Overexpression of recombinant human antithrombin III in Chinese hamster ovary cells results in malformation and decreased secretion of recombinant protein

Overexpression of Recombinant HumanAntithrombin III in Chinese HamsterOvary Cells Results in Malformation andDecreased Secretion ofRecombinant Protein

Martin Schroder, Peter Friedl

Technische Hochschule Darmstadt, Institut fur Biochemie, D-64287Darmstadt, Germany; Telephone: 49-6151-163655; fax: 49-6151-164759;e-mail: [email protected]

Received 1 March 1996; accepted 26 July 1996

Abstract: Overexpression of recombinant proteins in ani-mal cells is commonly achieved by using gene amplifi-cation techniques. Gene amplified cells possess up toseveral thousand genes coding for the target protein.Constitutive expression of these genes leads to high lev-els of the corresponding mRNA species and the imma-ture protein in the cell. Inefficient processing of theseprecursors may result from their great abundance in thecell. To study the influence of elevated intracellular levelsof a recombinant protein on its maturation and secretion,we examined the maturation and secretion of human an-tithrombin III (hATIII) in Chinese hamster ovary (CHO)cells at different levels of gene amplification. No loss ofvitality was caused by elevated secretion of hATIII. As theintracellular hATIII content increased, the efficiency ofhATIII secretion decreased steadily. The state of intracel-lular hATIII from the different cell lines was studied bydetermining the specific heparin cofactor activity ofhATIII. Intracellular hATIII from the highest amplified cellline displayed a lowered specific heparin cofactor activityindicating the presence of malfolded, only partiallyfolded, or incompletely or incorrectly posttranslationallymodified hATIII in this cell line. Thus, the ability of CHOcells to fold and/or introduce posttranslational modifica-tions and subsequently to secrete the recombinant pro-tein becomes saturated, and therefore these processesmay become limiting for protein secretion at highly el-evated expression levels. This limitation was not due to ageneral exhaustion of the secretory capacity of the cellsbecause hATIII constituted only a minor fraction of thesecreted proteins, even at high expression levels. © 1997

John Wiley & Sons, Inc. Biotechnol Bioeng 53: 547–559, 1997.

Keywords: recombinant protein production; protein fold-ing; protein secretion; human antithrombin III; Chinesehamster ovary cells; serum-free culture

INTRODUCTION

Various hosts have been used to express heterologous pro-teins. The most prominent among them are bacteria such as

Escherichia coli,yeasts such asHansenuela polymorphaand Saccharomyces cerevisiae,and mammalian cells suchas baby hamster kidney and Chinese hamster ovary (CHO)cells. Gene amplification is commonly used to express het-erologous proteins in mammalian cells. Cells are cotrans-fected with a gene encoding an amplifiable selection markerand a gene encoding a heterologous protein. After selectionfor initial transformants, the transfected genes are amplifiedby the stepwise increase of the concentration of the selectiveagent in the culture medium. Up to several thousand copiesof the transfected genes can be achieved. Among the avail-able amplification systems (Kaufman, 1990, 1993; Kellems,1991), the most commonly used is dihydrofolate reductase(DHFR). Using DHFR several heterologous proteins havebeen successfully expressed in CHO (Bonthron et al., 1986;Gentry et al., 1987; Kaetzel et al., 1985; Kaufman et al.,1985, 1988; Oprian et al., 1987; Seahill et al., 1983; Wasleyet al., 1987; Wurm et al., 1986; Zettlmeissl et al., 1987). Theeffect of cloned gene dosage on secretion of recombinantproteins has been studied inE. coli and mammalian cells.For E. coli it is well established that the overall gene ex-pression efficiency is maximal at an optimal plasmid copynumber per cell and then decreases as the number of plas-mid copies per cell increases (Aiba et al., 1982; Bailey et al.,1983; Seo and Bailey, 1985, 1986; Yamakawa et al., 1989).The plasmid content is dependent on the growth rate ofE.coli, and high plasmid copy numbers inhibit the formationof proteins and the growth ofE. coli (Bailey et al., 1983;Seo and Bailey, 1985, 1986; Yamakawa et al., 1989). Incontrast, only limited information is available for the effectof cloned gene dosage on the production of heterologousproteins in mammalian cells. Guarna et al. (1995) showedthat secretion of activated protein C (APC) was maximal ata cDNA dosage of approximately 300 copies per cell inCHO cells. A further increase in cDNA copy number led toa significant decrease in APC secretion (Guarna et al.,1995). Secretion of the viral-like particle hepatitis B surfaceantigen by CHO cells increased only fivefold whereas itsCorrespondence to:Peter Friedl

© 1997 John Wiley & Sons, Inc. CCC 0006-3592/97/060547-13

cDNA gene copy number increased 40-fold (Pendse et al.,1992). This discrepancy was caused by inefficient secretionof the viral-like particle.

Inefficient production of recombinant glycoproteins byamplified mammalian cells has only been reported for bloodcoagulation factor VIII (Kaufman et al., 1988). Intracellularfactor VII is found mostly in stable association with theheavy chain binding protein (BiP) (Dorner et al., 1987),which correlated with the inefficient secretion of factor VIIIby CHO cells. In contrast, a stable association of smallerglycoproteins with BiP, such as tissue-type plasminogenactivator (Dorner et al., 1987) and macrophage colony-stimulating factor (Dorner et al., 1992), was not detected.Even glycoproteins of similar size, e.g., von Willebrandfactor (Dorner et al., 1987), and of similar domain structuresuch as factor VIII, e.g., factor V (Pittman et al., 1994),were not detected in stable association with BiP. However,these studies did not address the influence of overexpressionof glycoproteins on their secretion. Thus, to date no data isavailable about the expression efficiency of recombinantglycoproteins at elevated expression levels in mammaliancells.

To identify activities that become limiting for proteinsecretion in highly amplified mammalian cells, we com-pared the secretion of human antithrombin III (hATIII) byCHO cells at different levels of gene amplification. hATIIIis a plasma serine protease inhibitor that acts primarily onthrombin (Jordan et al., 1980; Rosenberg and Damus,1973), but can inactivate other plasma serine proteases, in-cluding factor IXa, Xa (Kurachi et al., 1976), XIa (Damus etal., 1973), and XIIa (Stead et al., 1976) with lower effi-ciency. It is a 58-kDa glycoprotein (Nordenman et al., 1977)and possesses three intramolecular disulfide bonds (Pe-tersen et al., 1979). Four biantennary N-linked carbohydratechains account for 10–15% of the molecule by weight(Franzen and Svensson, 1980; Mizuochi et al., 1980).hATIII forms an equimolar covalent complex with plasmaserine proteases (Owen, 1975). The rate of complex forma-tion is enhanced approximately 10,000-fold through heparin(Hoylaerts et al., 1984; Jordan et al., 1980). Binding ofheparin to hATIII induces a conformational change in thehATIII molecule (Einarsson and Andersson, 1977; Fish andBjork, 1979; Olson et al., 1981; Li et al., 1976; Rosenbergand Damus, 1973; Villaneuva and Danishefsky, 1977),which accelerates the reaction between hATIII and throm-bin (Rosenberg and Damus, 1973) or tightens the heparin–antithrombin III complex (Olson et al., 1981; Peterson andBlackburn, 1987). The cDNA for hATIII was cloned (Bocket al., 1982; Chandra et al., 1983; Colau et al., 1985;Prochownik et al., 1983) and stable expression in CHO cells(Wasley et al., 1987; Zettlmeissl et al., 1987) yielded bio-logically active hATIII (Zettlmeissl et al., 1987). hATIIIproduced in CHO cells is very similar to plasma hATIII, butshows an altered glycosylation pattern (Zettlmeissl et al.,1989) and affinity for heparin (Bjo¨rk et al., 1992).

In this study we examined the influence of overexpres-sion on maturation and secretion of hATIII in CHO cells by

comparing secretion rates of hATIII and the intracellularhATIII level of four cell lines that are resistant to increasingconcentrations of the competitive inhibitor of DHFR,methotrexate (MTX). Specific activities of secreted and in-tracellular hATIII were compared.

MATERIALS AND METHODS

Materials

The DHFR deficient CHO cell line DUKXB1 (Urlaub andChasm, 1980) and hATIII secreting cell lines A11-A2, A11-A27, A11-A279, and A11-A279-C7 (Zettlmeissl et al.,1987) were provided by Dr. G. Zettlmeissl (Behringwerke,Marburg, Germany). Cell culture media and fetal calf serum(FCS) were purchased from Gibco BRL (Eggenstein, Ger-many). Human plasma fibronectin was prepared as de-scribed by Engvall and Ruoslahti (1977).holo-Transferrin,polyclonal anti-hATIII-antibody, and hATIII purified fromhuman plasma were from Behringwerke (Marburg, Ger-many). Pentex Ex-Cyte was from Bayer Diagnostic (Mu-nich, Germany). Tryptone soya broth was from Unipath(Wesel, Germany). Human plasma was from Deutsche Blut-spendedienst (Frankfort/Main, Germany). Trypsin, testcombinations for ammonia andL-lactate,L-lactate dehydro-genase (l-LDH), andD-biotinoyl-e-aminocapronyl-N-hydroxysuccinimide were from Boehringer Mannheim(Mannheim, Germany). Peroxidase conjugated goat-anti-rabbit-IgG and peroxidase-streptavidin conjugate were fromDianova (Hamburg, Germany). Bovine insulin, fetuin, soy-bean trypsin inhibitor, porcine gelatin, MTX, all otherchemicals, and Kodak X-Omat X-ray film were from Sigma(Deisenhofen, Germany). Tissue culture plastics and en-zyme-linked immunosorbent assay (ELISA) plates werefrom Greiner (Frickenhausen, Germany). Dialysis tubingwas from Roth (Karlsruhe, Germany). Ultrafiltration tubeswere from Amicon (Witten, Germany) and nitrocellulosesheets (pore size 0.45mm) were from Schleicher & Schuell(Dassel, Germany).

Cell Culture

Cultures dishes were coated with gelatin and 5mg/cm2 hu-man plasma fibronectin. The serum-free medium was me-dium CHO T1 (Noe et al., 1994) with minor modifications.Concentrations of proteins were 5mg/mL for insulin, 5mg/mL for transferrin, 10mg/mL for fetuin, and 20mg/mLfor lipoproteins; 5 g/L peptone were used. HEPES wasomitted from the medium and medium pH was bufferedwith a 10% (v/v) CO2 atmosphere. Hypoxanthine and thy-midine were not present in the medium for recombinant celllines. Cells were counted as described previously (Warbur-ton and James, 1993). Cell vitality was assayed at the be-ginning the the end of the experiments by reduction of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide (MTT) for 30 min (Mosman, 1983). For determination

548 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 53, NO. 6, MARCH 20, 1997

of the wet weight, cells were washed with phosphate buff-ered saline (PBS), trypsinized, counted, and centrifuged for10 min at 110g. The cells were resuspended in PBS, cen-trifuged for 5 min at 110g, and resuspended in PBS. Cellswere 1:2 serially diluted in PBS 6 times, centrifuged for 5min at 110g, the supernatant aspirated, and the cellsweighed. The weight was plotted against the number ofcells, extrapolated to zero cells, which corresponds to theweight resulting from residual moisture in the tubes, calcu-lated from they axis intercept, and subtracted from thedetermined weight of the cells.

Preparation of Samples for Analysis ofhATIII Content

Conditioned media were centrifuged for 5 min at 110g toremove cell debris, frozen in liquid nitrogen, and stored forlater analysis at −80°C. Cells were lysed in lysis buffer (50mM Tris-HCl, pH 8.4, 7.5 mM EDTA, and 0.05% (v/v)Triton X-100). After incubation at 4°C for 1 h the lysate wascentrifuged for 2 min at 12,000g. The supernatant was fro-zen in liquid nitrogen and stored at −80°C for later analysis.The pellet was extracted with 10× lysis buffer [50 mMTris-HCl, pH 8.4, 7.5 mM EDTA, and 0.5% (v/v) TritonX-100] as described above. Another round of extraction wasdone with a triple-detergent lysis buffer [50 mM Tris-HCl,pH 8.4, 7.5 mM EDTA, 0.1% (w/v) SDS, 0.5% (w/v) so-dium deoxycholate, and 1.0% (v/v) Nonidet P-40]. Concen-tration of samples by ultrafiltration was done according tothe manufacturer’s instructions.

Determination of hATIII

ELISA plates were coated overnight at 4°C with 2.5mg/mLpolyclonal anti-hATIII-antibody in PBS containing 0.05%(w/v) sodium azide, blocked with blocking buffer [0.1%(v/v) Tween-20 in PBS] for 30 min, and incubated withsample or standard (diluted in blocking buffer) for 2 h.hATIII purified from human plasma was used as the stan-dard in a range from 0.39 to 25 ng/mL. Detection of boundhATIII was done for 1 h each using first a biotinylatedpolyclonal anti-hATIII-antibody (50 ng/mL) and streptavi-din-peroxidase conjugate (100 ng/mL), both diluted inblocking buffer. Detection of bound peroxidase was donewith 0.1 mg/mL 3,38,5,58-tetramethylbenzidine in 100 mMsodium acetate, pH 4.0, adjusted with 20% (w/v) citric acid,to which 0.004% (w/v) hydrogen peroxide was added priorto use. The color reaction was allowed to proceed for 10min. All incubations were done at room temperature. Be-tween each step plates were rinsed 3 times with PBS. Ab-sorbance was measured at 690 nm using an ELISA reader(SLT Labinstruments, Crailsheim, Germany). Biotinylationof anti-hATIII-antibody was done according to the manu-facturer’s instructions.

Gel Electrophoresis and Immunoblotting of hATIII

Proteins were separated according to Scha¨gger and vonJagow (1987) on 16% (w/v) T, 3% (w/w) C (Hjerte´n, 1962)

separation gels with a 10% (w/v) T, 3% (w/w) C spacer geland a 4% (w/v) T, 3% (w/w) C stacking gel. Silver stainingwas done by the method of Hochstrasser as described byRabilloud (1992). For immunoblotting, samples were sepa-rated by SDS-PAGE on a 7.5% (w/v) T, 3.33% (w/w) Cseparation gel and a 4% (w/v) T, 3.33% (w/w) C stackinggel using the electrophoresis system of Laemmli (1970).Immunoblotting onto nitrocellulose sheets (0.45mm) wasdone using the transfer system of Kyhse–Andersen (1984).All subsequent steps were done at room temperature inblocking buffer. Membranes were blocked for 1 h, incu-bated with 25 ng/mL polyclonal anti-hATIII-antibody for 1h, washed 4 times for 15 min, incubated with 80 ng/mLperoxidase conjugated goat-anti-rabbit-IgG for 1 h, washed4 times for 15 min, and finally equilibrated in 100 mMTris-HCl, pH 8.5, for 15 min. Membranes are then incu-bated for 1 min in the dark with gentle agitation in 100 mMTris-HCl, pH 8.5, 1.25 mM luminol, 0.2 mM p-coumaricacid, and 0.0087% (w/v) H2O2, and exposed to X-ray filmfor up to 2 min. The molecular size standard transferred tothe membrane was stained with amido black 10 B accordingto Kyhse–Andersen (1984).

Determination of In Vitro Activity of hATIII

Two activities of hATIII in vitro, the progressive and theheparin cofactor activities, were determined. The progres-sive activity of hATIII was determined in conditioned me-dia and cell extracts after dialysis against 50 mM Tris-HCl,pH 8.4, 295 mM sodium chloride, 15 mM potassium chlo-ride, and 7.5 mM EDTA as described by Abildgaard et al.(1976). A final concentration of 50 mg/L polybrene wasadjusted in the dialysates to inhibit heparin. Heparin cofac-tor activity of hATIII was determined in conditioned mediaand cell extracts after dialysis against 100 mM Tris-HCl, pH8.4, 330 mM sodium chloride, 30 mM potassium chloride,and 15 mM EDTA using a commercially available chromo-genic assay that measures inhibition of thrombin by hATIII(Immuno AG, Heidelberg, Germany). Prior to assays dialy-sates were diluted with 1 vol water and a final concentrationof 3 × 103 international units (IU)/L heparin was adjusted.Progressive and heparin cofactor activities of hATIII werestandardized using the international hATIII reference prepa-ration (Kirkwood et al., 1980). Specific activities were cal-culated by dividing volumetric hATIII activities by hATIIIconcentrations determined by an ELISA specific forhATIII. Linear regression was used to calculate productionrates (mg hATIII/106 cells/24 h).

Determination of Metabolites, L-LDH, and Protein

L-lactate and ammonia were determine using commerciallyavailable test combinations.L-glutamine (Schrimpf et al.,1994),D-glucose (Kunst et al., 1984), andL-LDH (Vassault,1987) were determined as described previously. The frac-tion of cells lysed during culture was calculated as the ratioof L-LDH activity found in the culture medium toL-LDH

SCHRODER AND FRIEDL: PRODUCTION OF RECOMBINANT hATIII 549

activity found in the cell extracts. Multiplication of this ratiowith the amount of intracellular hATIII yielded the amountof hATIII released from the cells by cell lysis. Total proteinin dialyzed conditioned media was quantitated using thebicinchoninic acid assay of Smith et al. (1985) and was usedas an internal standard for total protein determination inpreviously collected conditioned media by the method ofBradford (1976) as modified by Read and Northcote (1981)and Bearden (1978).

RESULTS

Determination of hATIII Secretion Rates

CHO cells were seeded at 2 × 104 cells/cm2 in 35-mmdishes and grown to monolayers in serum-free mediumCHO T1. Medium was replaced and secretion of hATIIImonitored for 24 h. No loss of viability of A11-A2, A11-A27, and A11-A279 cells was observed over a period of 24h whereas a slight decrease in the viability of A11-A279-C7cells was observed. However, this decreased viability wasstill comparable to the viability of the other cell lines (Fig.1). At various times after medium replacement, the condi-tioned media were collected, cells counted, and cell extractsprepared. Secreted hATIII was determined by immunoblot-ting and an ELISA. No hATIII could be detected in mediaconditioned by DUKXB1 cells. Immunoblotting of condi-tioned media (Fig. 2A) revealed two forms of hATIII withapparent molecular masses of 58 and 54 kDa. Both werefound to be present in equal amounts. Secretion of hATIII

was linear up to 24 h in serum-free medium (Fig. 3). Se-cretion rates were calculated from the slope of lines gener-ated by linear regression (Table I). Increasing resistance toMTX lead to a steady increase in hATIII secretion.

Recently it was shown that cell lysis may be a majorproblem when producing hATIII in serum-free media.Large amounts of unglycosylated hATIII were detected inconditioned media, which obviously was liberated from thecells by lysis (Teige et al., 1994). Therefore, we determinedthe L-LDH activity in conditioned media to estimate theamount of hATIII liberated by cell lysis. However, noL-LDH activity was detected in conditioned media, whereasthe detection limit of the assay was 0.023 U/mL.L-LDH

Figure 1. Vitality of monolayer cultures. hATIII secreting CHO cellsA11-A2 (resistant to 0.1mM MTX), A11-A27 (resistant to 1mM MTX),A11-A279 (resistant to 10mM MTX), and A11-A279-C7 (resistant to 100mM MTX) were grown to monolayers and medium was replaced. Thevitality of monolayers was determined directly after medium replacement(solid bars) and 24 h after medium replacement (hatched bars) and isexpressed as the absorbance at 550 nm/106 cells.

Figure 2. Immunoblotting of hATIII in (A) conditioned media and (B)cell extracts. hATIII secreting CHO cells were grown to monolayers, me-dium was replaced, conditioned media were collected, and cell extractsprepared as described under Materials and Methods. (A) hATIII secretedfrom 1.5 × 104 cell each. In lanes 1, 3, 5, 7, and 9 media were conditionedfor 12 h; in lanes 2, 4, 6, 8, and 10 media were conditioned for 24 h byDUKXB1 cells (lanes 1 and 2), A11-A2 cells (lanes 3 and 4), A11-A27cells (lanes 5 and 6), A11-A279 cells (lanes 7 and 8), and A11-A279-C7cells (lanes 9 and 10). (B) Intracellular hATIII from 1 × 104 cells. Lanes1–3 are A11-A2 cells, 4–6 are A11-A27 cells, 7–9 are A11-A279 cells,10–12 are A11-A279-C7 cells at 0, 12, and 24 h after medium replacement.


activity was readily detectable in cell extracts. The amountof intracellularL-LDH per 106 cells was found to be 3.33 ±0.07 U for A11-A2 cells, 5.98 ± 0.21 U for A11-A27 cells,4.13 ± 0.12 U for A11-A279 cells, and 0.79 ± 0.02 U forA11-A279-C7 cells. From these data it was calculated thatless than 1% of the cells lysed during a 24-h cultivation andthus less than 0.1% of hATIII found in conditioned mediawas liberated from the cells by lysis.

Intracellular Steady-State Levels of hATIII

Cell extracts from the same experiment as for the determi-nation of hATIII secretion rates were analyzed for theirhATIII content by immunoblotting and an ELISA (Table I).Immunoblotting showed that intracellular hATIII possessed

an apparent molecular mass of 53 kDa (Fig. 4), which isslightly lower than that of secreted hATIII. In extracts ofA11-A279 and A11-A279-C7 cells a minor band of 45 kDawas detected (Fig. 2B), which may represent unglycosylatedhATIII. Control experiments demonstrated that extractionof hATIII was quantitative. After extraction with lysisbuffer, the remaining unsoluble material was reextractedwith lysis buffers of increasing stringency, namely 10× lysisbuffer and a triple-detergent lysis buffer (cf. Materials andMethods section). Whereas 2.1mg hATIII were extractedfrom A11-A279-C7 cells with lysis buffer, reextraction ofinsoluble material with 10× lysis buffer and triple detergentlysis buffer yielded only a further 0.02mg hATIII. Thus,more than 99% of intracellular hATIII was extracted withlysis buffer. Similar results were obtained for A11-A2, A11-A27, and A11-A279 cells (data not shown). No hATIIIcould be detected in cell extracts of DUKXB1 cells. No lossof intracellular of secreted hATIII could be detected undervarious experimental conditions, including incubation ofhATIII in conditioned media at 37°C for 24 h, repeatedfreezing and thawing of conditioned media and cell extracts,and incubation of cell extracts at 4°C for 5 days.

Comparison of hATIII Secretion Rates withIntracellular Steady-State hATIII Levels

Secretion rates were related to the intracellular hATIIIsteady-state level by calculating a secretion efficiency de-fined as micrograms secreted hATIII per micrograms intra-

Figure 3. Accumulation of hATIII in serum-free medium. hATIII secret-ing CHO cells (d) A11-A2, (s) A11-A27, (j) A11-A279, and (m) A11-A279-C7 were grown to monolayers, and the medium was replaced andsecretion of hATIII monitored. Conditioned media were collected at thetimes indicated after medium replacement. Secreted hATIII was deter-mined by an ELISA. Lines were generated by linear regression.

Table I. Secretion rates for hATIII and intracellular steady-state levels ofhATIII.

Cellline

MTX(mM)

hATIIIsecretion rate(mg hATIII/

106 cells/24 h)

IntracellularhATIII

(mg hATIII/106 cells)

hATIIIsecretionefficiency

(mg secretedhATIII/ mg

intracellularhATIII/24 h)

A11-A2 0.1 0.35± 0.01 0.025± 0.002 14.0± 1.2A11-A27 1.0 1.9± 0.1 0.22± 0.03 8.5± 1.2A11-A279 10 2.2± 0.1 0.46± 0.04 4.9± 0.5A11-A279-C7 100 7.4± 0.3 2.11± 0.19 3.5± 0.3

CHO cells A11-A2, A11-A27, A11-A279, and A11-A279-C7 weregrown to monolayers, and the medium was replaced, and secretion ofhATIII monitored over a period of 24 h. At the times indicated in Figure3, conditioned media were collected, cells counted, cell extracts prepared,and hATIII determined as described under Materials and Methods. Secre-tion rates were calculated by linear regression from Figure 3, and secretionefficiencies were calculated from secretion rates and intracellular hATIIIamounts.

Figure 4. Comparison of secreted recombinant hATIII with (A) plasma-hATIII and (B) intracellular recombinant hATIII by SDS-PAGE and im-munoblotting: (A) 10 ng plasma-hATIII (lane 1) and 2.5 ng secretedhATIII from each cell line (lane 2); (B) 2.5 ng secreted hATIII from eachcell line (lane 1) and 2.5 ng intracellular hATIII from each cell line.


cellular hATIII per day (Table I). With increasing resistanceto MTX, the secretion rate and intracellular amount ofhATIII increased. However, the latter increased to a greaterdegree, resulting in a decreasing secretion efficiency(Table I).

Influence of Overexpression of hATIII on SpecificGrowth Rates

Overexpression of hATIII may result in a loss of viability ofthe cell lines, which might decrease the secretion efficiencyof hATIII. This was shown by Pendse et al. (1992) forhepatitis B surface antigen. The viability of the cell lineswas described by two parameters: reduction of MTT and themaximum specific growth rate. Reduction of MTT showedthat all cell lines possess a comparable viability (Fig. 1).The maximum specific growth rates for all cell lines weredetermined from their growth curves and are shown inTable II. No significant decrease of the maximum specificgrowth rate was observed. Even A11-A279-C7 cells (resis-tant to 100mM MTX) showed a maximum specific growthrate that was comparable to that of the cell line DUKXB1.Thus, the decrease in the secretion efficiency of hATIII didnot coincide with a decrease in viability of the recombinantcells.

Influence of Key Metabolites on Secretionof hATIII

Depletion ofD-glucose from culture medium inhibits pro-tein secretion by induction of BiP (Pouysse´gur, et al., 1977;Shin et al., 1977) and inhibits initiation of translation(Sonenshein and Brawerman, 1977; van Venrooij et al.,1970, 1972), whereas ammonia influences the glycosylationpattern of glycoproteins (Gawlitzek et al., 1995, 1996) butnot secretion rates (Thorens and Vassalli, 1986). Further-more, it was shown that secretion of glycoproteins is ATP-dependent (Dorner et al., 1990; Pittman et al., 1994) and themain sources for energy metabolism of cultured mammaliancells wereD-glucose (Levington and Eagle, 1961; Warburg,1956) andL-glutamine (Donnelly and Scheffler, 1976). To

examine whether utilization ofD-glucose andL-glutamineby the various cell lines could account for the observeddecrease in secretion efficiency, we analyzed the consump-tion of D-glucose andL-glutamine by the different cell lines.The concentration ofD-glucose remained above 25 mM dur-ing the 24-h observation period and that ofL-glutamineabove 4 mM. Thus, protein synthesis and secretion had notbeen disturbed by depletion ofD-glucose orL-glutamine orthe accumulation of ammonia orL-lactate. Linear regressionwas used to calculate the utilization ofD-glucose andL-glutamine and the formation ofL-lactate and ammonia bythe different cell lines (Table III). All cell lines showed avery similar utilization of D-glucose. Approximately 66–75% of the utilizedD-glucose was converted toL-lactate asindicated by the yield coefficient (Table III). Utilization ofL-glutamine and formation of ammonia by A11-A279-C7cells were three- to fourfold higher than by the other celllines, whereas the yield of ammonia fromL-glutamine by allcell lines was similar.

Estimation of Total Protein Secretion Rate

Total protein concentrations were determined in condi-tioned media collected for analysis of hATIII secretion, andtotal protein secretion rates were calculated by linear regres-sion and expressed as micrograms secretion protein per 106

cells per day (Table IV). No decreases of the amount ofproteins present in the culture medium, namely insulin, fe-

Table II. Maximum specific growth rates of DUKXB1 and hATIII se-creting cells.

Cell lineMax. sp. growth rate

(h−1)

DUKXB1 0.067± 0.001A11-A2 0.069± 0.007A11-A27 0.052± 0.007A11-A279 0.055± 0.003A11-A279-C7 0.062± 0.012

To determine the maximum specific growth rates, CHO cells wereseeded at 1z 104 cells/cm2 in 35-mm dishes. Medium was replaced every24 h to avoid depletion of nutrients. To monitor growth of cells, cells werecounted every 12 h as described under Materials and Methods. Maximumspecific growth rates were calculated from the maximum slope of thegrowth curves.

Table III. Utilization of key metabolites by various cell lines.

Cell line

D-glucoseutilization(mmol/106

cells/24 h)

L-lactateformation(mmol/106

cells/24 h)Yield

coefficient

A11-A2 11.2± 2.0 14.0± 1.7 1.3± 0.2A11-A27 13.4± 3.0 18.2± 2.7 1.4± 0.2A11-A279 10.6± 2.0 16.0± 2.0 1.5± 0.2A11-A279-C7 12.9± 4.5 19.3± 3.9 1.5± 0.4

Cell line

L-glutamineutilization(mmol/106

cells/24 h)

Ammoniaformation(mmol/106

cells/24 h)Yield

coefficient

A11-A2 0.7± 0.2 0.5± 0.1 0.7± 0.2A11-A27 1.8± 0.4 1.9± 0.3 1.1± 0.2A11-A279 1.2± 0.5 1.5± 0.2 1.3± 0.5A11-A279-C7 4.2± 1.2 4.4± 0.9 1.1± 0.2

CHO cells A11-A2, A11-A27, A11-A279, and A11-A279-C7 weregrown to monolayers, and the medium was replaced and secretion ofhATIII monitored over a period of 24 h. At times indicated in Figure 3,conditioned medium was collected and analyzed forD-glucose,L-lactate,L-glutamine, and ammonia as described under Materials and Methods.Linear regression was used to calculate the utilization ofD-glucose andL-glutamine. Yield coefficients are the absolute ratios ofL-lactate forma-tion to D-glucose utilization and ammonia formation toL-glutamine utili-zation, respectively. Standard errors were also calculated by linear regres-sion.


tuin, and transferrin, was detected (data not shown). Totalprotein secretion rates increased about threefold from A11-A2 cells to A11-A279-C7 cells, whereas hATIII secretionincreased about 20-fold (Table I). In all cell lines hATIIIrepresented only a minor fraction of the secreted protein(Table IV).

Protein secretion may be influenced by the size of cells.Larger cells may provide a greater amount of enzymes thatare required for the processing of secretory proteins, thusleading to higher secretion rates per cell. To take differentsizes of cells into account, the wet weight of the differentcell lines was determined: 3.0 ± 0.5 mg/106 cells for A11-A2, 3.6 ± 0.9 mg/106 cells for A11-A27, 4.0 ± 0.9 mg/106

cells for A11-A279, and 2.8 ± 0.7 mg/106 cells for A11-A279-C7 cells. As all cell lines possessed comparable wetweights, no qualitative difference was found when proteinsecretion was related to biomass instead of cell number.

Specific Activity of hATIII In Vitro

Cells were grown to monolayers and medium was replaced.At 12 and 24 h after medium replacement, conditioned me-dia were collected and cell extracts prepared. hATIII wasquantitated by an ELISA and the progressive and heparincofactor activity in vitro in conditioned media and cell ex-tracts were determined. Specific activities were calculatedby dividing the activity found in samples by the amount ofhATIII present in the sample. Specific progressive and hep-arin cofactor activity of secreted hATIII were slightlyhigher than published specific activities for plasma hATIII(Zettlmeissl et al., 1987) (Table V). The specific heparincofactor activity of intracellular hATIII of A11-A2, A11-A27, and A11-A279 cells was close to that of secretedhATIII. In contrast, the specific heparin cofactor activity ofintracellular hATIII of A11-A279-C7 cells was 3.6 IU/mgsmaller than that of secreted hATIII and 2 IU/mg smallerthan that of intracellular hATIII of A11-A2, A11-A27, andA11-A279 cells. A11-A279-C7 cells also exhibited the

highest amount of intracellular hATIII compared to theother cell lines (Table I). Specific progressive activities ofintracellular hATIII from all cell lines were significantlylower than that of secreted hATIII. Only minor differencesof specific progressive activities of intracellular hATIIIfrom the different cell lines were detected, except for intra-cellular hATIII from A11-A279 cells.

Control experiments showed that progressive and heparincofactor activity of hATIII are stable under various experi-mental conditions, including incubation of hATIII in con-ditioned media at 37°C for 24 h, dialysis of samples at 4°Cfor 24 h, and repeated freezing and thawing of conditionedmedia, dialyzed samples, and cell extracts. Triton X-100 didnot affect the progressive or heparin cofactor activity ofhATIII (data not shown). Furthermore, neither media con-ditioned by DUKXB1 cells nor extracts of DUKXB1 cellsexhibited progressive or heparin cofactor activity or inhib-ited thrombin. In the absence of thrombin from the assay nocleavage of chromogenic substrates by dialyzed cell extractsor conditioned media was observed. Thus, assay conditionswere specific for hATIII and thrombin.

Table V. Specific activity of secreted and intracellular hATIII.

Cellline

Specific progressiveactivity(IU/mg)

Specific heparincofactor activity

(IU/mg)

Secreted Intracellular Secreted Intracellular

A11-A2 7.5± 0.2 1.4± 0.4 7.1± 0.1 6.7± 0.3A11-A27 6.5± 0.3 1.7± 0.4 8.1± 0.1 6.9± 0.7A11-A279 7.2± 0.3 4.4± 0.3 6.6± 0.2 6.8± 0.4A11-A279-C7 8.4± 0.1 2.1± 0.4 8.5± 0.1 4.8± 0.2

Cell line

Specific progressive activityof intracellular hATIII(% secreted hATIII)

Specific heparincofactor activity

of intracellular hATIII(% secreted hATIII)

A11-A2 18.9± 5.5 (p 4 1.27z 10−5) 88.2± 6.1A11-A27 23.0± 5.5 (p 4 2.22z 10−5) 90.8± 10.4A11-A279 59.5± 5.2 (p 4 1.43z 10−3) 89.5± 7.1A11-A279

-C7 28.4± 5.6 (p 4 9.10z 10−4) 63.2± 4.2 (p 4 1.97z 10−3)

hATIII secreting CHO cells were grown to monolayers and medium wasreplaced. At 12 and 24 h after medium replacement conditioned mediawere collected and cell extracts prepared. hATIII was quantitated by anELISA. Progressive and heparin cofactor activity in conditioned media andcell extracts were determined as described under Materials and Methods.Specific activities are expressed in international units (IU) as defined byKirkwood et al. (1980) per mg hATIII. Values are the means of twoindependent experiments. Specific activities of intracellular hATIII wereexpressed as % of the specific activity of secreted hATIII. The averagevalues for the specific activities of secreted hATIII were calculated to be7.4± 0.4 IU/mg for the specific progressive activity and 7.6± 0.4 IU/mgfor the specific heparin cofactor activity. A one-sidedt test was performedto test whether the specific activity of intracellular hATIII was significantlylower than that of secreted hATIII. Significant differencesp values statethe probability that the average values for the specific intracellular andextracellular activity are indistinguishable.

Table IV. Total protein secretion rates of hATIII secreting CHO cellsunder serum-free conditions.

Cell line

Secretion ratefor total protein

(mg/106 cells/24 h)

Fraction ofsecreted hATIII of

secreted total protein (%)

A11-A2 23.2± 4.5 1.5± 0.3A11-A27 31.0± 5.2 6.0± 1.1A11-A279 38.3± 3.4 5.9± 0.6A11-A279-C7 67.9± 8.0 10.9± 1.3

CHO cells A11-A2, A11-A27, A11-A279, and A11-A279-C7 weregrown to monolayers, and the medium was replaced and secretion ofhATIII monitored over a period of 24 h. At times indicated in Figure 3,conditioned medium was collected, hATIII determined by ELISA, andtotal protein determined by binding of Coomassie brilliant blue G-250 asdescribed under Materials and Methods. Secreted total protein was esti-mated by subtracting the protein concentration found in the medium fromthat found in conditioned media. Secretion of hATIII and total protein werecalculated as described in Table I.


DISCUSSION

Heterologous proteins have been expressed at high levels inmammalian cells by the use of gene amplification. It isconceivable that the expression level may influence the ef-ficiency of translation of specific mRNAs and the matura-tion and secretion of proteins. Secretion of a geneticallyengineered mutant of tPA, called tPA-3x, in which the threeN-linked glycosylation sites have been abolished, wasshown to be influenced by expression level (Dorner et al.,1987). tPA-3x was efficiently synthesized and secreted atlow expression levels, whereas high expression levels wereassociated with inefficient secretion of the molecule. How-ever, tPA-3x cannot be glycosylated so it is prone to interactwith BiP in the rough endoplasmic reticulum (RER), whichresults in inefficient secretion. We were interested in wheth-er the expression level also influences secretion of structur-ally intact glycoproteins in mammalian cells.

hATIII secreting CHO cells were derived by Zettlmeisslet al. (1987) using DHFR as an amplifiable selectionmarker. Secretion of hATIII by these cell lines in serum-freemedium CHO T1 is linear for up to 24 h, indicating that thecells do not suffer from adverse effects, such as depletion ofnutrients or the accumulation of waste products to toxiclevels. Comparison of hATIII secretion rates of A11-A2cells (resistant to 0.1mM MTX) and A11-A279-C7 cells(resistant to 100mM MTX) indicated a 20-fold increase insecretion of hATIII with increasing resistance to MTX.However, intracellular hATIII levels increased about 80-fold from A11-A2 to A11-A279-C7 cells. Therefore, secre-tion rates were related to the amount of intracellular hATIII,yielding a parameter called ‘‘secretion efficiency.’’ Thissecretion efficiency decreased steadily with increased resis-tance of cells to MTX (Table I). Thus, the efficiency ofhATIII secretion decreased with increasing expressionlevel. The steady decrease of the secretion efficiency fromA11-A2 to A11-A27, A11-A279, and A11-A279-C7 cellsindicates that this is primarily an effect of the amount ofintracellular hATIII.

Immunoblotting of conditioned media revealed twoforms with apparent molecular masses of 58 and 54 kDa,which were present in equal amounts (Fig. 2A). IntracellularhATIII was found to possess an apparent molecular mass of53 kDa (Fig. 2B) and was slightly smaller than secretedhATIII (Fig. 4). Thus, most of the intracellular hATIII is notcompletely posttranslationally modified. Terminal glyco-sylation of hATIII late in the batch process may be inhibitedby accumulation of ammonia in the culture medium, as hasbeen shown for the secretion of immunoglobulins by plasmacells (Thorens and Vassalli, 1986). This may lead to thesecretion of incompletely posttranslationally modifiedhATIII, which might be structurally very similar to the ma-jor population of intracellular hATIII. However, the oligo-saccharide side chains of secreted hATIII might be partiallycleaved in the culture supernatant by glycosidases, thus re-sulting in two forms of secreted hATIII. The release of sialicacid from glycoproteins by an extracellular CHO cell siali-

dase is well documented in the literature and cannot becompletely ruled out here (Gramer et al., 1995; Warner etal., 1993). Cell extracts of A11-A279 and A11-A279-C7cells displayed an additional minor band at 45 kDa, whichmay represent unglycosylated hATIII. However, the natureof this band has to be elucidated by further experiments.

It was shown by other investigators that secretion of gly-coproteins is ATP dependent (Dorner et al., 1990; Pittmanet al., 1994) and that depletion ofD-glucose from the culturemedium induces BiP (Pouysse´gur et al., 1977; Shiu et al.,1977) and inhibits initiation of translation (Sonenshein andBrawerman, 1977; van Venrooij et al., 1970, 1972). There-fore, we studied the energy metabolism of the differenthATIII-secreting CHO cell lines. We found only minor dif-ferences in the utilization ofD-glucose, the conversion ofD-glucose toL-lactate, the utilization ofL-glutamine, and theactivity of mitochondrial dehydrogenases. The cells showedgreater differences in the intracellular level ofL-LDH, butthe pattern of variation between the cell lines did not cor-relate with inefficient secretion of hATIII at high expressionlevels. However, a higher demand for the energy of cellswith high expression levels than those with lower expres-sion level is suggested by the three- to fourfold higher uti-lization of L-glutamine and the increased reduction of MTTby A11-A279-C7 cells compared to the other three celllines. The decreased amount of intracellularL-LDH in A11-A279-C7 suggests that the required additional energy isgenerated by oxidation ofL-glutamine instead of aerobicglycolysis.

hATIII constituted only a minor portion of secreted pro-tein from all cell lines (Table IV). Further, secretion ratesfor total protein increased steadily from A11-A2 to A11-A279-C7 cells and the increase was greater than the in-crease in hATIII secretion at every step of gene amplifica-tion (cf. Tables I, IV). Therefore, the observed inefficientsecretion of hATIII is confined to the recombinant protein,perhaps because other proteins secreted by CHO cells differsignificantly in their demands for posttranslational modifi-cation prior to secretion, e.g., glycosylation.

The specific heparin cofactor activity of secreted hATIIIfrom all cell lines was slightly greater than that of plasmahATIII (Zettlmeissl et al., 1987). Differences in the N-linked glycosylation pattern of hATIII secreted by CHOcells and hATIII purified from plasma may account for thisdifference (Fig. 4A). It was shown that a minor portion ofplasma hATIII, called hATIIIb, lacked one of the four N-linked oligosaccharides normally found on plasma hATIII,called hATIIIa, and displayed a threefold higher heparincofactor activity than hATIIIa (Bashkov et al., 1989; Bren-nan et al., 1987; Peterson and Blackburn, 1985). Bjo¨rk et al.(1992) reported that increased glycosylation of recombinanthATIII produced in BHK and CHO cells decreased its af-finity for heparin. Immunoblotting of conditioned mediashowed two forms of hATIII with molecular masses of 58and 54 kDa, respectively (Fig. 2A). Both were present inequal amounts. Whereas the form with a molecular mass of58 kDa was similar to plasma hATIII (Fig. 2A), the other


was slightly smaller than plasma hATIII (Fig. 4) and thusmight be responsible for the increased heparin cofactor ac-tivity of recombinant hATIII when compared to plasmahATIII. Similar results were obtained for other glycopro-teins of mammalian origin, e.g., tPA (Einarsson et al., 1985;Spellman et al., 1989; Zettlmeissl et al., 1991), erythropoi-etin (Yamaguchi et al., 1991), anda1-proteinase inhibitor(Guzdek et al., 1990).

The specific heparin cofactor activity of intracellularhATIII of A11-A2, A11-A27, and A11-A279 cells did notdiffer significantly from that of secreted hATIII (Table V).So the major part of intracellular hATIII in these cell linesis active, although intracellular hATIII appears to be slightlysmaller than secreted hATIII (Fig. 2). Terminal posttrans-lational processing of hATIII is not necessary for completeheparin cofactor activity of the molecule. Therefore, matu-ration of hATIII to complete heparin cofactor activity oc-curs early in the secretory pathway, because only the post-translational modifications that constitute the major part ofintracellular hATIII with an apparent molecular mass of 53kDa are necessary for complete heparin cofactor activity ofhATIII. This observation is in concordance with publisheddata. First, Bjo¨rk et al. (1992) showed that the removal ofsialic acids and the degree of antennarity of N-linked oli-gosaccharides did not affect the heparin cofactor activity ofhATIII. Second, the transit of secretory proteins from theRER to Golgi apparatus was demonstrated to be the rate-limiting step of secretion in human hepatoma HepG2 cells(Lodish et al., 1983). Thus, the major part of any secretoryprotein within the secretory pathway should be located inthe RER.

Intracellular hATIII of A11-A279-C7 cells exhibited aspecific heparin cofactor activity that was 2 IU/mg lowerthan that of intracellular hATIII of A11-A2, A11-A27, andA11-A279 cells. A greater fraction of intracellular hATIII inA11-A279-C7 cells was inactive or only partially activecompared to the other three cell lines. Therefore, maturationof hATIII in A11-A279-C7 cells was slower or incomplete.Thus, maturation of hATIII becomes a limiting activityabove a certain threshold concentration of intracellularhATIII, which is somewhere between 0.5 and 2.1mghATIII/106 cells in this system (Table I). A posttranslationalmodification of hATIII must occur to some extent to yieldactive protein, as can be concluded from unsuccessful at-tempts to express active hATIII in prokaryotic and lowereukaryotic hosts (Bro¨ker et al., 1987; Dingermann et al.,1991; Colau et al., 1985; Prochownik et al., 1983). Thisindicates that in A11-A279-C7 cells the capability for per-forming early posttranslational modifications is limited tocertain intracellular amounts of a recombinant protein.

A comparison of the specific heparin cofactor activity ofhATIII secreted by A11-A279-C7 cells and intracellularhATIII of A11-A279-C7 cells shows that the intracellularhATIII exhibited a specific heparin cofactor activity thatwas 3.6 IU/mg smaller than that of secreted hATIII. Inactiveor only partially active hATIII is not secreted by the cell linebut is retained in the cell. There it matures to the active form

or is degraded. Again, maturation of hATIII late in thesecretory pathway cannot be ruled out but seems unlikely.First, because the rate-limiting step of secretion is the transitof secretory proteins from RER to the Golgi apparatus (Lo-dish et al., 1983), most of the intracellular hATIII should bewithin the RER. Second, intracellular hATIII of A11-A2,A11-A27, and A11-A279 cells is fully active, which pre-cludes maturation of hATIII late in the secretory pathway,and differences in the location of maturation steps of hATIIIin the secretory pathway between the cell lines seem to beunlikely, too. Third, late posttranslational modificationssuch as the addition of sialic acids have been shown to notaffect the affinity of hATIII for heparin (Bjo¨rk et al., 1992).

The specific progressive activity of secreted hATIII by allcell lines was found to be 6–8 IU/mg (Table V) and wastherefore slightly higher than that of plasma hATIII (Zet-tlmeissl et al., 1987). The same cause as discussed for theheparin cofactor activity of hATIII may be applicable here.Intracellular hATIII from all cell lines exhibited a signifi-cantly lower specific progressive activity than secretedhATIII. This is in contrast to the observations made for thespecific heparin cofactor activity of hATIII and suggeststhat intracellular hATIII is associated with proteins of thesecretory apparatus that were not dissociated from hATIIIduring cell lysis and masked the progressive activity ofhATIII. Dissociation of these proteins may have occurredafter addition of heparin to the cell extract. Upon binding toheparin a conformational change of the hATIII molecular isobserved (Einarsson and Andersson, 1977; Fish and Bjo¨rk,1979; Olson et al., 1981; Li et al., 1976; Rosenberg andDamus, 1973; Villaneuva and Danishefsky, 1977), whichmay release proteins bound to hATIII from the hATIII mol-ecule. However, we cannot rule out that the progressive andheparin cofactor activities of hATIII are generated by twoindependent maturation steps. In this case heparin cofactoractivity would be generated prior to progressive activity,and it could be speculated whether progressive activity ofhATIII is generated late in the secretory pathway or is therate-limiting step of maturation of the hATIII molecule.

Proteins of the secretory pathway bound to intracellularhATIII from A11-A279-C7 cells may also mask its heparincofactor activity. However, this was not observed for all celllines, as the heparin cofactor activity of intracellular hATIIIfrom A11-A2, A11-A27, and A11-A279 cells was indistin-guishable from that of secreted hATIII. Thus, if proteins aretightly bound to hATIII in A11-A279-C7 cells a structuralcause related to the decreased heparin cofactor activity ofthese hATIII molecules has to exist.

Summarizing the data so far, it is obvious that the ob-served decrease in secretion of hATIII in A11-A279-C7cells is at least partially due to incomplete maturation ofhATIII in these cells as shown by the decreased specificheparin cofactor activity of intracellular hATIII from thesecells. Two topics are of special interest: what is responsiblefor the decreased specific heparin cofactor activity of intra-cellular hATIII from A11-A279-C7 cells, and what is re-sponsible for increasing retention of hATIII with increasing


amounts of intracellular hATIII? The observed decrease inspecific heparin cofactor activity of intracellular hATIIImust be related to a structural change in the molecule. Post-translational modifications performed on the hATIII mol-ecule include the cleaving of the N-terminal signal peptide,the formation of disulfide bridges, the addition and modi-fication of N-linked oligosaccharides, and the folding of thepolypeptide chain. From these only the latter three can beresponsible for the decreased heparin cofactor activity, be-cause no band representing pre-hATIII at approximately 48kDa was detected on immunoblots of cell extracts (Fig. 2B).The correct formation of disulfide bonds is necessary forbinding of heparin by hATIII (Ferguson and Finlay, 1983;Sun and Chang, 1989), and thus incorrect formation of di-sulfide bonds could explain the decreased heparin cofactoractivity of intracellular hATIII from A11-A279-C7 cells.We showed that only early steps in the formation and matu-ration of N-linked oligosaccharides are responsible for thedecreased heparin cofactor activity of intracellular hATIIIfrom A11-A279-C7 cells. Minor differences in the glyco-sylation pattern of hATIII linked to the transit of hATIIIfrom the RER to the cis-, medial-, and trans-Golgi compart-ments are unlikely to cause a decrease in heparin cofactoractivity for several reasons. First, the major part of intra-cellular hATIII should be located to the RER (Lodish et al.,1983). Second, glycosylation is directly involved in neitherinhibition of thrombin by hATIII (Owen, 1975), nor bindingof heparin to hATIII. In the latter reaction mainly basicamino acids (van Boeckel et al., 1991; We et al., 1994) areinvolved and major changes in glycosylation of hATIII arenecessary to influence its heparin cofactor activity (Bashkovet al., 1989; Brennan et al., 1987; Peterson and Blackburn,1985, Bjork et al., 1992). A minor band at 45 kDa wasdetected in cell extracts of A11-A279-C7 cells in immuno-blotting experiments (Fig. 2B), which might representunglycosylated hATIII. Complete lack of N-linked glyco-sylation may result in malfolding and retention of the mol-ecule in the cell as has been shown for factor VII andtPA-3x (Dorner et al., 1987); thus, perhaps accounting forthe decrease in heparin cofactor activity and retention ofhATIII in the cell. Last, the polypeptide chain might beincorrectly folded, thus explaining the decreased heparincofactor activity. Kozutsumi et al. (1988) demonstrated thatthe presence of malfolded proteins in the RER elevates theexpression of BiP and results in retention of the malfoldedprotein in association with the BiP. Current research fromBraakman et al. (1992) and Marquardt and Helenius (1992)showed that either the inhibition of N-linked glycosylationby tunicamycin or depletion of ATP, which in turn inhibitsthe action of BiP in the folding reaction, produces disulfide-bonded aggregates of malfolded proteins within the RER.Therefore, one limiting posttranslational activity will prob-ably suffice to produce these aggregates, and it would be ofminor importance whether the formation of disulfide bonds,the addition of N-linked carbohydrates, or the folding of thepolypeptide chain becomes limiting. All these processeswere shown to result in retention of the aggregates in the

cell. hATIII present in these aggregates will possess a de-creased heparin cofactor activity that explains the observeddecreased heparin cofactor activity of intracellular hATIIIfrom A11-A279-C7 cells. Further, we were also able toshow that incompletely processed hATIII was not secreted.

Secretion efficiency decreased steadily from A11-A2 toA11-A279-C7 cells and was already significantly decreasedin A11-A27 and A11-A279 cells. Only minor differences inspecific heparin cofactor activity of intracellular and se-creted hATIII from these cell lines were observed. No dif-ferences of intracellular hATIII from the different cell lineswere detected by immunoblotting (Fig. 2B). Transport ofsecretory proteins from the RER to the cell surface wasshown to occur by bulk flow (Wieland et al., 1987) and thesecretory activity increased steadily from A11-A2 to A11-A279-C7 cells as judged by the steady increase of totalprotein secretion from A11-A2 to A11-A279-C7 cells,which was not due to an increase in biomass of the cells.Thus, hATIII has to be retained in A11-A27 and A11-A279cells to a greater degree than in A11-A2 cells to explain thedecreased secretion efficiency in the presence of elevatedsecretory activity of the cells. The cause for retention orslower secretion of hATIII in A11-A27 and A11-A279 cellswas not the presence of inactive or not fully active hATIIIin the cell but instead seemed to be the amount of intracel-lular hATIII.

A steady increase of the total protein secretion rate fromA11-A2 to A11-A279-C7 cells was observed, whereas thebiomass of the cells remained constant. Therefore, the in-crease of protein secretion from A11-A2 to A11-A279-C7cells was due to an elevated activity of the secretory path-way of A11-A279-C7 cells and not an increase in the massof the secretory pathway. However, the secretion efficiencyfor the recombinant glycoprotein dropped steadily fromA11-A2 to A11-A279-C7 cells and at least partially inactivehATIII was present only in A11-A279-C7 cells. This dem-onstrates that the elevated secretory activity of A11-A279-C7 cells did not compensate the accumulation of immaturehATIII precursors in the secretory pathway. High intracel-lular levels of the recombinant protein may saturate onlysome activities of the secretory pathway that then limit thematuration of hATIII in the cell. Only a minor part of thehomologous proteins secreted by the cells may require theseactivities. This would explain the increase in the total pro-tein secretion rate as secretion efficiency for hATIIIdropped.

The data presented here show that secretion of a glyco-sylated secretory protein decreases as the intracellularamount of the glycoprotein increases. Furthermore, matu-ration of the glycoprotein to its active form becomes inef-ficient above a certain threshold concentration. This thresh-old concentration may be somewhere between 0.5 and 2.1mg hATIII/106 cells. This observation is in accordance withcalculations that predict that aggregation of proteins pre-vails over protein folding a high expression rates (Kiefhaberet al., 1991). Through this threshold concentration the hostcell may confine production levels that can be achieved by


gene amplification techniques. It is tempting to speculatewhether different mammalian cells will exhibit differentthreshold concentrations or whether the threshold concen-tration can be manipulated by genetic engineering. It hasbeen shown that the expression level of BiP correlates withthe efficiency of secretion of different glycoproteins (Dor-ner et al., 1988, 1992; Robinson and Wittrup, 1993) andtherefore BiP may represent at least one factor that mayregulate this threshold concentration.

Other expression systems show similar limitations for theproduction of recombinant heterologous proteins. Overex-pression of heterologous proteins inE. coli often results inthe formation of aggregate and denatured protein in so-called inclusion bodies (Marston, 1986; Schein, 1989;Williams et al., 1982), and elevated expression levels arelinked to the formation of inclusion bodies in at least somecases (Botterman and Zabeau, 1985; Gribskov and Burgess,1983; Williams et al., 1982; Wittrup et al., 1988). Theseinclusion bodies have been shown to be formed by off-pathway aggregations of folding intermediates (Haase–Pettingell and King, 1988) and chaperones have been shownto increase the solubility ofE. coli (Ellis and van der Vies,1991; Gaitanaris et al., 1990; Skowyra et al., 1990) andforeign proteins (Lee and Oiins, 1992). Further, BiP hasbeen reported to elevate the secretion of foreign proteins inS. cerevisiae.Thus, all expression systems seem to be lim-ited by their capability to process nascent polypeptide prop-erly when expressed at elevated levels.

We thank Dr. G. Zettlmeissl (Behringwerke, Marburg) for kindlyproviding the CHO cell lines, polyclonal anti-hATIII-antibody,and hATIII purified from human plasma. M. S. was supported bya grant from the Fonds der Chemischen Industrie (Frankfort/Main, Germany). This work is part of the Ph.D. thesis of M. S.at the Technische Hochschule Darmstadt (D17).

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Overexpression of recombinant human antithrombin III in Chinese hamster ovary cells results in malformation and decreased secretion of recombinant protein