Use of Hyphenated Liquid-Phase AnalysesâMass Spectrometric

Chapter 23

Downloaded by UNIV MASSACHUSETTS AMHERST on October 14, 2012 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1995-0619.ch023

Use of Hyphenated Liquid-Phase Analyses—Mass Spectrometric Approaches for the Characterization of Glycoproteins Derived from Recombinant DNA 1

1

1

1,3

2

A. Apffel , J. Chakel , S. Udiavar , W. S. Hancock , C. Souders , and E. Pungor, Jr. 2

1

Hewlett-Packard Laboratories, 3500 Deer Creek Road, MS26U-R6, Palo Alto, CA 94304 Berlex Biosciences, 430 Valley Drive, Brisbane, CA 94005 2

The analysis of recombinant Desmodus Salivary Plasminogen Activator (DSPAα1), a heterogeneous glycoprotein, is demonstrated through the use of High Performance Liquid Chromatography (HPLC), High Performance Capillary Electrophoresis (HPCE), Electrospray Liquid Chromatography Mass Spectrometry. (ES-LC/MS) and Matrix Assisted Laser Desorption Ionization - Time of Flight Mass Spectrometry (MALDI-TOF MS). The protein is analyzed at three specific levels of detail: the intact protein, proteolytic digests of the protein and fractions from the proteolytic digest. A method for "on-column" collection of HPLC fractions for subsequent transfer and analysis by HPCE and MALDI-TOF is shown. For many "real world" applications in bioscience no single technique, however powerful, is sufficient to provide the total picture. In these cases, the challenge is to be able to exploit the strengths of various complimentary techniques to develop a complete, reliable and robust solution to a given problem. A further challenge is to effectively integrate the data produced by these analytical techniques into purity and product consistency information suitable for the characterization of a protein pharmaceutical. The transformation of the data from a mass of unrelated facts derived from both the intact molecules as well as series of enzymatically produced fragments to a coherent understanding of the nature of the pharmaceutical product is not a trivial one, rather an essential part of the development of improved analytical methodology. 3

Corresponding author 0097-6156/95/0619-0432$17.00/0 © 1996 American Chemical Society

In Biochemical and Biotechnological Applications of Electrospray Ionization Mass Spectrometry; Snyder, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


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It is interesting to note the evolution of the concept of multidimensional or hyphenated techniques. Ten years ago, gas chromatography/mass spectrometry (GC/MS) was considered a "multidimensional" analysis; five years ago, liquid chromatography/mass spectrometry (LC/MS) was "hyphenated"; now these are at best a single part of a higher dimensional approach to analyses. Similarly, a few years ago, the application of Electrospray LC/MS to peptide mapping was a revolutionary approach supplying a great deal of information in an astonishingly short time when compared to the earlier approaches of sequencing each and every peak collected from an High Performance Liquid Chromatography (HPLC) peptide map. Now, such an analysis is routine, and the more challenging problems are the characterization of post-translational modifications of proteins which introduce significant heterogeneity and therefore cannot be solved in a single step. While such maps have become the primary quality control (QC) method for the characterization of small proteins, it has recently been realized that the mapping approach has limitations. For example, the analysis of a much larger protein, e.g. fibrinogen (MW of 350 kDa), heterogeneous glycoproteins or antibodies (MW of 150 kDa) is hindered by the complexity of the range of peptides generated by an enzymatic digestion. The limitations of the map are particularly evident with the analysis of a glycoprotein in which many hundreds of glycoforms have the potential of adding an enormous number of peaks in the map. Often, however, such peptides are not observed because of limitations of the detection system or due to the inability of the separation method to discriminate between closely related species. Table 1 illustrates the enormous heterogeneity that can be present in a typical glycoprotein. This table is derived from Tissue Plasminogen Activator (rTPA) which has three sites of glycosylation. Thefirstsite contains mainly high mannose forms while the second and third sites contain complex structures. With the additional factor of optional glycosylation at the second site, one can calculate that this glycoprotein will contain over 11,000 major forms. With such substantial microheterogeneity, it is often observed that the abundance of glycopeptides is close to the detection limit, and the small glycopeptide peaks are obscured by the non-glycosylatedfragments.Figure 1 shows a typical situation where the intensity of a major set of glycopeptides is found to be l/5th to l/40th of the level of a non-glycosylated sample. Such complexity makes a single reversed phase HPLC separation combined with on-line U V detection of limited utility. The advent of commercially available combined HPLC and Electrospray Ionization Mass Spectrometry (ESI LC/MS) systems compatible with conventional HPLC methodology has increased the power of peptide mapping considerably . ESI LC/MS in combination with in-source collisionally induced dissociation (CID) has been used effectively to identify sites of N - and O-linked glycosylation ' ' . However, even this technique is limited by insufficient resolution resultingfromthe large number of very similar peptides caused by variable protein glycosylation and enzymatic digests of moderately sized glycoproteins. It is therefore necessary to employ a range of techniques with orthogonal selectivity in order to characterize such samples. A 1,2

3 4 5



434

BIOLOGICAL AND BIOTECHNOLOGICAL APPLICATIONS OF ESI-MS

Table 1: Possible glycoforms for rTPA Site I

a

Total: 10

Hybrid: 4

High Mannose: 6

Site Π

Complex Complex Tetraantennary Triantennary: 8 : 10

Total: 24 Complex Biantennary :6

Site ΠΙ

Complex Complex Tetraantennary Triantennary: 8 : 10

Total 24 Complex Biantennary :6

rTPA

b

(24*24* 10)*2

Total: 11520

a. Derivedfromdata presented by A. Guzzetta . b. The factor of two is due to partial glycosylation of the second site.


23. APFFEL ET AL.


435


significant issue is to efficiently utilize such multidimensional data in a way that multiple samples can be characterized for a product development program. We have, therefore, investigated the use of combinations of HPCE, HPLC, ESI LC/MS and MALDI-TOF MS to allow for characterization of enzymatic digests of underivatized glycoprotein samples. The separation selectivity of HPLC and CE is sufficiently orthogonal to yield a great deal of comparative information. Although ESI LC/MS is extremely powerful for peptide mapping, when dealing with intact proteins the mass analyis is limited to relatively homogeneous proteins. Due to the complexity of the ion envelope generated by ESI-LC/MS, which is observed for a protein with substantial heterogeniety, spectra become difficult, if not impossible, to interpret In addition, as the mass range is limited to · »•

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460

Figure 10: Detail of Contour Map for DSPAal ArgC digest. 78 pmol total protein injected. CapEx Voltage=100V. Data for Signal >2000 counts shown


23.

APFFEL ET AL.


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DSPAal map, the 2D map shows that the latter digest is much more complex as would be expected from the greater number of glycosylation sites in DSPAal. The more heterogeneous the sample is (as in the case of DSPAal), the more glycoforms over which the ion current will be distributed. Unlike the UV chromatogram in which the different unresolved glycoforms contribute to a moderately intense, relatively broad chromatographic peak, the contour map has a single point for each mass state present for the glycoform. Figure 10 shows a contour for a section of the ArgC map of DSPAal from 40-50 minutes. The circled area shows signal due tofragmentT15[l 11-123] which contains complex sialylated biantennary structures with three different charge states (+1, +2 and +3) for variable degrees of sialylation. The visual pattern is striking because the higher the charge state, the closer the pattern and the greater the slope of the diagonal (due to the effective compression of the mass range by multiple charging). We are investigating the application of contour plots and multivariate statistical techniques to the examination of different lots of the drug substance as we believe such an approach may have value in demonstrating consistency in glycosylation and for the detection of significant levels of host cell contaminants. The use of in-source collisionally induced dissociation in the analysis of DSPAal is illustrated in Figure 11. The CID mass spectral acquisition was carried out in Selected Ion Monitoring mode to increase the sensitivity. The marker ions which were monitored are shown in Table 3. The reproducibility of the HPLC separation is sufficiently good that areas identified in the SIM analysis can be accurately compared with separate full spectral acquisitions conducted at lowerfragmentationenergy. This allows the parent glycopeptide to be identified. The success of this technique can be compared to the identification of glycopeptidesfromthe standard ESI chromatogram. At low collision energies (no CID), extensive microheterogeneity in the glycoforms results in a low abundance of all of the individual ions. Often, the intensities may be difficult to distinguishfrombackground. Note, however, that the sensitivityfromthe SIM CID data is sufficient since all of the different chromatographically unresolved glycoforms will contribute fragment ions to the electrospray signal of the glycomarkers. The summation of the signals due to the marker ions results in relatively broad looking chromatographic peaks. Based on the CID data, it is possible to infer some glycostructure differentiation. For example, all N-linked structures will generate a m/z 204 ion corresponding to the HexNAc in the backbone structure. All complex N-linked glycopeptides will generate a m/z 366 ion corresponding to the HexNAc+Hex structure indicative of the branching in multi-antennary structures. Only sialyted glycopeptides will generate a m/z 292 ion. Figure 11 relates the major structure of the glycopeptide T15 to thefragmentsthat could be detected in the CID experiment. It can be seen that the CID data in the Tl5 region (around 45 minutes) would predict structures that would be low in fucose, but contain a substantial amount of sialic acid.



Complex Biantennary

I 1

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b) m/z 147.00: Fucose

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In Biochemical and Biotechnological Applications of Electrospray Ionization Mass Spectrometry; Snyder, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996. 20.00

30.00

40.00

time (minutes)

50.00

60.00

Figure 11 : CID detection of Glycopeptides. 78 pmol total protein injected, a) Total ion chromatogramfromscan acquistion (CapEx=100 V). b) through e) from selected ion monitoring acquisition (CapEx=200 V). b) m/z 147 Da. c) m/z 204 Da. d) m/z 292 Da. e) m/z 366 Da

10.00

»'» • ι '

a) Scan TIC


70.00


464


Table 3: Glycomarkers Sugar

Symbol

Chemical Formula

Ion Monitored (M+l) 146.1 147.1 162.1 163.1 203.2 204.2 291.3 292.1

MW

1+

Fucose Hexose N-Acetymexosamine N-Acetymeuraminic Acid Complex Branch Unit

Fuc Hex HexNAc NANA HexNAc + Hex

C0H C 06H C 0 NHi5 Cn0 NH 6

5

13

6

8

12

6

9

19

365.3 366.3


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APFFEL ET AL.


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HPLC Fractionation of the Digest In many cases, the complexity of the mixture generated by an enzymatic digest of a glycoprotein is too high for all components to be completely resolved by a single dimensional analytical technique. Recently, Jorgenson introduced two dimensional separations based on the combination of HPLC and HPCE. Only a smallfractionof the LC effluent is analyzed in the second dimension and thus a pre-separation fluorescent derivatization was required. While this approach produces excellent 2D displays, the analysis is not able to chemically characterize any differences that may be observed in lot-to-lot comparisons. For this reason, we chose to transfer samples from an LC separation to subsequent analysis in a semi-off-line technique. Fractions eluting from the reversed-phase HPLC separation were selectively transferred onto disposable hydrophobic collection columns after the effluent was diluted with a non-elutropic solvent. Because of the highly reproducible retention times of the LC separation, this process could be repeated, loadingfractionsfrommultiple LC runs on single collection columns. The hardware setup employed used a 14 port, 6 position valve which would allow 4 collection columns to be randomly accessed in addition to a bypass path. Following fraction collection, the fractions were eluted from the collection column with a rapid gradient. Since thefractionelution solvent doesn't have to be the same as the solvent system used in the peptide map, buffer transfer or desalting can also be accomplished in this step. This had the advantage of concentrating the samples for subsequent analysis and of great flexibility in transferring the sample fraction to subsequent analytical techniques.


19

This technique is particularly useful in transferring samples to subsequent HPCE separation. As mentioned above, HPCE suffers from rather poor concentration sensitivity due to relatively small injection volumes and short path length UV detection. While on-line sample focusing and preconcentration techniques such as sample stacking and capillary isotachophoresis (CITP) are extremely useful in this respect, the low concentration of individual glycopeptides requires additional concentration to fully characterize the sample. 20

21,22

The technique is also useful for transferring samples to inherently static techniques such as MALDI-TOF, in which an on-line flow based approach would be difficult. The fractionation step itself is very important in reducing the complexity of the mixture to a point at which most of the components can be identified mass spectrally by MALDITOF. The desalting characteristics of the fraction elution step can be very useful in increasing signal yields and thefractionof components observed. For the purpose of illustration, we will focus on a singlefractioncollectedfromthe HPLC peptide map as illustrated in Figure 12. Thisfractionwas chosen because of the high degree of glycosylation present in these areas as shown by the CID studies on glycomarkers. The benefits of the approach are not only the simplification of the analytical sample viafractionation,but a concentration step of approximately 10 fold as well. Thefractionwas analyzed by HPCE using the same method as had been used for the total digest. The electropherogram of thefractionis shown in Figure 13 a. The real strength of using CE to reanalyze HPLCfractionslies in the orthogonality of the separation mechanism. To fully exploit this separation power, a method is needed to



0

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400000

300000

200000

S 100000

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3000000

4000000

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40

50

I

60

Figure 12: Area of HPLC Fraction Collection. Identification of regions from analytical separation (76 pmol). a) Scan Acquisition Total Ion Current (CapEx= 100V). b) CID SIM Acquisition Total Ion Current (CapEx = 200 V). c) UV at 214nm. The actualfractioncollections were done with 312 pmol injected

10

b) MS CID SIM (GlycoMarkers) (Summation of Multiple SIM Traces)

a) MS TIC: SCAN

Collected Fraction


70


0-

5-

10-

15-

20-

10

12.5

17.5

Figure 13 :

15

22.5 time (min)

25

27.5

a) HPCE of collectedfractions30 nL injected

20

30

a) HPCE


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ON 00

Downloaded by UNIV MASSACHUSETTS AMHERST on October 14, 2012 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1995-0619.ch023 In Biochemical and Biotechnological Applications of Electrospray Ionization Mass Spectrometry; Snyder, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

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APFFEL ET AL.

Glycoproteins DerivedfromRecombinant DNA

469

couple the CE separation with mass spectrometry, either directly via electrospray CE/MS or in an off-line manner running collected CEfractionsby MALDI-TOF. This approach has been used to characterize growth hormone tryptic peptides in a CE separation and other laboratories for other protein samples. These approaches are currently under investigation, and will be reported in a future publication. Alternately, UV spectra could be used to track the elution order of a CE separation transferred from an HPLCfraction,analogously to peak tracking in HPLC peptide mapping . This approach requires that the HPLC separation was already sufficiently resolved to obtain pure spectra or that pure peptide standards exist. 23


24

The collectedfractionwas also analyzed by MALDI-TOF as shown in Figure 13b. This is particularly useful combination because the sensitivity of MALDI-TOF allows clear unequivocal spectra which actually exhibit a selectivity closer to HPCE than HPLC, being based on mass/charge (as can be seen in the similarity of profiles in Figure 13a and Figure 13b). The orthogonality of MALDI-TOF relative to HPLC generates extremely useful information (Figure 13b relative to Figure 12). In analyzingfractionscollected from the total digest, the sample complexity has been reduced sufficiently so that the MALDI-TOF spectrum is a good representation of the components present in the fraction. Contrast this with the data obtained for the total digest, in which only afractionof the components are represented by strong signals in the spectrum. Thus a more complete and accurate picture is obtained by a combination of the two approaches. Conclusions

This study has shown that there is considerable potential in the analysis of complex glycoprotein samples by hyphenated liquid phase separations and mass spectrometry. Such information should prove invaluable in reducing the approval barriers for the pharmaceutical use of glycosylated proteins produced by mammalian fermentation systems. For example, a high capacity two dimensional analytical method can be used to demonstrate consistency of carbohydrate structures produced under different scale fermentations. The use of electrospray mass spectrometry on-line with reversed phase HPLC has greatly expanded the power of peptide mapping to identify carbohydrate structures that are attached to asparagine, serine or threonine residues. One can use in-source CID to scan the map for regions with a high concentration of glycopeptides. The presence of certain glycoforms can be achieved by extracted ion monitoring and used to monitor distribution of sialic acid in complex carbohydrate structures. We have started to explore the use of contour maps (m/z vs retention time) as a facile approach to rapid 2D mapping of complex samples. Such maps are readily available from the data generated by an LC/MS analysis and can give valuable information about glycosylation patterns and product consistency. A problem with current 2D methods is that it is difficult to explain differences that may be observed in comparison of samples. An approach to this problem was described in this report where an automated fraction collection system was developed for the RP-HPLC as first


470


dimension separation. The collection system was designed to concentrate the samples in a buffer suitable for the second dimension CE analysis. The third dimension of this analysis was illustrated by the application of off-line MALDI-TOF to the CE fractions. This analytical approach has great potential as it allows the combination of chemical characterization with a multidimensional analysis approach.


Acknowledgments

The authors would like to acknowledge Sally Swedberg and Bob Holloway at Hewlett-Packard Laboratories for help with capillary electrophoresis, Julie Sahakian and James Kenny at Hewlett-Packard Protein Chemistry Systems for N-terminal sequencing, Steve Fischer at Hewlett-Packard Bay Analytical Operation for aid in mass spectral interpretation, Thabiso M'Timkulu and Joanne Johnson at Berlex Biosciences for carbohydrate analysis, Ray-Jen Chang and Maria Johnson at Berlex Biosciences for N-terminal sequencing, Peter Murakami and Peter Sandel at Berlex Biosciences for amino acid analysis and BaiWei Lin and Joe Traîna at Berlex Biosciences for mass spectrometry. Literature Cited 1

2

Ling, V., Guzzetta, A.W., Canova-Davis, E., Stults, J.T., Hancock, W.S., Covey, T.R. and Shushan, B.I., Anal.Chem., 63 (1991) 2909-2915.

Guzzetta, A.W., Basa, L.J., Hancock, W.S., Keyt, B.A. and Bennet, W.F., Anal.Chem., 65 (1993) 2953-2962.

3

Carr, S.A., Huddleston, M.J. and Bean, M.F., Protein Science, 2 (1993) 183-196.

4

Huddleston, M.J., Bean, M.F. and Carr, S.A.,Anal.Chem.,65 (1993) 877-884.

5

Conboy, J.J. and Henion, J.D., J.Am.Soc.Mass Spectrom. 3 (1992) 804-814.

6

Kraetschmar, J., Haendler, B., Langer, G., Boidol, W., Bringman, P., Alagon, Α., Donner, P. and Scheuning, W.D., Gene 105 (1991) 229-237.

7

Witt, W., Maass, B., Baldus, B., Hildebrand, M., Donner, P. and Scheuning, W.D., Circulation, 90 (1994) 421-426.

8

Witt, W., Baldus, B., Bringmann, P., Cashion, L., Donner, P.and Scheuning, W.D., Blood, 79 (1992) 1213-1217.

9

Apffel, Α., Fischer, S., Goldberg, G., Goodley, P.C. and Kuhlmann, F.E., J.Chromatogr,, Submitted for publication, 1994.

10

Gusev, A.I., Wilkinson, W.R., Proctor, A. and Hercules, D.M., Anal.Chem., 67 (1995) 1034-1041.

11

Apffel, A. Chakel, J. Udiavar, S., Hancock, W.S., Souder, C and Pungor, E., J.Chromatogr.Submitted for publication (1995)

12

Oroszlan, P., Wicar, S., Teshima, G., Wu, S.-L., Hancock, W.S. and Karger, B.L., Anal.Chem., 64 (1992) 1623-1631.


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14

M'Timkulu, T. (unpublished results).

15

Guzzetta, A.W. and Hancock, W.S., Recent Advances in Tryptic Mapping, CRC Series in Biotechnology, CRC Press, Boca Raton, FL, in press (1994).

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Marshall, R.D., Ann. Rev. Biochem. 41 (1972) 673-702.

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Aubert, J.P., Biserte, G., and Loucheux-Lefebre, M-H., Arch. Biochem. Biophys. 175 (1976) 410-418.

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Spellman, M.W. Basa, L.J., Leonard, C.K., Chakel, J.A., O'Connor, J.V., Wilson, S.W., and van Halbeek, H., J.Biol.Chem. 264 (1989) 14100-14111.

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Chein, R.L. and Burgi, D.S., J.Chromatogr. 559 (1991) 141-152.

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Mikkers, F.E.P., Thesis Eindhoven University, Eindhoven, 1980.

22

Foret, F., Szoko, Ε. and Karger, B.L., J.Chromatogr. 608 (1992) 3-12

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24

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Use of Hyphenated Liquid-Phase AnalysesâMass Spectrometric

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