i —
`
`VOL. 705 NO. 1
`
`23 JUNE 1995
`
`.
`
`_
`Analytical Biotechnology
`
`ISSN 0021 -9673
`
`\th‘
`
`I
`
`'l‘
`
`I, — 5‘“
`— l.
`a
`(3)391-”
`
`r-:\AL OF
`
`!.UDING ELECTROPHORESIS AND OTHER SEPARATION METHODS
`
`I-IROMATOGRAPHY A
`
`EDITORS
`
`U.A.Th. Brinkman (Amsterdam)
`FLW. Giese (Boston. MA)
`J.K. Haken (Kensington, N.S.W.)
`C.F. Pooie (London)
`L.Fl. Snyder (Orinda, CA)
`S. Terabe (Hyogo)
`
`EDITORS. SYMPOSIUM VOLUMES.
`E. Heflrnann (Orinda. CA). 2. Deyl (Prague)
`
`EDITORIAL BOARD
`DW. Armslrong (Hulla. MO)
`WA. Aua (Halifax)
`P, Booak (Brno)
`PW. Carr (Minneapolis) MN)
`.1 Crornrnan (Liéga)
`MA. Davankov (Moscow)
`G,J, do Jung (Weasp!
`Z. Deyl (Prague)
`5. Dilli (Kensinglon, N.S.W.)
`2. El Floss: (Sullwaler. 0K)
`Hi Engelharoi (Saamrflcken)
`MBV Evans (Hatfield)
`Sr Farrah (Rome)
`GA. Guiochon (Knoxville) TN)
`PH. Had-dad (Hobart. Tasmania)
`LM Hais (Hradac Krélové)
`W.S. Hancock (Palm Alla. CA)
`S. Hiaflén (Uppsala)
`S. Honda (Higashinsalna)
`Cs‘ HOrvélh (New Haven) CT)
`J.F.K. Huber (Vienna)
`J. Janak (Brno)
`P. Jandera (9ardubice)
`BL Korea: (Boston) MA)
`J.J. Kirkland (Newport. DE)
`E7 327 Kovéts (Lausannel
`C S) Lee (Am. IA)
`Kr Masai (Prague)
`AJP. Martin (Cambridge)
`ED. Morgan (Kaela)
`Hi Pmpe (Armlerdam)
`PISA Flighatn (Milan)
`P. Schoenrnakers (Amsterdam)
`RI Schwarzenbach (Dfibandorl)
`REA Shoup (Wasl Lalayetle. IN]
`FIR Slnghal (Wichita. KS)
`AMI Sioulfl (Marseille)
`DJ. Sirydom (Boston. MA)
`T, Takagi (Osaka)
`Ni Tanaka (Kymo)
`K.K. Unger (Mainz)
`P. van 200mm (Bilmuvan)
`R Varpoorte (Leiden)
`Gy. Vigh (College Station) TX)
`J To Watson (East Lansing, MI]
`B‘D. Weslorlund (Uppsala)
`
`EDITORS. BIBLIOGRAPHY SECTION
`
`2. DEVI (Prague). Jr Janak (Brno), V Sci-marl (Prague)
`
`Pfizer EX.1007
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`ELSEVIER
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`JOURNAL OF CHROMATOGRAPHY A
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`INCLUDING ELECTROF’HORESIS AND OTHER SEPARATION METHODS
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`

`. VOL. 705, NO. 1
`
`JOURNAL OF CHROMATOGRAPHY A
`
`23 JUNE 19.95
`
`CONTENTS
`
`(Abstracts! Contents Lists published in Analytical Abstracts, Biochemical Abstracts, Biological Abstracts, Chemicai Abstracts. Chemi-
`gcai' Titles, Chromatography Abstracts. Current Awareness in Biological Sciences ('CABS). Current Contents! Life Sciences. Current
`ContentsfPhysicai, Chemical «lit Earth Sciences, Deep-Sea Research/Part B: Oceanographic Literature Review. Excerpta Medica,
`Index Medicus, Mass Spectrometry Bulletin, PASCAL-CNRS. Referativnyi Zhumai, Research Alert and Science Citation Index)
`
`TOPICAL ISSUE ON: ANALYTICAL BIOTECHNOLOGY
`
`Preface
`
`by G.C. Davis and R.M. Riggin (Indianapolis, IN, USA) ...............................
`
`High-speed high-performance liquid chromatography of peptides and proteins
`by H. Chen and Cs. Horvéth (New Haven, CT, USA) .................................
`
`1
`
`3
`
`Protein mass spectrometry: applications to analytical biotechnology (Review)
`by D.N. Nguyen, G.W. Becker and R.M. Riggin (Indianapolis, IN. USA) ....................... 21
`
`Chemical methods of protein sequence analysis (Review)
`by l.M. Bailey (Duarte, CA, USA) ........................................... 47
`
`Isoelectric focusing as a tool for the investigation of post-translational processing and chemical modifications of proteins
`(Review)
`by E. Gianazza (Milan. Italy) .............................................. 67
`
`fluorophore-assisted carbohydrate electrophoresis. Technology and applications (Review)
`by G.-F. Hu (Boston. MA, USA)
`........................................... 89
`
`Host cell contaminant protein assay development for recombinant biopharmaceuticals (Review)
`by LC. Eaton (Kalamazoo, MI, USA) ......................................... 105
`
`Moisture content in proteins: its effects and measurement (Review)
`by .l .K. Towns (Indianapolis. IN, USA) ........................................ 115
`
`Processing of C—terminal lysine and arginine residues of proteins isolated from mammalian cell culture (Review)
`by RJ. Harris (San Francisco, CA, USA) ....................................... 129
`
`Capillary electrophoresis of S. nuclease mutants
`by F. Ktilman, S. Ma. RD. Fox and Cs. Horvéth (New Haven. CT. USA) ...................... 135
`
`Analysis of recombinant human growth hormone in Escherichia coii fermentation broth by micellar high-performance liquid
`chromatography
`by M.A. Strege and AL. Lagu (indianapolis, IN. USA) ................................ 155
`
`Detection of neu differentiation factor with a biospecific affinity sensor during chromatography
`by H.S. Lu, D. Chang, D. Brankow and D. Wen (Thousand Oaks. CA, USA) .................... 163
`
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`
`
`
`
`lOURNAI. 0F
`CHROMATOGRAPHY A
`
`
`Journal of Chromatography A, 705 (1995) 129— 134
`
`Review
`
`Processing of C-terminal lysine and arginine residues of
`proteins isolated from mammalian cell culture
`
`L
`
`IIIyricai Chemistry Department. Genentech, Inc. (#62). 460 Point San Bruno Bouievard. South San Francisco. CA 94080,
`USA
`
`Reed J. Harris
`
`'_ terminal Lys or Arg residues whose presence was expected based on gene sequence information are often
`Us 1 in proteins isolated from mammalian cell culture. This discrepancy is believed to be due to the activity of
`or more basic carboxypeptidases. Internal Argi'Lys residues that become C-termjnal upon proteolysis or
`._v=Iogen activation. such as in the two—chain form of tissue plasminogen activator, may also be removed from the
`",;I
`' protein. Charge heterogeneity results when this type of processing is incomplete; such heterogeneity can be
`-d by isoelectric focusing or ion—exchange chromatography. The absence of C-terminal basic residues is not
`y a regulatory concern, as plasma-derived proteins are often similarly processed.
`
`tents
`
`129
`II oduction ....................................................................................
`1!
`130
`in rimentai conditions ...........................................................................
`...‘-‘:
`130
`-'1. CNBriC4 assay ..............................................................................
`130
`L2. Cation-exchange chromatography ................................................................
`130
`j 3. Materials ...................................................................................
`130
`_- ults .........................................................................................
`130
`I :1. Antibodies and antibody-related proteins ..........................................................
`L2. Two~chain tPA ................................................................................. 131
`_I:
`Ton ......................................................................................
`132
`'5“ » ledge-meats .................................................................................
`134
`-s ........................................................................................
`134
`
`a.“ inaction
`
`VEI theory, the characterization of recombinant
`i was is a straightforward matter,
`as
`the
`finite genetic engineering provides an ex-
`,{d amino acid sequence, with potential sites
`@Iodification identified based on known con—
`
`in practice, however, a
`sensus sequences [1].
`number of variations from the expected structure
`can be found. Variants may result from either
`known or novel types of in vivo (posttranslation-
`31) modification [2] or from spontaneous (non~
`enzymatic)
`protein
`degradation,
`such
`as
`methionine oxidation [3]. diketopiperazine for-
`
`7529673l95i$29.00 © 1995 Elsevier Science B.V. All rights reserved
`a: 0021-9673(94)01255-5
`
`
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`

`130
`
`R]. Harris 1’ J. Chromatogr. A 705 (1995) 129—134
`
`mation [4], aspartate isomerization and deamida-
`tion of asparagine residues [5]. or succinimide
`formation [6,7].
`This review will cover a type of posttransla-
`tional processing that
`is becoming a common
`analytical protein chemistry experience: the re-
`moval of Lys or Arg residues from the C-ter-
`minus of a protein obtained through mammalian
`cell culture. Several examples of this type of
`processing have been reported, and some suc~
`cessful approaches for identifying such variants
`are reported herein.
`
`2. Experimental conditions
`
`2.1. CNBr/C4 assay
`
`Samples were exchanged into 0.1% formic
`acid by dialysis, then 88% formic acid was added
`to bring the samples to a final solution of 20%
`formic acid. CNBr (Pierce) was dissolved in 20%
`formic acid at a concentration of 50 mg! ml, then
`added to sample(s) at a 5:1 (CNBrzprotein)
`weight ratio. After stirring 20 h at room tem-
`perature in the dark,
`the CNBr was removed
`under a nitrogen stream. Samples were reconsti-
`tuted in 20 pl 88% formic acid, then diluted with
`water to 300 pl final. C—terminal CNBr frag-
`ments were resolved using a Vydac C4 (250 X 2.1
`mm) column. The system was equilibrated for 20
`min at 100% solvent A [0.1% trifluoroacetic acid
`(TFA)
`in water],
`then, 6 min after sample
`injection, a linear gradient
`to 25% solvent B
`(0.1% TFA in acetonitrile) was developed over
`50 min by a Hewlett-Packard 1090 system. The
`flow-rate was 0.20 mi/min. with a constant
`
`temperature of 40°C.
`
`2.2. Cation-exchange chromatography
`
`A Pharmacia MonoS column (50 x 5 mm) was
`equilibrated with 95% solvent A (20 mM sodium
`phosphate. pH 6.9)-5% solvent B (solvent A +
`100 mM NaCl) at 40°C with a flow-rate of 1.0
`ml/min. Upon injection of 72 pg from three lots
`of rhuMAb HERZ. a gradient from 5 to 40%
`
`solvent B was developed over 40 min to -
`peak fractions.
`
`2.3. Materials
`
`Tissue plasminogen activator (tPA) p tag;
`from Chinese hamster ovary (CHO) cells II.1:_.=
`fected with the human tPA gene was producedu:
`Genentech (Activase). Bowes melanoma
`.11.-
`was provided by Desire Collen (University-7;;
`Leuven).
`rhuMAb HERZ is
`a
`ICCOII'Ibn11
`humanized antibody produced in transfect«.g_':
`CHO cells [3]. TFA was purchased from Pierce,
`while acetonitrile was from Burdick and Jackso
`
`Vydac C4 (214TP52) and C18 (218TP58) HM
`umns were purchased from The Separatif'u
`Group.
`.
`
`3. Results
`
`3.1. Antibodies and antibody-related proteins
`
`rCD4- lgG is a recombinant chimeric 11ouin
`dimcric protein secreted from transfected CH
`cells with the CH1 and CH 2 regions of a hum
`lgGI heavy chain replaced by residues 1— 180I
`the human CD4 receptor [9]. A CNBr cleavJ.-
`C4 RP-HPLC method was developed to idea
`the C-terminus of the mature CHO«wiexprfi-
`protein; in that study, the expected C-te u_ IJ‘
`
`Lys residues were found to be completely an “it
`[10]. This approach can be used for any prof I'
`with human IgG heavy chains such as rhu
`HERZ [11] as shownin Fig. 1.1n this exam‘7
`CNBr cleavage after Met4
`liberated C—te
`I “i3
`peptides that were isolated by RP-HPLC'
`t'_.I'
`characterized Peak A contains the ex-'
`heavy chain Cterminal CNBr peptide (rend
`432—450: HEALHNHYTQKSLSLSPGK).
`the major product
`(peak B)
`is a des-LYS
`peptide (residues 432—449: HEALHNH *
`SLSLSPG). Minor additional peaks were '_
`tained that
`resulted from formylation 0f
`peptide during the CNBr/formic acid incubati -'-r__
`We and others have reported this typeffJ
`processing with antibody and antibodylike “-—.;
`reins from a variety of sources (Table l)-
`
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`

`RJ. Harris J J. Chromatogr. A 705 (1995) 129434
`
`131
`
`as
`
`
`
`4.31
`
`450
`
`from lgG heavy chains causes charge
`idues
`heterogeneity. as forms with 0,
`1 or 2 Lys
`3‘31“"memw'c‘mmmm
`"::- ncmchfipevfldw WBWMW residues will result. Such charge variants can be
`misuse (—Lys'lSUi
`.
`resolved by cation-exchange chromatography
`[14,15]. For example, rhuMAb HER2 shows five
`charge species (Fig. 2). The main peak (peak 3)
`has no Lys“50 residues, while the more basic
`peaks 4 and 5 have one or two Lysm residues,
`respectively (data not shown). The more acidic
`peaks 1 and 2 are deamidated at Am” in one
`light chain; peak 1 has no Lysm residues, while
`peak 2 has one ”5450 residue.
`
`filmyllud
`. i
`
`3.2. Two-chain (PA
`
`A
`
`
`46
`
`44
`
`Tinctmin)
`
`an
`
`53
`
`
`in
`
`
`' lation of C-tenninal CNBr peptides from 25 mg of
`IgG, antibody by C4 RP-HPLC. Peak A: residues
`( + Lysm). Peak B: residues 432—449 (- Lys“).
`
`The activation of serine protease zymogens,
`including many of the coagulation/fibrinolytic
`proteins, occurs by proteolysis of arginyl bonds,
`converting the zymogen to a two—chain form
`whose polypeptide chains remain associated by
`disulfide bonds. Tissue plasminogen activator
`(tPA) is synthesized as a single-chain 527—residue
`polypeptide; depending on the cell culture con~
`ditions emplo ed, proteolytic cleavage can occur
`between Arg 75 and Ilezm, converting tPA to a
`two~chain form. Two GHQ-expressed recombi-
`nant
`forms were produced at Genentech, a
`primarily two-chain product and a primarily
`single-chain product (the latter is licensed as
`Activase).
`tPA isolated
`from the Bowes
`melanoma cell line is largely a two~chain prod-
`uct.
`
`
`
`
`.
`. of the C-terminal Lys residues from
`
`'7
`teins is not due to cloning errors, but,
`appears to be due to the action of car-
`
`tidase(s). The
`penultimate
`residues
`
`T do not appear to be removed, suggesting
`I_"_ela
`responsible carboxypeptidase(s)
`are
`" fer basic residues. Plasma-derived anti-
`also typically lack the heavy chain C-
`: Lys residues [12,13].
`plete removal of C-terminal Lys res-
`
`
`
`
`
`
`
`j of C-terminal Lyszrg processing
`
`Susceptible
`residue
`
`Cell lineisource
`
`Ref.
`
`Lys
`Lys
`Lys
`Lys
`Lys
`Lys
`Lys
`Arg
`Arg
`
`Arg
`Arg
`
`Transfected CHO
`Transfected CHO
`Hybridoma I ascites
`Hybridoma/cell culture
`Hybridoma! ascites
`Hybridomafcell culture
`Transfected SPZIO
`Transfected CHO
`Bowes melanoma
`
`Human urine
`Transfected CHO
`
`[10]
`[ll]
`[14]
`[14]
`[15]
`[15]
`[16]
`
`[1?]
`[17]
`
`
`
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`

`[32
`
`RJ. Harris I J. Chromatogr. A 705 (1995) 129434
`
`
`
`Nativeabombmat215nm
`
`
`
`minutes
`
`Fig. 2. Cation-exchange chromatography of three lots of rhuMAb HER2. Chromatographic conditions are given in Section 2.}.
`
`‘ _
`
`Tryptic peptide mapping of the two CHO-
`expressed forms showed peptides with or without
`Argm (residues
`268—275: QYSQPQFR and
`268—274: QYSQPQF, respectively) as shown in
`
`A
`
`128
`[BE
`38
`
`EB
`
`EB
`
`'13
`
`D
`
`mummnmm
`
`Fig. 3. The 268—274 (ties-mg”) peptide -
`dominates in the two-chain form, whereas a-a
`single-chain material gave the 268—275 pep'W',‘“
`almost exclusively Evidently, cleavage of
`I:
`Argm—Ile:7":J bond in the two-chain forms #9
`poses Arg27S to basic carboxypeptidase(s) in
`cell culture fluid. No evidence for further «41.:-
`
`the C-tenninus of the heavy Hr.
`cessing at
`(residues 1—274/275) was evident
`in WI”
`from the tryptic map. In both single-chain :4:
`twochain IPA the light chain C-tenninus (-
`.79
`Arg--52Pro) is unprocessed (unpublished dam
`The predominantly two-chain melanoma-deri— '1}
`[PA shows roughly equivalent
`levels of the f
`Argz'peptides (Fig. 4). Figs. 3 and 4 d'i “*
`slightly because different RP-I-IPLC col
`..=«--
`(albeit from the same vendor) were used.
`
`
`
`121a
`1.3
`
`B
`
`an
`an
`48
`22
`a
`
`
`34
`36
`33
`4B
`
`Time(nlin)
`
`4. Discussion
`
`Fig. 3. Detail showing differences between single-chain and
`two-chain GHQ-expressed tPA tryptic maps. Chromato-
`graphic details are given in Ref. {18]. (A) Two-chain tPA
`digest.
`(B) Single-chain IPA digest. The peaks marked
`+ Argm and —Arg”’
`contain
`residues
`268—275
`(OYSQPQFR) and 263—274 (QYSQPQF), respectively.
`
`'1 ,
`When derived from mammalian cell cult
`'ff_
`proteins that might be expected to term'
`with Arg or Lys residues may be processed*1 "-
`that
`these residues are absent
`in the purifi
`product. A number of antibody structural stu
`
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`RJ. Harris I J. Chromatogr. A 705 (1995) 129—134
`
`133
`
`
`
`Lys/Arg side-chain usually provide two basic
`groups for tryptic peptides. The number of basic
`groups is reduced by one in peptides that result
`from basic carboxypeptidase processing; as a
`consequence, the processed form(s) of C-ternii-
`nal peptides may be overlooked unless the inves-
`tigators actively look for any potentially minor
`des-Argx‘Lys singly protonated form. Automated
`methods for C-terminal analysis [21] may also
`assist in C-terminal processing studies.
`Basic carboxypeptidases are known to regulate
`peptide hormonal activity (e.g. bradykinin,
`the
`enkephalins and anaplylatoxins) [22]. However,
`no biological effect(s) have been reported for the
`C—terrninal processing of the proteins described
`in this report. The presence or absence of C-
`terminal Lys residues on antibody heavy chains
`is not likely to influence antigen binding, which
`is mediated by the distant complementarity-de-
`termining regions [23], nor are binding to the
`Fey receptor or complement Clq likely to be
`affected, as these involve residues in the hinge-
`CHZ region and CH2 domains,
`respectively
`[24,25].
`In general, the absence of C-terminal Lys or
`Arg residues is unlikely to be considered a cause
`for concern, as similar processing affects plasma-
`or urinary-derived proteins such as antibodies
`[12,13] and huEPO [17]. Variation in the extent
`of C-terrninal processing can lead to production
`lots with different charge distributions. The
`charge variants generated by incomplete process-
`ing of the Lys/Arg residue(s) may be isolated by
`cation-exchange chromatography; assaying these
`fractions for potency or clearance will help assess
`the appropriate level of concern for this type of
`heterogeneity.
`The responsible carboxypeptidasds) have not
`yet been identified. The lack of processing
`beyond the Lyszrg residue(s) suggests that an
`enzyme similar to one or more of several known
`basic carboxypeptidases may be responsible;
`such basic carboxypeptidases include pancreatic
`carboxypeptidase B, plasma carboxypeptidase N,
`membrane-bound (extracellular) carboxypepti-
`dase M, and carboxypeptidase H of secretory
`granules [22]. Plasma plasminogen-binding and
`urinary basic carboxypeptidases have also been
`
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`34
`
`'
`
`36
`Tlmetmh'tl
`
`38
`
`43
`
`
`
`‘
`_
`._.Detail showing differences between Bowcs melanoma
`~'-'..-
`single-chain CHO-expressed tPA tryptic maps.
`
`1
`tographic details are given in Ref.
`[17].
`(A)
`
`.-.
`lPA digest.
`(B) Singleuchain CHO [PA digest.
`- marked + Mg” and —Argm contain residues
`' (QYSOPOFR) and 268—274 (QYSQPQF).
`respec—
`
`
`
`
`--"- demonstrated the absence of heavy chain
`I...” '- al Lys residues [11,14—16]. The absence
`
`expected C-terminal Arg residue from
`
`and
`recombinant
`(CHO-expressed)
`__ erythropoeitin (huEPO) has also been
`
`,d [17]. Similarly, as
`shown for
`tPA,
`,rsion of serine protease zymogens to two-
`
`forms upon cleavage after Arg or Lys
`
`'-: during cell culture may allow basic
`xypeptidase processing to occur at
`the
`
`exposed Arg/Lys C—terminus. Incomplete
`ing of basic residues will cause charge
`
`neity that can be detected by ion—ex-
`
`_ chromatography or isoelectric focusing
`although additional factors (e.g., deamida-
`
`Qhosphorylation or sialic acid variability)
`'80 contribute to the overall charge heterm
`
`rminal processing may also be detected by
`d characterization of peptide maps using
`
`5 LC—MS approach (peptide digestion fol-
`
`. _.:-. by RP-HPLC separation with on-line mass
`I Metric detection)
`[19].
`In electrospray
`
`spectrometry,
`the major observed ion
`
`3) corresponds to the number of basic
`
`2"» [20];
`the N-terminus and a C-terminal
`
`
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`134
`
`RJ. Harris I J. Chromatogr. A 705 (1995) 129—134
`
`identified [26,27]. Basic carboxy—peptidase activi—
`ty has been reported for hybridoma cell culture
`supernatants and ascites fluid [14,15]; antibodies
`purified from ascites
`tend to be completely
`processed.
`It
`is also possible that
`the activity
`seen during cell culture results from release of a
`cytosolic enzyme from damaged cells, as is the
`case for the sialidase isolated from CHO cell
`
`culture [28]. Isolation and characterization of the
`responsible
`carboxypeptidase(s)
`should allow
`susceptible proteins to be cultured in the pres-
`ence of inhibitors, generating unprocessed ma-
`terial that could be used for investigations as to
`the effect(s) of the C—terminal processing.
`
`Acknowledgements
`
`The author thanks Madelyn Marine and Brent
`Larsen for developing the cation—exchange meth-
`od shown in Fig. 2, and Karen Wagner for
`contributing to the characterization of the ion-
`exchange peak fractions.
`
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`
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