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Quantitative MALDI-TOF-MS Using Stable-isotope Labeling: Application to theAnalysis of N-glycans of Recombinantα-1 Antitrypsin Produced Using DifferentCulture ParametersVéronique Blanchard a , Matthias Kaup a , Susann Eigel a , Silke Riecka , Volker Sandig b , Uwe Marx b , Rudolf Tauber a & Markus Berger aa Glycodesign and Glycoanalytics, Central Institute of LaboratoryMedicine and Pathobiochemistry, Charité Medical University,Charitéplatz 1, 10117, Berlin, Germanyb ProBioGen AG, Goethestraße 54, 13086, Berlin, GermanyVersion of record first published: 09 Nov 2011.

To cite this article: Véronique Blanchard , Matthias Kaup , Susann Eigel , Silke Rieck , Volker Sandig ,Uwe Marx , Rudolf Tauber & Markus Berger (2011): Quantitative MALDI-TOF-MS Using Stable-isotopeLabeling: Application to the Analysis of N-glycans of Recombinant α-1 Antitrypsin Produced UsingDifferent Culture Parameters, Journal of Carbohydrate Chemistry, 30:4-6, 320-333

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Journal of Carbohydrate Chemistry, 30:320–333, 2011Copyright C© Taylor & Francis Group, LLCISSN: 0732-8303 print / 1532-2327 onlineDOI: 10.1080/07328303.2011.605194

Quantitative MALDI-TOF-MSUsing Stable-isotope Labeling:Application to the Analysisof N-glycans of Recombinantα-1 Antitrypsin Produced UsingDifferent Culture Parameters

Veronique Blanchard,1 Matthias Kaup,1 Susann Eigel,1

Silke Rieck,1 Volker Sandig,2 Uwe Marx,2 Rudolf Tauber,1

and Markus Berger1

1Glycodesign and Glycoanalytics, Central Institute of Laboratory Medicine andPathobiochemistry, Charite Medical University, Chariteplatz 1, 10117 Berlin,Germany2ProBioGen AG, Goethestraße 54, 13086 Berlin, Germany

It is usually difficult to compare quantitatively different sets of glycan samples thathave slight differences among themselves using MALDI-TOF-MS. To overcome thisproblem, 13C-labeled glycans were used as internal standards during the measurementof the N-glycan samples permethylated with 12C-methyl iodide. This method was usedto analyze the N-glycans from human α-1 antitrypsin (A1AT) that was recombinantlyproduced using various culture parameters. Several pH values, dissolved oxygen con-centrations, and temperatures were used to produce A1AT using a batch process sys-tem, and we investigated the extent to which the glycosylation pattern of A1AT wasinfluenced by these parameters.

Keywords N-glycosylation; α-1 Antitrypsin; Human neuronal cell line/labeling/fermentation parameters

Received May 4, 2011; accepted July 9, 2011.Address correspondence to Veronique Blanchard, Glycodesign and Glycoanalytics, Cen-tral Institute of Laboratory Medicine and Pathobiochemistry, Charite Medical Univer-sity, Chariteplatz 1, 10117 Berlin, Germany. E-mail: veronique.blanchard@charite.de

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INTRODUCTION

Glycans influence the biological properties of glycoproteins such as solubility,[1]

protection against proteases,[2,3] antigenicity,[4] biological activity,[5] and serumhalf-life,[6] hence the activity of recombinant therapeutics. In order to producerecombinant glycoproteins with an optimal set of glycans, it is therefore neces-sary to develop accurate analytical methods similar to the ones of the field ofproteomics suited to compare the N-glycans of recombinant proteins expressedunder different conditions.

High-throughput techniques have been established in the field of pro-teomics that allow quantifying changes of protein expression in biologicalsystems[7]: labeling with 15N during cell culture,[8] labeling with 18O duringtryptic digestion,[9] stable isotopic labeling using amino acids,[10] or chemicallabeling.[11] Although the analysis of glycans is more complex than that ofpeptides, robust analytical methods have been established. N- and O-glycansare usually terminated with acidic residues that disable efficient ionizationin MALDI-TOF mass spectrometers and may even prevent any MS detection.Therefore, a chemical derivatization such as permethylation is recommendedprior to MALDI-TOF-MS analyses because sialic acids are stabilized.[12] Bothneutral and acidic glycans are chemically derivatized by permethylation andare detected using the same conditions. Permethylated oligosaccharides ionizein a more uniform way than their native forms because of their hydropho-bicity.[13] In 2007, Kang et al.[14] introduced a method of stable-isotope la-beling prior to MS analyses that involves permethylating samples and mix-ing them with a sample that has been deuteriomethylated. In the same year,Alvarez-Manilla et al.[15] used the same principle but with 13C-labeled methyliodide.

During fermentation, glycosylation of recombinant glycoproteins is af-fected by culture parameters.[16] For instance, an excess of ammonia can leadto a decrease in glycoprotein sialylation on both N- and O-glycans.[17,18] As itis of paramount importance to characterize the glycosylation profiles of recom-binant therapeutics, it is equally relevant to investigate and to understandhow cell culture parameters affect N-glycosylation of the final product. To thisend, we chose human α-1 antitrypsin (A1AT) and the novel human neuronalcell line (AGE1.hn) as a model system.[19] A1AT, which belongs to the familyof serine protease inhibitors, is involved in the protection of lungs from neu-trophil elastase enzyme that drastically affects tissue integrity. Therefore, re-combinant A1AT is of high interest for the treatment of patients suffering fromhereditary A1AT deficiency. The glycoprotein consists of a single polypeptidechain of 394 amino acids[20,21] and is N-glycosylated at Asn-46, Asn-83, andAsn-247.

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MATERIALS AND METHODS

All chemicals were purchased from Sigma-Aldrich, MO, unless stated other-wise.

Production of Recombinant Human α-1 AntitrypsinThe cloning, expression, and purification of biologically active A1AT were

performed as described elsewhere.[19] Cells were grown in the EX-CELL R©

VPRO Serum-Free Medium containing 2 mM glutamine using eight fer-menters, each having a total capacity of 800 mL (DasGip, Germany). Dissolvedoxygen (DO) was set automatically at the desired value by the fermenter us-ing a combination of compressed air and pure oxygen. At the beginning ofthe fermentation, DO, pH, and temperature were set at different values thatwere maintained until the end of the process. Cell culture supernatant aliquotswere harvested regularly during the course of the fermentation as described inTable 1.

Purification of Recombinant Human α-1 AntitrypsinCell culture supernatants, preconcentrated using a Vivaspin 20 MWCO

30000 column (Sigma-Aldrich, MO), were purified first by anion-exchangechromatography using a Mono Q 50/5 HR column (GE Healthcare, Piscataway,NJ), then by size-exclusion chromatography using a Superdex 200 column300/10 HR (GE Healthcare, Piscataway, NJ). The A1AT fraction was collectedand the protein content was subsequently quantified using a bicinchoninic acidprotein assay kit (Pierce Biotechnology, Inc., Rockford, IL). The purity of A1ATwas checked by discontinuous SDS-PAGE according to Laemmli.[22] The reduc-ing gel was composed of a 4% stacking gel at pH 6.8 and a 10% separatinggel at pH 8.8. The bands were visualized using the Coomassie Blue stainingmethod.

Table 1: Summary of the different culturing parameters that were used in this study.Each fermenter was run using the same parameters (pH 7.1, 37◦C, 50% DO) exceptone, which varied in pH, temperature, or DO

Parameter Values Harvesting (day)

pH 6.9, 7.1, 7.3 0, 6, 12Temperature (◦C) 33, 35, 37 0, 6, 10, 17Dissolved oxygen (DO,%) 10, 50, 100 0, 10

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Preparation of Asialylated N-glycans from α-1Acid Glycoprotein (AGP)AGP, 200 µg dissolved in 50 mM phosphate buffer pH 7.0, was digested

with trypsin overnight at 37◦C. The solution was deactivated for 5 min at95◦C. Then, the tryptic digest was incubated overnight with 200 mU PNGaseF (EC 3.5.1.52; Roche Applied Science, IN) at 37◦C. N-glycans were isolatedfrom tryptic peptides using C18 cartridges and desalted on graphitized carboncolumns (Alltech, Deerfield, IL).[23] N-glycans were dissolved in 3M acetic acidand incubated for 90 min at 80◦C in order to cleave off sialic acids. After the re-action, the sample was neutralized with 25% aqueous NH3 and was lyophilizedthree times before being permethylated using either 12CH3I or 13CH3I.

Release and Isolation of N-glycans from A1ATA1AT, dissolved in 0.5 × PBS, was denatured in the presence of 1% sodium

dodecyl sulfate (w/v) and 10% 2-mercaptoethanol for 5 min at 95◦C. The gly-coprotein solution was then diluted 1/10 and digested with PNGase F (1mU/µg A1AT, EC 3.5.1.52; Roche Applied Science, IN) overnight at 37◦C inthe presence of 1% NP-40. Detergents were removed using Calbiosorb beads(Calbiochem, San Diego, CA).[24] N-glycans were isolated and desalted ongraphitized carbon columns. Typically, about 3 µg N-glycans were obtainedfrom 30 µg A1AT and were subsequently permethylated.

Permethylation and Mass SpectrometryPermethylation was carried out according to standard protocols.[15,24] A1AT

or AGP N-glycans, dissolved in NaOH/DMSO, were permethylated with ei-ther 12CH3I or 13CH3I (CortecNet, France). After the reaction, chloroformwas added and the chloroform phase was washed with MilliQ water untilthe pH was neutral. Samples were lyophilized and dissolved in 10 µL 75%aqueous acetonitrile and the internal standard, consisting of A1AT or AGPN-glycans permethylated with 13CH3I, was added. MALDI-TOF mass spec-tra were recorded on an Ultraflex III mass spectrometer (Bruker Daltonics)equipped with a Smartbeam laser and a LIFT-MS/MS facility. Calibration wasperformed on a glucose ladder for N-glycans (Bruker Daltonics, Germany).Each spectrum consisted of 4,800 laser shots and was recorded in reflectorpositive ionization mode in the mass range m/z 1,000 to 5,000 Da; 0.5 µLpermethylated N-glycans were mixed on the ground steel target in a ratio of1:1 with the matrix consisting of D-arabinosazone (5 mg/mL) dissolved in 70%aqueous ethanol.[25]

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RESULTS AND DISCUSSION

Quantification of Permethylated N-glycans Using 13C LabelingIn order to compare accurately a maximum number of glycan samples

that have a very similar content within the shortest time, we chose the ap-proach of stable-isotope labeling using 12CH3I and 13CH3I in combination withMALDI-TOF-MS measurements. We first tested the linearity of the method onN-glycans isolated from AGP. To this end, N-glycans, released and isolatedfrom AGP, were permethylated with either 12CH3I or 13CH3I. MALDI-TOFmass spectra are presented in Figure 1A and B. N-glycans were observed intheir sodiated forms. Stable-isotope labeling with 13CH3I increments masseswith 1 Da per methylated free hydroxyl group compared to a standard 12C-permethylation. For example, a diantennary digalactosylated structure has 29methylated hydroxyl groups, and a shift of 29 Da is observed between the 12C-and the 13C-labeled isotopic cluster peaks. Therefore, none of the 13C-labeledand 12C-labeled MS peaks overlap among themselves. 12C- and 13C-labeledsamples were subsequently mixed in the following ratios: 10:1, 3:1, 1:1, 1:3, and1:10, and MALDI-TOF mass spectra were recorded. The intensity of each peak,measured in duplicate, was determined as the sum of the intensities of eachisotopic cluster. The comparison between theoretical and measured 12C/13C ra-tios is presented in Figure 1C for the diantennary digalactosylated N-glycan atm/z 2070.0 (12C) and 2099.0 (13C). A linear response was obtained in the rangethat we examined (r = 0.9995), indicating that the measured ratios were coin-ciding with the theoretical values. Finally, we decided to choose a 12C/13C ratioof 3/1 for further experiments because the 13C-labeled methyl iodide is morecostly than the 12C-labeled one.

Analysis of A1AT N-glycosylation Using Stable-isotope LabelingThe clone producing the highest A1AT levels (about 300 mg/L) was grown

for 10 to 17 d in eight fermenters that were run in parallel. Each fermenterwas run using the same parameters (pH 7.1, 37◦C, 50% DO) except one, whichvaried in pH, temperature, or DO (see Table 1). Sample aliquots were takenover the course of the fermentation at the time points specified in Table 1. Asa cell culture needs some adaptation time, samples harvested at “day 0” weretaken from the preculture (inoculum) and not from the fermenter itself.

Sample aliquots were first concentrated by ultracentrifugation and werethen purified by anion-exchange chromatography followed by size-exclusionchromatography on Superdex 200. The purity of rA1AT was checked on 10%reducing SDS-PAGE stained with Coomassie Blue (Fig. 2).

Typically, A1AT (30 µg) was digested with 30 mU peptide-N4-(N-acetyl-β-glucosaminyl) asparagine amidase F (PNGase F) in the presence of SDS,

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Influence of Culture Parameters on A1AT N-glycans 325

2-mercaptoethanol, and NP-40. The efficiency of the de-N-glycosylation wasanalyzed by SDS-PAGE (Fig. 2). After removing detergents using Calbiosorbbeads, N-glycans were desalted by solid-phase extraction on carbographcolumns. After permethylation, samples were mixed in a ratio of 3/1 (sam-ples/internal standard) with an internal standard consisting of A1AT N-glycans that were permethylated with 13CH3I instead of 12CH3I using the same

Figure 1: MALDI-TOF-MS spectra of desialylated N-glycans from AGP measured in thepositive-ion mode after permethylation with 12C-labeled CH3I (A) and 13C-labeled CH3I (B).All molecular ions are present in sodiated form [M+Na]+. Gray circle, Man; white circle, Gal;black square, GlcNAc; black triangle, Fuc. (C) Comparison between the theoretical andmeasured 12C/13C ratios (10:1, 3:1, 1:1, 1:3, 1:10) of the diantennary structure at m/z 2070.0and 2099.0, respectively. A correlation coefficient of 0.9995 was obtained. Samples weremeasured in duplicate. (Continued)

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Figure 1: (Continued)

Figure 2: 10% SDS-PAGE of purified A1AT stained with Coomassie Blue. Lanes 1 and 2 showrecombinant A1AT before and after PNGase F digestion, respectively.

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Figure 3: (A) Representative MALDI-TOF spectra of permethylated N-glycan pools releasedfrom A1AT that was produced using the reference parameters (7.1, 37◦C, 50% DO) andharvested on day 10. Measurements were carried out in the positive-ion mode. Allmolecular ions are present in sodiated form [M+Na]+. Gray circle, Man; white circle, Gal;black square, GlcNAc; black triangle, Fuc; black diamond, Neu5Ac. The displayed structuralcartoons imply neither specific triantennary N-glycan isomer nor assignment of an LeX todistinct antennae. Relative intensities were determined and data are presented accordingto the degree of sialylation (B) and antennarity (C).

standard conditions. The stock of 12CH3I A1AT N-glycans was prepared froma pool of A1AT that was produced under non-modified conditions. An initialexperiment was carried out in quadruplicate using our reference parameters(7.1, 37◦C, 50% DO) and a representative set of data is given in Figure 3.The N-glycosylation pattern of A1AT expressed in the human neuronal cellline AGE1.hn was recently reported by our group.[19] Core-fucosylated di-, tri-,and tetra-antennary N-glycans bear 0-4 N-acetyl-neuraminic acid (Neu5Ac)

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Table 2: Permethylated N-glycans from recombinant A1AT were analyzed andquantified by MALDI-TOF mass spectrometry in the positive-ion mode using thestable-isotope labeling method

m/z (12C) m/z (13C)

Composition Calculated Measured Calculated Measured

dH3H5N4 2592,3 2592,1 2626,3 2626,2dH1H5N4S1 2605,3 2605,1 2640,3 2640,2dH2H5N4S1 2779,4 2779,2 2816,4 2816,3dH1H5N4S2 2966,5 2966,3 3006,5 3006,4dH3H6N5 3041,5 3041,4 3081,5 3082,4dH1H6N5S1 3054,5 3054,4 3095,5 3096,4dH4H6N5 3215,6 3215,4 3257,6 3258,5dH2H6N5S1 3228,6 3228,5 3271,6 3271,5dH3H6N5S1 3402,7 3402,6 3447,7 3447,7dH1H6N5S2 3415,7 3415,6 3461,7 3460,7dH2H6N5S2 3589,8 3589,7 3637,8 3637,8dH1H6N5S3 3776,9 3776,8 3827,9 3827,9dH1H7N6S2 3864,9 3864,9 3916,9 3917,0dH2H7N6S2 4039,0 4039,0 4093,0 4093,1dH1H7N6S3 4226,1 4226,1 4283,1 4283,2dH2H7N6S3 4400,2 4400,2 4459,2 4459,4dH1H7N6S4 4587,3 4587,5 4649,3 4649,6

dH, deoxyhexose; H, hexose; N, N-acetylhexosamine; S, sialic acid (Neu5Ac).

residues. Part of the N-glycan pool contains the Lewis X epitope. A monosialy-lated difucosylated diantennary N-glycan at m/z 2779.2 and a disialylated di-antennary core-fucosylated glycan at m/z 2966.3 are the two major structuresof A1AT also observed in the present study. In order to examine the influenceof pH, DO, and temperature on the N-glycans of recombinant A1AT in termsof antennarity and degree of sialylation, the 17 most abundant N-glycan struc-tures of A1AT listed in Table 2 were quantified. The data shown in Figure 4represents an average of two measurements.

Effect of the DOA constant supply of oxygen is required during a fermentation process be-

cause oxygen has a low solubility in aqueous media. Fermenters were run atDO concentrations set to 10%, 50%, and 100%. After 10 days, the relative in-tensities of diantennary N-glycans increased from 73% to over than 83% forthe three tested concentrations (Fig. 4A), whereas the sialylation degree re-vealed differences for the DO values that were tested. The relative intensityof asialylated N-glycans decreased from 20% to 17% over the course of thefermentation, whereas it increased to 21% and 25% for 10% and 50% DO, re-spectively (Fig. 4D). Although it is quite costly to run a process using 100% DO,it allows maximizing the degree of sialylation. Chotigeat et al.[26] also reported

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Influence of Culture Parameters on A1AT N-glycans 329

Figure 4: Influence of fermentation parameters on the N-glycans of recombinant A1AT: (A)and (C) DO; (B) and (D) pH; (C) and (F) temperature. Relative intensities of diantennary andasialylated N-glycans were added. Experiments were performed in triplicate.

that the sialylation degrees of human follicle-stimulating hormone producedin CHO cells were highest was 100% DO were used. The increase of DO wassimultaneously correlated with an increase in sialyltransferase activity.

Effect of the pHExtracellular pH affects cell metabolism by modifying intracellular pH and

glucose transport among others.[27,28] Therefore, glycosylation changes are ex-pected to happen as the pH is modulated. Samples were harvested on days 0,6, and 12. For the three pH values that were tested, namely, 6.9, 7.1, and 7.3,an increase in diantennary N-glycans of about 5% was observed after 6 days.After 12 days, the fraction of diantennary N-glycans was reduced except for the

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fermenter that was set to pH 7.1 (Fig. 4B). A decrease in asialylated N-glycanswas observed after 6 d for the pH values that were investigated; the lower thepH value was, the stronger the effect was observed (Fig. 4E). On days 6 and 12,the lowest value (12%) for asialylated N-glycans was observed at pH 6.9. Thisvalue is therefore the optimum of the three pH values regarding the degree ofsialylation in our model system. Borys et al.[29] have shown using SDS-PAGEthat the expression of glycans on mouse placental lactogen was fairly constantbetween pH 6.9 and 8.2 and was decreased outside this range, but the methodemployed might have failed to detect such slight changes as observed in thisstudy.

Effect of the TemperatureThe relative intensity of diantennary structures increases from 80% to

about 84% to 86% in the first 6 d of the fermentation at the three working tem-peratures 33◦C, 35◦C, and 37◦C (Fig. 4C). Then, at 33◦C and 37◦C, a decreaseis observed from day 10 on, whereas a maximum of 90% is reached after 10 dat 35◦C, which is followed by a slow decrease at day 17. The degree of asialy-lation is constant at 14% for the first 6 d at 33◦C and 37◦C, whereas it risesto 25% at 35◦C (Fig. 4F). At day 10, an increase in asialylated structures isobserved, especially for the temperatures 33◦C and 37◦C, which is followed bya decrease at day 17. The degree of asialylation increases over the course of thefermentation at 35◦C to end at 39% at day 17. Globally, over the course of theprocess, the lowest temperature tested here (33◦C) correlates with the high-est degree of sialylation. Nabi et al.[30] reported that a low temperature wasslowing down the transit of nascent glycoproteins in the Golgi. Temperaturesas low as 20◦C were used, which resulted in the formation of polylactosamineon lysosomal membrane glycoproteins expressed in epithelial cells. Similarly,the higher sialylation observed in this work could be attributed to the reducedtemperature.

In conclusion, the present work shows that the newly established methodof glycan quantification using stable-isotope labeling can be used for quanti-tative comparison of the N-glycosylation profile of recombinant glycoproteinsexpressed under different cell culture conditions. However, it can be inferredfrom the data that changes of pH, temperature, and DO affect the degree ofsialylation and N-glycan branching of recombinant A1AT.

Abbreviations

A1AT, α-1 antitrypsin; AGE1.HN, human neuronal cell line; AGP, α-1 acidglycoprotein; Asn, asparagine; CH3I, methyl iodide; dH, deoxyhexose; DO,dissolved oxygen; Fuc, fucose; Gal, galactose; GlcNAc, N-acetylglucosamine;

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Influence of Culture Parameters on A1AT N-glycans 331

H, hexose; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; Man, mannose; MS, mass spectrometry; N, N-acetyl hexosamine;Neu5Ac, N-acetylneuraminic acid; PAGE, polyacrylamide gel electrophore-sis; PBS, phosphate buffered saline; PNGase F, peptide-N4-(N-acetyl-β-glucosaminyl) asparagine amidase F; S, sialic acid; SDS, sodium dodecylsulfate.

ACKNOWLEDGEMENTS

We acknowledge Detlef Grunow and Dennis Mewis for their technical assis-tance. This work was supported by the Bundesministerium fur Bildung undForschung (InnoProfile 03IP511) and by the Sonnenfeld foundation.

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