CE in Biotechnology: Practical Applications for Protein and Peptide Analyses

102 Pages·2001·6.069 MB·English

by  A. B. Chen, E. Canova-Davis (auth.), Anthony B. Chen, Wassim Nashabeh, Timothy Wehr (eds.)

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An International Journal for Rapid Communication in Chromatography, Electrophoresis, and Associated Techniques
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Volume 53, Supplement 2001
Scientific Editors T. A. Berger K.-S. Boos H. Engelhardt
Berger Instruments Inc. Institut fur Klinische Chemie Instrumentelle Analytik - Umweltanalytik
123A Sandy Drive Klinikum der Universitat M un chen Universitat des Saarlandes,
Newark, DE 19713, USA 81366 Munchen 66041 Saarbriicken, Germany
Technical Editors E. R. Adlard, Delryn, Vicarage Lane, I. W. Davies, 78 Blinco Grove
Burton, South Wirral CH64 5TJ, UK Cambridge CB 1 7TS, UK
K. D. Altria, Analytical Evaluation Gp. R. Stock, 23 Highbury Road,
Glaxo R&D, Ware, Herts SG 12 ODP, UK Keyworth, N otts., N G 12 5JB, UK
Editorial Advisory Board
K.Albert C. A. Cramers P. R. Haddad A. Mosandl R.M. Smith
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Honorary Editorial Board
L. S. Ettre G. Machata c. S. G. Phillips B. Sebille E. Smolkova-Keulemansova
Bridgeport, CT, USA Vienna, Austria Oxford, UK Thiais, France Prague, Czech Republic
W. Haerdi R. L. Munier L. Rohrschneider P.A. Sewell L. Szepesy
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K.-P. Hupe H.M. McNair G. Schomburg C. F. Simpson K. Tesarik
Baden-Baden. Germany Blacksburg, VA, USA Mulheim. Germany London. UK Bmo. Czech Republic
E.Jellum
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Editorial OHice M.Schaub,Manager H. Weinheimer, Publisher
Vieweg Publishing
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An International Journal for Rapid Communication in Chromatography, Electrophoresis, and Associated Techniques
Contents Supplement Vol. 53, 2001
Chromatographia CE Series 4 Lse of Capillary Electrophoresis to Determine
Series Editor: Kevin D. Altria. Analytical Evaluation the Dilute Protein Concentration in Formulations
Group, Glaxo R&D. Ware. Herts SG 12 ODP. UK Containing Interfering Excipients . . . . . . . . . . . . . . . .. S -34
S. S. Park I A. Cate I B. S. Chang
CE in Biotechnology: Practical Applications for Protein
4.1 Abstract........ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. S -34
and Peptide Analyses
4.2 Introduction................................... S -34
Edited by Anthony B. Chen,
4.3 Material and Methods .......................... S -35
Genentech Inc. South San Francisco, CA, USA
4.4 Result and Discussion. . . . . . . . . . . . . . . . . . . . . . . . . .. S -35
Wassim Nashabeh 4.5 Conclusion.................................... S -38
Genentech Inc. South San Francisco, CA, USA 4.6 Acknowledgements............................. S -38
4.7 References..................................... S-38
Timothy Wehr
Bay Bioanalytical Laboratory. Richmond, CA, USA
5 Effects of the Solution Environment on the
Resolution of Recombinant Human
Preface ........................................... . S-5
Deoxyribonuclease Variants in Capillary
Zone Electrophoresis. . . . . . . . . . . . . . . . . . . . . . . . . . .. S -39
Capillary Electrophoresis in the Development
C. P. Quan I E. Canova-Davis I A. B. Chen
of Recombinant Protein Biopharmaceuticals ....... . S -7
5.1 Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. S -39
A. B. Chen I E. Canova-Davis
5.2 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. S -39
1.1 Summary .................................... . S-7
5.3 Materials. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. S -39
1.2 Introduction ................................. . S-7
5.4 Methods ...................................... S -40
1.3 Capillary Electrophoresis Reviews .............. . S-8
5.5 Results and Discussion. . . . . . . . . . . . . . . . . . . . . . . . .. S -40
1.4 Reviews of CE U sed for the Analysis
5.6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. S -44
of Recombinant Proteins ....................... . S-II
5.7 Acknowledgements...... ..... .............. .... S-44
1.5 Recent CE Literature .......................... . S -13
5.8 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. S -44
1.6 Quality Control and Validation ................. . S-15
1.7 DevelopmentsinCE-MS ....................... . S -15
6 Recent Advances in Capillary Isoelectric Focusing
1.8 References .................................... . S-16
T. Wehr I R. Rodriguez-Diaz 1M. Zhu ............ S-45
6.1 Introd uction .................................. . S-45
2 Selection of Buffers in Capillary Zone Electrophoresis:
6.2 Theory and Practice of clEF .................... . S-45
Application to Peptide and Protein Analysis ........ . S -18
6.3 clEF in Microchannels ......................... . S-47
G. M. Janini/H. J. Issaq
6.4 Imaging clEF ................................. . S-48
2.1 Introduction .................................. . S-18
6.5 clEF-Mass-Spectrometry ....................... . S-49
2.2 Buffer Parameters ............................. . S -18
6.6 Capillary Isoelectric Focusing of Monoclonal
2.3 Buffers Used for the Separation of Peptides
Antibodies .................................... . S-50
and Proteins .................................. . S-23
6.7 Capillary Isoelectric Focusing of Hemoglobin
2.4 Acknowledgements ............................ . S-25
and Hemoglobin Variants ...................... . S-54
2.5 References .................................... . S-25
6.8 Capillary Isoelectric Focusing of Erythropoietin ... . S-57
6.9 Novel Applications of clEF ..................... . S-57
3 Reverse-Phase High Performance Liquid
6.10 References .................................... . S-58
Chromatography and Capillary Zone
Electrophoresis Separation of Protegrin Analogs ..... S-27
7 Separation of Enbrel® (rhuTNFR: Fc) Isoforms
J. Chen I J. Fausnaugh-Pollitt
by Capillary Isoelectric Focusing ................. . S-59
3.1 Abstract ...................................... . S-27
C. Jochheim I S. Novick I A. Balland I
3.2 Introduction .................................. . S-27
J. Mahan-Boyce I W.-c. Wang I A. Goetze I
3.3 Experimental ................................. . S-28
W. Gombotz
3.4 Results and Discussion ......................... . S-29
7.1 Abstract ...................................... . S-59
3.5 References .................................... . S-33
7.2 Introduction .................................. . S-59
7.3 Experimental ................................. . S-60
S-2 Chromatographia Supplement Vol. 53,200 I Contents
7.4 Results and Discussion ......................... . S-61 9.3 Capillary Isoelectric Focusing (clEF) ............ . S-81
7.5 Acknowledgements ............................ . S-64 9.4 Capillary Zone Electrophoresis (CZE) ........... . S-83
7.6 References .................................... . S-65 9.5 Carbohydrate Analysis ......................... . S-84
9.6 Methods ..................................... . S-86
8 Optimization, Validation, and Use of Capillary Gel 9.7 Acknowledgements ............................ . S-89
Electrophoresis for Quality Control Testing 9.8 References .................................... . S-89
of Synagis®, a Monoclonal Antibody .............. . S-66
M. A. Schenerman I S. H. Bowen 10 Sensitive and High Resolution CEIMS/MS for
8.1 Abstract ...................................... . S-66 Protein Identification in Complex Mixtures ........ . S-90
8.2 Introduction .................................. . S-66 W. Tong I J. R. Yates
8.3 Methods ..................................... . S-67 10.1 Introduction .................................. . S-90
8.4 Regulatory Guidance .......................... . S-67 10.2 Approaches to Improve the Sensitivity
8.5 Optimization Strategy .......................... . S-68 ofCE/MS/MS for Protein Identification .......... . S-90
8.6 Validation .................................... . S-68 10.3 Approaches to Improve the Resolution
8.7 Validation Parameters for CGE ................. . S-68 of CE/MS/MS for Protein Identification .......... . S-94
8.8 Acknowledgements ............................ . S-72 10.4 Other Approaches ............................. . S-96
8.9 References .................................... . S-72 10.5 Conclusions .................................. . S-97
10.6 References .................................... . S-98
9 Analysis of Protein Therapeutics by Capillary
Electrophoresis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. S -75 Subject Index .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. S -100
S. Ma/W. Nashabeh
9.1 Introduction................................... S-75 Abbreviations ...................................... S -102
9.2 Capillary Electrophoresis - Sodium Dodecylsulfate
(CE-SDS) ..................................... S-75
Contents Chromatographia Supplement Vol. 53, 2001 S-3
1..d.1
Vol. 53, Supplement 2001
,I
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Chromatogram on front page: Gas chromatographic separation of gasoline hydro carbons (selected section of
chromatogram) with glass capillary column.
S-4 Chromatographia Vol. 53, Supplement 2001
Preface
The continuing development of capillary regulatory approval for the routine analy to hold the CE Symposium (in his words,
electrophoresis (CE) is evident by the in sis of marketed recombinant protein the time was right), the California Separa
creasing number of journal articles, text pharmaceuticals. This meeting demon tion Science Society for sponsoring the
books and symposia devoted to CE. This strated that CE has found a niche in the symposium, and Michael Kunitani and
monograph is part of a continuing series routine analysis of proteins and peptides SungAe S. Park who were also on the or
covering individual areas of CEo A list of in an industrial setting. The editors of this ganizing committee. We would like to
topics already published in this series in monograph, along with the series editor thank the symposium participants for
cludes: Analysis of nucleic acids by capil (Kevin Altria) were of the opinion that their efforts in preparing manuscripts.
lary electrophoresis (edited by Christoph the proceedings of that meeting could pro The editors greatly appreciate the efforts
Heller), Analysis of pharmaceuticals by vide the basis for theoretical and practical of Amy Greene-Dittz who reviewed all
capillary electrophoresis (written by Ke issues covering the analysis of proteins the figures for the manuscripts, scanned in
vin Altria), Analysis of carbohydrates by and peptides by CEo This supplement images when electronic copies were not
capillary electrophoresis (written by Aran therefore consists of some of the practical available and modified some images to in
Paulus and Antje Klockow-Beck), Capil applications that were presented at the crease their legibility. We would also like
lary Electrophoresis: instrumentation and symposium and several review chapters, to thank Angelika Schulz for her patience
operation (written by Wim Kok). including 'CE in the development of re and help in assembling this monograph.
The Symposium 'CE in Biotechnology: combinant protein biopharmaceuticals',
Practical Applications for Protein Analy 'Selection of buffers in CZE: application
sis' was held in San Francisco, California, to peptide and protein analysis' and 'Re
USA on August 18, 1999. The sympo cent advances in clEF'. Anthony B. Chen
sium highlighted practical applications, The editors would like to acknowledge Wassim Nashabeh August 2000
including methods which had obtained Dr Barry L. Karger, who encouraged us Timothy Wehr
Preface Chromatographia Supplement Vol. 53,2001 S-5
CHROMATOGRAPHIA CE Series
Edited by Kevin D. Altria, Glaxo Wellcome R&D, UK
There are currently a number of general • Volume 1: C. Heller (Ed.), Analysis of
textbooks covering Capillary Electro Nucleic Acids by Capillary Electro
phoresis where information on a range of phoresis
applications and techniques can be found.
• Volume 2: K. D. Altria, Analysis of
Readers who are interested in a specific
Pharmaceuticals by Capillary Electro
area of CE struggle to find truly compre
phoresis
hensive treatments of their areas of inter
est. The CHROMATOGRAPHIA CE • Volume 3: A. Paulus / A. Klockow
series has been established to allow com Beck, Analysis of Carbohydrates
prehensive books to be produced covering by Capillary Electrophoresis
individual topics. The books are written
• Volume 4: W. Kok, Capillary Electro
by well known authors in their specialist
phoresis: Instrumentation and Operation
application areas and cover CE topics
such as DNA analysis, analysis of phar • Volume 5: A. B. Chen / W. Nashabeh /
maceuticals, chiral separations, MECC, T. Wehr (Eds), CE in Biotechnology:
carbohydrate analysis, biomedical appli Practical Applications for Protein and
cations and troubleshooting in CEo Peptide Analyses
S-6 Chromatographia Supplement Vol. 53, 200 I
Capillary Electrophoresis in the Development
of Recombinant Protein Biopharmaceuticals
A. B. Chen 1 / E. Conovo-Dovis2
Departments of Quality Control I and Analytical Chemistr/, Genentech Inc. One DNA Way, South San Francisco, CA 94080, USA
of the protein during various stages of
1.1 Summary production including the crude cell paste
or cell culture fluid to determine the yield
The understanding of the physicochemical properties of proteins in solution is required to un of the production process. During produc
derstand the separations taking place in the capillary. For example, during isoelectric focusing, tion, various post-translational modifica
it is important to ensure the lack of protein precipitation during fOCUSing and mobilization
tions can occur such as deamidation, oxi
otherwise artifacts occur and the true resolution of charge variants is not achieved. Similarly,
dation, isoaspartate isomerization, and
buffer conditions, including additives, composition of buffer, and pH can influence the resolu
proteolytic cleavages. Hence, it may be ne
tion of product variants. With diligent efforts one can achieve desired separations. As knowl
cessary to analyze for size, charged, or hy
edge of buffer conditions and use of these techniques increase, the methods will be improved.
drophobic variants, especially if these var
MECC can be useful for the characterization of recombinant proteins and monoclonal anti
iants have different biological potencies
bodies. However, the relatively long analyte migration times may be a deterrent for routine use
than the desired product. According to
in quality control situations. Pharmaceutical companies will employ new techniques which pro
the ICH guideline, Q6B 'Guidance on
vide advantages over current technology. CE has advantages in certain applications. Applica
Specifications: Test Procedures and Ac
tions which use slab gel techniques, IEF and SDS-PAGE can be replaced by the CE methods for
ceptance Criteria for Biotechnologicall
formulation development, characterization of charge isoforms, and for purity determinations.
Biological Products' [I], a product variant
CZE and clEF can be used for rapid identity tests which do not require peptide mapping. CZE
which does not have the same safety and
has a tremendous advantage in analyzing basic proteins, and CZE and CE-SDS can be used
efficacy profile as the desired product is
to quantify product in the presence of interfering excipients. It is the responsibility of the analyti
termed a product-related impurity. For
cal chemist to determine applications which are appropriate for CEo It is apparent that these are
example, isomerization of a heavy chain
currently being defined for the biotechnology industry, and the usage of CE should continue to
aspartate residue of a therapeutic mono
increase.
clonal antibody to isoaspartate resulted in
a variant with low potency [2]. The manu
facturer may choose to remove these im
purities during the purification process or
1.2 Introduction sulin, growth hormone (rhGH), recombi monitor their concentration during lot re
nant human erythropoietin (rhEPO), re lease.
Since the cloning and expression of insu combinant tissue plasminogen activator Product characterization is required
lin, the biotechnology industry has experi (rt-PA), and a number of monoclonal throughout the development of a recombi
enced a boom in the number of products antibodies, the required characterization nant product. In early preclinical stages, it
that have been developed, tested in clinical and process controls for the approval of a is necessary to confirm ifthe product man
trials, and approved for unmet medical protein product are becoming more estab ufactured is the desired product prior to
needs for many indications. The develop lished. After the protein has been cloned beginning pharmacokinetic and toxicol
ment of proteins as pharmaceuticals is and expressed in eukaryotic or prokary ogical studies. In the case of glycoproteins,
somewhat different than the development otic cells, there are usually several rounds there could be heterogeneity due to vary
of traditional small molecule pharma of purification, product characterization, ing degrees of glycosylation and occu
ceuticals. These differences are mainly due evaluation of the production process, and pancy of glycosylation sites. These varia
to the size and complexity of proteins. preclinical and toxicology studies leading tions could affect the intrinsic biological
With the experience gained after the ap to the filing of an IND to begin clinical activity of the protein or the pharmacoki
proval of recombinant proteins such as in- trials. Assays are required for quantitation netic profiles of these proteins. For exam-
Original Chromatographia Supplement Vol. 53,2001 S-7
0009-5893/00102 7-II $ 03.0010 © 2001 Friedr. Vieweg & Sohn Verlagsgesellschaft mbH
A. B. Chen et al. (eds.), CE in Biotechnology: Practical Applications for Protein and Peptide Analyses
© Friedr. Vieweg & Sohn Verlagsgesellschaft mbH, Braunschweig/Wiesbaden 2001
pie, it has been shown that glycosylation is decylsulfate polyacrylamide gel electro formed by three on-line methods. In the
necessary for the antibody-dependent cell phoresis (SDS-PAGE) and isoelectric fo first. sample self-stacking was used to de
mediated cytotoxic and complement-de cusing (lEF) methods have been used ex termine trace concentrations of recombi
pendent cytotoxic activities of monoclonal tensively for the analysis of recombinant nant interleukins [20]. The second proce
antibodies [3]. Carbohydrate moieties play proteins. CE analyses add to the reper dure utilizes solid-phase extraction on a
a role in the clearance ofrt-PA [4] and bio toire of available analytical methods and cartridge containing Spherisorb CI8. with
logical activity; e. g. type II rt-PA with an will be discussed as they support the re the concentrated pep tides or proteins
unoccupied glycosylation site displays quirements for product development. being released with acetonitrile. The latter
more in-vitro activity than type I rt-PA method was used to analyze a tryptic di
which has all three glycosylation sites oc gest of bovine serum albumin. The third
cupied [5]. In these instances, it may be ne 1.3 Capillary method for preconcentration used a semi
cessary to monitor glycosylation to de Electrophoresis Reviews permeable hollow fiber between the sam
monstrate consistent man ufacture of a ple vial and the capillary inlet. An injec
quality product. When the results of clini Several excellent reviews have been writ tion current is applied which concentrates
cal trials are promising. more extensive ten in the use of capillary electrophoresis the proteins into the hollow fiber. Subse
characterization is usually performed for for the analysis of proteins including bio quently, an electric field is applied
regulatory filings to demonstrate that the technology-derived proteins. These re through the hollow fiber to begin the elec
manufacturing process is well understood views have provided information based trophoretic separation. This method can
in order to avoid surprises when full pro upon the individual capillary electrophor lower the detection limit by a factor of
duction occurs. Some of the methods that esis (CE) techniques such as capillary zone 1000 [21]. Because the walls of fused silica
are used in characterization are validated electrophoresis (CZE, [6-8]), capillary capillaries carry negative charges due to
for final product lot release testing. Ana isoelectric focusing (clEF, [9, 10]), capil ionized silanol groups, some proteins may
lytical characterization is also required to lary electrophoresis using sodium dodecyl adsorb to the walls during electrophoresis.
support methods for use in the quality sulfate (CE-SDS, [11]) and micellar elec Either dynamic (buffer additives) or static
control system. Formulation studies are trokinetic chromatography (MEKC, also (permanently coated) wall coatings are
performed to optimize the excipients and called micellar electrokinetic capillary used to reduce adsorption to capillary
conditions necessary for the stability of chromatography [MECC], [12]). Reviews walls. For improving the selectivity of a
the recombinant protein over a proposed have also been written for the analysis of CZE method, optimizing the composition
shelf-life. Formulation studies are also recombinant proteins in general [13-15], of the background electrolyte (BGE) is
performed to determine the modes of de antibodies [16], and glycoproteins [17, 18]. usually a more effective procedure with
gradation of the protein molecule. Thus, adjusting the pH of the BGE as the first
formulation studies normally include ac choice. A considerable amount of re
celerated stability studies of the protein 1.3.1 CZE search has been done in describing buffer
molecule under various conditions. Fol additives for CZE which minimize pro
lowing successful clinical trials and the fi The simplest mode of capillary electro tein-capillary interactions, improve selec
nalizing of the manufacturing process, phoresis is that of free solution capillary tivity and resolution. and control electro
validation of the methods to be used in the electrophoresis or CZE. In this mode. the osmotic now (EOF). Corradini [22] has
quality control of the product is initiated. intrinsic properties of the molecule, its summarized these additives which include
The control system for lot release is deter charge and size are used to characterize its neutral polymers, ionic salts and zwitter
mined based upon product characteriza electrophoretic behavior, determine its ions. amine modifiers, surfactants, and
tion and manufacturing history. Tests for purity, and as a means of quantification. ion-pairing agents used in protein CZE
identity, strength, purity, and potency are In Dolnik's review [6] of the CZE of pro methods. In some cases cylcodextrins
among those required for final product lot teins, he addressed modeling the migra have been successfully used to resolve pro
release. The primary identity test used re tion behavior of proteins, sample pretreat tein mixtures. Thus, carboxymethylated
quires peptide mapping to confirm the ments such as preconcentration and der ~-cyclodextrin was used to improve the
amino acid sequence of the protein. Iden ivatization to increase sensitivity, meth separation of a model mixture of proteins
tity tests are also used to confirm the prod ods to reduce interactions with the capil (::i-chymotrypsinogen A, cytochrome c,
uct's identity, for example during packag lary wall, and ways to improve the selec lysozyme, and ribonuclease A) in the cat
ing. or transfer of product to collaborators tivity of the method. He also briefly re ionic mode at pH 2.5.
and contract labs. Stability studies are re viewed CE-SDS using cross-linked and A fundamental requirement in the
quired on bulk and final product to con non-crosslinked gels. When more sensitive analysis of a recombinant protein phar
firm the shelf life of the recombinant pro methods than UV detection are required, maceutical is the need to demonstrate
duct. laser-induced fluorescence (LIF) or mass identity with respect to its primary amino
The traditional chromatographic spectrometry methods have been used. acid sequence by peptide mapping. The
methods such as reversed-phase high per The detection limit for bovine serum albu method may also be used to show. by
formance liquid chromatography (RP min (BSA) decreases from 0.5 ~lg mL -1 to comparison with an appropriate reference
HPLC). size-exclusion chromatography 25 ng mL -1 when UV absorption is re material that changes in the primary ami
(SEC), ion-exchange chromatography placed with argon-laser-induced fluores no acid sequence have not occurred, con
(both cation and anion), hydrophobic in cence [19]. In his subsequent review, Dol firming product consistency and/or genet
teraction chromatography, and gel elec nik [7] updated the information on these ic stability [23]. The method requires con
trophoretic methods such as sodium do- same topics. Preconcentration can be per- siderable expertise for performing peptide
S-8 Chromatographia Supplement Vol. 53,2001 Original
analysis in a quality control environment tides. The peptide analog mobility fit the sample salt levels and protein concentra
[23]. At present, only peptide maps gener Offord [26] equation. RPHPLC exhibited tion, and the importance of ampholyte
ated by HPLC have been reported to be better separation of single D-amino acid composition depending upon the desired
used for regulatory filings. Considerable substitutions, while CZE was better for separation range. Coated capillaries are
progress has been made in the analysis of truncated analogs. Thus, RPHPLC and necessary to reduce electroosmotic flow
pep tides by CZE, and this is one of the CZE provided orthogonal methods for which would result in band broadening.
most promising areas for routine analysis analysis of these peptides. Righetti et al. Once the proteins are focused, they must
in quality control. Janini and Issaq in this [8] describe the CZE of peptides and pro be mobilized for detection. Several exam
monograph [24] provide the background teins in acidic, isoelectric buffers. These ples are provided for chemical, hydrody
for selection of a buffer to perform CZE buffers enable the use of high voltage gra namic, and electro osmotic flow mobiliza
analysis of proteins and peptides. This dients (up to 1000 V cm-1) in large bore tion used to monitor the charged iso
chapter provides a detailed description of capillaries which produce separations of a forms. Protein precipitation is described
buffer basics including temperature and few minutes with high resolution. Thus, as the major source of difficulty in clEF.
Joule's heat considerations, buffer type isoelectric aspartic acid (Asp) was used to Reducing the focusing time, lowering the
and concentration, and isoelectric buffers. generate a peptide map of trypsin digested protein concentration, and the addition of
The authors noted that in CZE some buf ~-casein. The pH of this isoelectric buffer protein-solubilizing agents such as organ
fer-capillary wall interactions can enable ranges from 3.36 to 2.80 as its concentra ic modifiers and surfactants are suggested
high resolution of pep tides and buffer tion is increased from 5 mM to 50 mM at for reducing protein precipitation. In that
analyte interactions can result in different 25°C. Isoelectric Asp (pI 2.77 at 50 mM review the authors presented the clEF of a
electrophoretic profiles for the same sam and 25°C) containing 7 M Urea and 0.5% mixture of standard proteins: rt-PA types
ple depending upon the buffer system hydroxyethyl cellulose, could be used to I and II, iron-complexed transferrin, and
used. The buffers that have been used to analyze storage proteins in cereals such as hemoglobin mutants. Wehr et al. [9] con
perform CZE of pep tides and proteins gliadins in wheat and zeins in maize. The cluded that clEF compared favorably
were presented, and buffer additives that hydroxyethyl cellulose probably provided with conventional gel IEF in terms of se
have been used to enhance resolution of a dynamic coating to prevent the proteins lectivity and analysis time with the added
CZE methods, control electroosmotic from adsorbing to the capillary wall. Ver benefit of complete automation. In a sub
flow, and reduce analyte adsorption to the zola et al. [28] describe a method for quan sequent review, Rodriguez-Diaz et al. [10]
capillary wall were described. MEKC for tifying protein adsorption to bare fused included a discussion on the ampholyte
peptide separation is also discussed. Jani silica and for determining the effect of composition, additives which influence
ni et al. [25] measured the electrophoretic amino quenchers added to the BGE. The the clEF of proteins, the use of internal
mobility of 58 peptides ranging in size method consists of flushing a fluores standards, and presented examples of
from 2 to 39 amino acids and varying in cently labeled protein into the capillary, clEF of immunoglobulins and rt-PA
charge from 0.63 to 7.82. Measurements equilibrated in Tris-acetate buffer, wherein many aspects of the methodology
were obtained with a polyacrylamide pH 5.0, until saturation of binding sites were reviewed in detail.
coated capillary using a 50 mM phosphate occurred. SDS micelles are then used to Liu et al. [29] described clEF as a tool
buffer, pH 2.5, at 22°C. The results indi electrophoretically desorb the bound pro for examination of pep tides and proteins
cated that the Offord model (26) which tein, which is quantified fluorometrically. of pharmaceutical interest. These authors
correlates electrophoretic mobility with Using fluorescein isothiocyante (FITC) provided a detailed review of flat-bed IEF
the charge-to-size parameter q/ M2/3 pro myoglobin to saturate protein-binding and clEF, including theory and funda
vided a good fit of the experimental data. sites, Verzola et al. [28] determined that mentals and a comparison of these meth
Endoproteinase Lys-C peptide maps of oligamines (spermidine, spermine, tetra ods. They discussed the chemical nature
melittin (26 amino acids) and horse myo ethylenepentamine) were more effective of carrier ampholytes, the formation and
globin were compared to theoretical simu than monoamines (triethylamine, trietha stability of pH gradients, and the ratio
lations of mobility. The melittin compari nolamine, ethylamine) and diamines (pu nale for their focusing properties and de
son was excellent with respect to line posi trescine, cadaverine, hexamethonium bro scribed resolution and peak capacity in
tion and relative area percent of the pep mide) in quenching interactions with the clEF. Liu et al. [29] provided examples of
tides. The agreement of the theoretical silica wall. Using this method, these inves the cIEF of hemoglobin, transferrin, and
e1ectropherogram of myoglobin although tigators also determined that electro monoclonal antibodies. Conti et al. [30]
not perfect agreed reasonably well with phoretic desorption with SDS micelles addressed the issue of protein solubility in
the experimental data. The chapter by (60 mM) was 100 percent effective for pro clEF, describing a family of protein solu
Chen and Fausnaugh-Pollitt [27] in this tein desorption, which was not the case bilizers (non-detergent sulfobetaines in
monograph compares the separation of for the commonly used washing proce concentrations up to 1 M) for preventing
33 polycationic protegrin analogs by dures of I M NaOH and 1 M HCI. protein precipitation and aggregation at
RPHPLC and CZE. These analogs (14 to the p/value and at low ionic strength. The
18 amino acids in length) consist of single proteins that were used in these studies
amino-acid substitutions, truncations, 1.3.2 clEF were L-aspartate oxidase, thermamylase
cyclic, and D-amino acid analogs. The (an et-amylase from Bacillus lichenifor
separation by CZE used a bare fused-sili Wehr et al. [9] presented an excellent re mis), and alcalase (an alkaline protease of
ca capillary and 100 mM sodium phos view on the capillary isoelectric focusing the subtilisin family). Conti et al. [30] sug
phate buffer, pH 2.6, and was achieved (clEF) of proteins. They described the in gested that common zwitterions such as
due to charge/mass differences of the pep- fluence of sample preparation including taurine and a few of Good's buffers such
Original Chromatographia Supplement Vol. 53,2001 S-9
as Bicine and CAPS were useful in acidic and physical gels. An example of a chemi crude E. coli fermentation broth of a re
pH gradients up to pH 8.0. Addition of up cal gel is cross-linked polyacrylamide. combinant protein were obtained using a
to 20'1<, of sugars. such as saccharose. sor This type of gel is attached to the capillary SO mM sodium borate buffer. pH 9.0. con
bitoL and sorbose. improved protein solu wall. Size separation of proteins up to taining 0.3% SDS. The recombinant pro
bility near the pI especially if the sugars 35 kDa in molecular weight have been re tein migrated after a large mass of cellular
were mixed with 0.2 M taurine. Tran et al. ported in chemical gels. An example of the components which demonstrates the un
[31] described a one-step clEF method for powerful capabilities of this system is the usual selectivities that can be obtained
resolving the glycoforms of recombinant separation of the two chains of insulin. with MEKC [35]. Another example is the
human immunodeficiency virus (HIY) en The disadvantage of the system is that use of MEKC to separate savinase (SAY.
velope glycoprotein. rgp 160sMN/LAI. only clean samples can be injected other a serine protease) and SA y* forms which
Separation was obtained with a polyvinyl wise the capillaries could clog and result differ from SA Y in a single residue where
alcohol (PYA) coated capillary using a in poor separation. Physical gels use non in methionine is replaced by serine [36]. If
mixture of narrow and wide-range pH cross-linked linear or slightly branched investigation must be performed on the
ampholytes. They determined that a mix polymers as the sieving matrix and are re native protein, MEKC can be conducted
ture of a sugar. saccharose and a zwit placed after each run. With the physical under non-denaturing conditions using
terion. 3( cyclohexylamino )-I-propanesul gel, pressure injection can also be used as zwitterionic or non-ionic surfactants. The
fonic acid was the most efficient additive a means of introducing the sample into work done by James et al. [37] on interfer
to avoid protein precipitation. The clEF the capillary. This option is not available on-gamma glycoforms using 400 mM bo
method was able to differentiate the rgp with the chemical gels. The noncross rate and 100 mM SDS. pH 8.5, demon
160 glycoform patterns of two sub-popu linked replaceable gel is becoming the pre strated that the glycoform migration
lations ofHIY-1. clEF is also reviewed in ferred method for performing size-separa times were inversely related to the amount
this monograph by Wehr et al. [32] who tion analyses with CEo These gels are com of associated carbohydrate. The separa
provide up-to-date information on am mercially available. Guttman [II] com tion efficiency was a result of the synergis
pholyte selection. mobilization tech pared SDS-PAGE and CE-SDS and con tic action of both the reduced electroos
niques. the use of internal standards. cluded that similar separation results and motic flow (EOF) and the higher surfac
methods for desalting and preventing pro precision can be obtained by either tech tant concentration of the MEKC method
tein precipitation, imaged cIEF, and de nique. With respect to molecular weigh as separate entities eliminating protein ad
tails of clEF coupled to mass spectrome determination. Guttman [II] presented a sorption. James et al. [37] suggested that
try. These authors also review in detail the table comparing slab gel electrophoresis the analytes with the shortest migration
clEF of monoclonal antibodies. hemoglo to CE-SDS on 65 individual proteins times had the highest carbohydrate con
bin and hemoglobin variants. and eryth ranging in molecular weight from 14- tent and thus the largest glycan structures.
ropoietin. 206 kDa. These data clearly showed that As the concentrations of borate and SDS
glycoproteins behave anomolously. and if in the buffers were increased, there was an
accurate molecular weights were required, increase in resolution observed. and three
1.3.3 CE-SDS the Ferguson method [33] described in main peak groups were resolved. Kats et
Guttman's review must be used. However, al. [38] used MECC to separate four major
Sodium dodecylsulfate polyacrylamide for recombinant proteins, the CE-SDS isoforms of the BR96 antibody following
gel electrophoresis (SDS-PAGE) has been method is used as a general purity test and heat treatment of the molecule in the pres
extensively used for the analysis of recom for analysis of production samples in a ence of 25 mM SDS in 12 mM sodium
binant proteins during formulation devel similar manner to the use of SDS-PAGE. borate buffer, pH 9.4. These analyses were
opment, purification of the product, and In this monograph, the chapter by Sche correlated to changes observed by circular
as a general purity test for quality control. nerman and Bowen [34] addresses the op dichroism spectropolarimetry.
CE methods based on similar principles timization, validation, and use ofCE-SDS Recently, Miksik and Deyl [39] pub
were reviewed by Guttman [II]. The CE in quality control testing. lished their investigation on separation of
technique has been called capillary so proteins and peptides in acid buffers con
dium dodecylsulfate gel electrophoresis or taining high concentrations of surfactants
capillary gel electrophoresis (CGE). The 1.3.4 MEKC (SDS). SDS was used because it has a
term. capillary electrophoresis SDS non washing effect on the capillary wall. Sec
gel sieving (CE-SDS-NGS), has also been In MEKC/MECC [12], surfactants such ondly, even under acidic conditions SDS
used when noncross-Iinked replaceable as SDS are added to the running electro can bind to protein analytes which then
matrices were used as the sieving medium. lyte at concentrations above the critical bear considerable negative charge that en
In this chapter. CE using SDS for size sep micellar concentration. The electro hances their separation. Apparently this
aration of proteins will be referred to sim phoretic migration of proteins is influ system is suitable for proteins which are
ply as CE-SDS. Guttman [II] described enced by protein-micelle interactions that soluble in acidic buffers. Separation of
the theory of size separation using CE can give a charged micellar characteristic four standard proteins. lactalbumin, cyto
SDS and evaluated the effects of opera to a neutral protein. A highly significant chrome c, carbonic anhydrase, and urease
tional variables such as field strength, benefit of the presence of surfactant is the was presented as well as the separation ob
temperature, capillary dimensions. and elimination of protein-wall interactions. served with a leucocyte lysate prepara
separation medium on the method. He re SDS may also induce protein denatura tion.
viewed the work performed using two tion, which could facilitate resolution of
types of gels that have been used. chemical individual components. Separations of a
S-lO Chromatographia Supplement Yol. 53,2001 Original