was
`monitored at 220 nm then was split 101 prior to
`ESIMS analysis Standard size column separations
`were carried out on a SpectraSystem Thermo Separa
`tion Products Mississauga ON consisting of a P4000
`quaternary pump system an AS 3000 autosampler and a
`UV6000LP diode array detector The column was an
`Aquapore RP 300 7 gm 250 x 46 mm id and was
`maintained at 50 °C Separations were carried out using
`
`gradient profiles 1 and 2 with the flow rate as indicated
`in Table II Chromatograms were monitored between
`200 and 350 nm In preparative runs 100 µL aliquots of
`HSA 2 mgml were injected and peaks were collected
`The solvent was removed by lyophilization
`
`CZE
`
`The CZE system consisted of a PACE 5500 Beckman
`Instruments Fullerton CA fitted with a variable wave
`length UV detector Fused silica capillaries were from
`Beckman Instruments Fullerton CA 57 cm x 50 gm
`id and Polymicro Technologies Phoenix AZ
`67 cm x 52 gm id Samples were injected hydrody
`on column at
`namically and peaks were detected
`200 nm Separation conditions were as previously re
`ported 18 The electrophoretic
`20 mM sodium phosphate5 mM putrescine pH 85 A
`rinse sequence of 1M HC1 3 minutes water 3 min
`NaOH 3 minutes water 3 minutes and
`utes 1
`buffer 5 minutes was carried out after each run A 2
`minute pre run equilibration was carried out with elec
`trophoretic buffer
`
`buffer consisted of
`
`ESIMS
`
`AutoSpecQ EBEqQ geometry Micromass Man
`chester LTK electrospray ion source spray housing at
`85 °C scan range 20001000 at 30 s dec1 needle volt
`age = 83 kV sampling cone = 42 kV skimmer lens =
`42 kV ring electrode = 41 kV and accelerating volt
`age = 40 kV vacuum sourceanalyzerinlet
`= 3 x
`1066 x 10802 mbar a variable position fused needle
`10 cm x 50 mm id flame drawn out = 15 pm id was
`used electrical contact was made via a SS union to the
`0127 mm id red Peek tubing transfer line purified air
`20 L hr1 was used as the nebulizer gas and UHP nitro
`gen 250 L hr1 as the bath gas Calibration was with
`
`S22
`
`Chromatographia Supplement I Vol 49 1999
`
`Original
`
`

`

`11AL1 A wg=uuna
`
`Kel=4bUd0AU 01 U1KANIACRALISUMIN
`
`1J
`
`e4
`11
`a
`
`a2o
`
`s
`
`A
`
`5
`IJAU1 ASig=zeut
`
`A
`A
`IteM45U30 Of h I HI t111 MAU3UMINI0
`
`I
`
`I
`
`1
`
`1
`
`4
`
`0
`
`laq
`
`i
`
`B
`
`mAU
`
`300
`
`250
`
`200
`
`50
`
`mAU
`
`300
`
`250
`
`Ise
`
`lop
`
`Figure 1
`RPHPLC analysis of Pentex HSA on an Aquapore RP 300 220 x 21 min id column partial chromatograms
`obtained with A gradient profile 1 Table I and B gradient profile 2 Table II In both cases the flow rate
`
`was that shown in Table I
`
`hen egg lysozyme molecular mass of 143034 ± 046 Da
`calculated value = 14305 Da
`
`Results and Discussion
`Analysis of HSA by RPHPLC
`
`Gradient elution using acetonitrile as organic modifier
`and TFA as ionpairing agent has been frequently used
`for the separation of proteins by RPHPLC 3 4 While
`the separation of non related proteins usually requires
`gradient elution over a wide range of organic modifier
`resolution of closely related species can be maximised
`using a stepwise shallow gradient
`a procedure that
`
`maximizes retention factor k differences 4 In pre
`liminary experiments we examined the elution of HSA
`
`on the moderately hydrophobic Aquapore RP 300 C8
`columns using a stepwise linear gradient over a wide
`range of Mobile Phase B 3060
`over 20 minutes
`Under these conditions HSA eluted off the column as a
`Mobile Phase B
`single peak in the range of 3540
`Optimization of the gradient conditions by incorporat
`ing a shallow step in that critical
`range led to the separa
`tion of HSA into several components as demonstrated
`in Figures 1 and 2 for narrowbore and conventional
`size columns respectively The column temperature was
`found to be an important factor
`in obtaining reproduci
`ble separations When runs were carried out at room
`shifts in peak
`retention
`temperature unpredictable
`times occurred a likely consequence
`of the nonspecific
`adsorption of HSA to the stationary phase At 50 °C re
`producible separations were obtained see below
`
`Original
`
`Chromatographia Supplement
`
`I Vol 49 1999
`
`S23
`
`

`

`csi
`
`A
`
`03
`
`10
`
`SO
`
`Time min
`
`02
`
`01
`
`AU
`
`nm
`
`220
`
`Absorbance
`
`oo
`
`4
`
`3
`
`1I 2I
`FRACTIONS
`
`10
`
`20
`
`30
`
`Time min
`
`Figure 2
`RPHPLC analysis of Pentex HSA on Aquapore RP 300 250 x 46 mmid using gradient profile 2 Table II
`Inset full chromatogram
`
`Chromatograms obtained with the narrowbore column
`Figure 1 featured two major peaks 1 and 2 and sev
`late eluting minor ones Most peaks were eluted off
`eral
`the column during the intermediate shallow gradient
`step The resolution was found to be highly sensitive to
`the steepness of the shallow step Figure 1A shows the
`chromatogram obtained with gradient profile 1 Ta
`ble I consisting of three linear gradient steps where the
`rate of change of the shallow step was 013 min in
`Mobile Phase B In gradient profile 2 Table II
`steepness of the shallow step was increase to 020 min
`which gave an increase in peak separation as seen in Fig
`ure 1B
`
`the
`
`Separations on standard size columns were carried out
`with the same gradient profiles after adjusting the flow
`rate to 07 mL min1 to account for the column size dif
`ference Peak profiles were similar to those obtained on
`narrow bore columns Figure 2 The two main peaks 1
`and 2 eluted off the column during the shallow gradient
`step as was the case with the narrowbore column The
`UV spectra taken at
`the apex of the two main peaks
`were typical of proteinaceous compounds with absorp
`tion maxima around 280 nm suggesting that they were
`related to HSA data not shown While the resolution
`
`between the two main peaks was acceptable the late
`resolved under
`eluting minor components were not
`these conditions and appeared as shoulders on the down
`slope of peak 2 As expected the overall decrease
`in
`resolution on conventional size columns was attributed
`to the increased dilution of the components as they tra
`verse the column a situation that generates larger peak
`volumes and increased peak widths Despite the re
`duced peak resolution the use of standard size columns
`remains advantageous as they can be used with most
`HPLC systems presently on the market They do not ne
`cessitate the use of specialized components such as de
`tector microcells
`
`Peak retention times were highly reproducible for runs
`made within the same series with relative standard de
`viations RSD of 07
`n = 6 for peaks 1
`and 04
`and 2 respectively Peak areas were less reproducible
`n = 6 for peaks 1
`with RSD values of 84
`and 138
`and 2 respectively For runs carried out on separate
`days while the variation in peak areas was essentially
`the same as that for same day experiments peak reten
`tion times varied by up to 23 minutes RSD > 10
`A comparison of commercial HSA preparations is pre
`sented in Figure 3 The HSA product
`used in the
`
`S24
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`Chromatographia Supplement
`
`I Vol 49 1999
`
`Original
`
`

`

`c HSA
`
`a
`
`NFraction
`
`1
`
`10
`
`20
`
`30
`
`10
`
`20
`
`30
`
`A
`
`02
`
`01
`
`00
`
`AU
`
`04 I
`
`03 H
`
`02
`
`01 H
`
`00
`
`nm
`
`220
`
`Absorbance©
`
`02
`
`01
`
`00
`
`10
`
`20
`
`30
`
`1300
`
`Time mm
`
`I
`
`I
`
`1
`
`2000
`
`Time min
`
`2800
`
`Figure 3
`
`Comparative RPHPLC analysis of A Pentex HSA B Armour
`HSA and C Sigma HSA Separation conditions as in Figure 2
`
`Figure 4
`CZE analysis of collected RPHPLC fractions from bottom to
`top fractions 1 2 and 3 and Pentex HSA 01 mg mL1
`
`method development Figure 3A was compared to two
`other commercially available products Although the
`three products contained peaks 1 and 2 as main compo
`nents substantial differences in peak widths and peak
`significant qualitative and quantita
`patterns suggested
`tive differences in their composition The product
`from
`Armour Figure 3B showed the best peak separation as
`well as the fewest shoulders on the down slope of peak
`2 On the other hand the product
`from Sigma Fig
`ure 3C had increased peak widths and numerous shoul
`ders a likely indication of the increased heterogeneity
`of this preparation Similar differences had previously
`been observed in the CZE analysis of
`these prepara
`tions 18
`
`Analysis of HSA Fractions by CZE
`
`Capillary zone electrophoresis separates compounds on
`the basis of mobility differences which are function of
`charge and size differences and as such it constitutes a
`technique to RPHPLC where hydro
`complementary
`phobicity is the major mode of separation In order to
`obtain further information on the identity of the HSA
`separated by RPHPLC fractions were
`components
`collected from consecutive HPLC runs as described in
`Figure 2 and were analyzed by CZE using a previously
`
`reported method 18 Figure 4 presents electrophero
`grams of the three collected fractions and that of Pentex
`HSA The electropherogram of Pentex HSA was char
`acterized by the presence of three major peaks a b and
`c and a number of unresolved minor peaks most of
`which migrated ahead of the major components Frac
`tion 1 which corresponded to a small early eluting
`shoulder by HPLC gave a broad unresolved peak by
`CZE On the other hand fraction 2 which corre
`sponded to HPLC peak 1 was composed mainly of peak
`b one of the three major peaks observed in the HSA
`electropherogram top The remaining two major CZE
`peaks a and c were found in the fraction corresponding
`to the HPLC peak 2 Several minor earlymigrating
`peaks were also found in the latter fraction These re
`sults indicate that RPHPLC can be effectively used to
`separate some of the major components present in HSA
`preparations
`
`ES MS Analysis of HSA HPLC Components
`
`The two major HPLC peaks
`1 and 2 resolved on the
`narrow bore column Figure IA were characterized by
`ESIMS Figure 5 21 The major component of peak 1
`Figure 5A was at 66655 Da which is 217 higher than
`the calculated average mass for intact HSA Peak 2 Fig
`
`Original
`
`Chromatographia Supplement I Vol 49 1999
`
`S25
`
`

`

`Peak 1
`
`117
`
`1482
`
`I
`
`1550
`
`1627
`
`1710
`
`1802
`
`1905
`
`1333
`
`1257
`
`JUJU
`
`1160
`
`12â0
`
`1360
`
`14611
`
`isOo
`
`1660
`
`1760
`
`181O
`
`1944
`
`ES Transform
`
`66100
`
`66200
`
`66300
`
`66400
`
`66500
`
`6640
`
`66700
`
`Mass
`
`MaxEnt Electrospray
`
`66659
`
`66550
`
`66810
`
`66920
`
`66101
`
`66Z01J
`
`66310
`
`664011
`
`66500
`
`dadoO
`
`thioO
`
`rdadOi
`
`MaxEnt Electrospray
`
`subtracted
`
`data
`
`66814
`
`661r00
`
`662100
`
`663100
`
`665b0
`
`666181
`
`667100
`
`66800
`
`11fas
`
`1547
`
`100
`
`80
`60
`
`40
`20
`
`0
`
`100
`80
`60
`
`ao
`
`20
`0
`
`100
`
`80
`60
`40201
`
`100
`
`80
`60
`
`40120
`
`100
`
`SO
`
`60
`
`ao
`
`20
`
`0
`
`100
`
`1014
`
`1
`
`Peak 2 B
`
`1233
`
`165
`
`1332
`
`1414
`
`11419
`
`I
`
`1584 4 1753
`4u4
`
`17
`
`19
`
`1846
`
`13óO
`
`iabo
`
`060
`
`itho 1740
`
`180C
`
`19k
`
`MaxEnt Mock Data
`
`LW 1548
`
`16A
`
`1903
`
`1959
`
`1
`
`II
`
`111
`
`1760
`
`isOo
`
`lobo
`
`wilz
`
`1416
`
`1359
`
`1 1
`
`11
`
`1280
`
`1160
`
`MaxEnt Transform
`
`66564
`
`66469
`
`601100
`
`66200
`
`43100
`
`66400
`
`6400
`
`6600
`
`66706766860
`
`66900
`
`60
`
`801
`40
`
`20
`01
`
`100 l
`SO
`
`60
`
`40
`
`20
`
`0
`
`Figure 5
`
`Continuum data from the narrow bore HPLCESIMS analysis of HSA A peak 1 and B peak 2 along with
`
`their respective Maximum Entropy data analysis
`
`ure 5B contains two main components at 66469 Da
`HSA and 66564 Da +126 The HSA peak was about
`31 Da or 005 `0 higher than the calculated maximum
`The bottom trace on Figure 5A represents the Maxi
`mum Entropy data analysis after the subtraction of the
`overlap region to eliminate most of the 66550 Da com
`ponent These findings are consistent with previous ob
`servations for HSA 22 231
`
`Conclusions
`
`The results of this study have demonstrated that HSA
`can
`be
`components
`separated
`by
`reproducibly
`RPHPLC under acidic ion pairing conditions using a
`shallow stepwise gradient profile and a moderately hy
`column C8 Component
`separation was
`drophobic
`sensitive to small modifications of the gradient profile
`particularly the steepness of the shallow step It also
`demonstrated the complementarity of RPHPLC and
`CZE for the characterization
`of HSA and some of its
`The resolved peaks were shown to be
`components
`HSArelated following analysis by ESIMS
`
`S26
`
`Chromatographia Supplement I Vol 49 1999
`
`Original
`
`

`

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`T A A M van de Goor Capillary Electrophoresis of Pro
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`ciples and Methods in Biotechnology
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`New York 1997 p 65
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`13 A Sugii K Harada K Nishimura R Hanaoka S Masuda
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`14 K Nishimura K Harada S Masuda A Sugii J Chroma
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`15 T Etoh M Miyazaki K Harada M Nakayama A Sugii
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`Palm Springs CA
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`horghe J Chromatogr B 6622 245 1994
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`Received Oct 8 1998
`Revised manuscript
`received Feb 1 1999
`Accepted Feb 10 1999
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`Int
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`2 3 4 56 7 89
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`10
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`11
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`12
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`Original
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`Chromatographia Supplement I Vol 49 1999
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`S27
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`nderlined in blue are linked to publications
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`on ResearchGate
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