Investigation of Protein Patterns in Mammalian Cells and Culture Supernatants by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Johanna H. M. van Adrichem, ²,§ K. Olaf Bo 1 rnsen,* ,‡ Horst Conzelmann, ‡ Marion A. S. Gass, ‡ Hans Eppenberger, § Gerhard M. Kresbach, ‡ Markus Ehrat, ‡ and Christian H. Leist ² Technical Cell Biology and BioAnalytical Research, Novartis Pharma AG, CH-4002 Basel, Switzerland, and Department of Cell Biology, ETH-Hoenggerberg, CH-8092 Zuerich, Switzerland The direct protein profiling of mammalian cells and bacteria has a growing influence in biotechnology as a high information bearing method for characterization of cells and cell states. Monitoring of proteins excreted in culture media not only serves to produce data on product yield and quality but provides important information on cell viability and nutrient supply that forms the basis for future process and expression optimization. Fast and simple MALDI mass spectrometry approaches were developed to efficiently characterize such complex biological sys- tems. Several mammalian cell lines including CHO DXB1 1 , CHOSSF3 , and hybridomas were investigated; the lysis process, the sample pretreatment, and the matrix preparation were optimized for MALDI conditions. Initial experiments to observe the success of protein translation in gene expression experiments were performed. Using MALDI-compatible detergents, it was possible to extend the mass range detectable by MALDI mass spectrometry from the current range of 16 000 to 75 000 Da. In this mass range, the data are complementary (offering a better mass accuracy) to those obtained by SDS-PAGE electro- phoresis experiments. These new methods were used to monitor a large-scale cultivation of hybridoma cells ex- pressing an antibody of the IgG type. The increase in whole antibody and antibody light-chain protein, 8 6 5 0 Da, and the decrease of insulin were followed during the monitoring period. Quantitative measurements of the IgG level during the cultivation compared favorably with those obtained by affinity HPLC. In the past decade, MALDI mass spectrometry has established its role as an important analytical tool for protein chemistry as well as for molecular biology. 1,2 Some key advantages of MALDI mass spectrometry render this technology especially suitable for the characterization of biologically relevant molecules in complex media. The absence of fragmentation, high sensitivity, and easy sample preparation are the main advantages of this analytical tool. This has led to the introduction of MALDI for the analysis of rather complex systems where classical approaches have proven to be complicated and tedious, including the rapid identification of pathogenic and nonpathogenic bacteria 3-6 and the determination of gluten in food. 7 The potential applicability of MALDI mass spectrometry for profiling biological cell reactions is investigated in this paper. Animal cell cultivation is becoming increasingly important for the production of pharmaceutically active proteins ranging from vaccines (measles, polio, hepatitis A) to proteins with therapeutic effects (TPA, TGF- , R-interferon, monoclonal antibodies). 8 Ex- actly defined and controlled cultivation procedures for cells, tissues, and organs are required to obtain and maintain a stable and high-quality production. MALDI mass spectrometry can easily detect and monitor subtle changes in the pattern of intracellular molecules and in the compound profile in cell cultivation liquids. Prerequisites for such applications are a careful selection of matrix compounds and solvents and especially a high reproducibility in the matrix embedding and crystallization process. 9 Compared to prokaryotic cells such as Escherichia coli, mammalian cells exhibit a far higher complexity. Composed of a wide variety of peptides, proteins, glycoproteins, oligonucleotides, lipids, and sugars, these cells offer a challenge to any kind of mass spectrometry, especially since larger amounts of inorganic salt and detergents are often present in the sample due to separation and stabilization requirements. This holds equally true for cell cultivation liquids. Up to now, MALDI mass spectrometry could only detect small proteins in cell lysates up to a molecular mass of 20 000 Daswhereas SDS- * Reprint requests: k olaf.boernsen@ pharma.novartis.com. † Technical Cell Biology, Novartis Pharma AG. ‡ Bioanalytical Research, Novartis Pharma AG. § ETH-Ho ¨ nggerberg. (1) Nuygen, D. N.; Becker, G. W.; Riggin, R. M. J. Chromatogr., A 1995 , 705, 21. (2) Kaufmann, R. J. Biotechnol. 1995 , 41, 155. (3) Krishnamurthy, T.; Ross, P. L.; Rajamani, U. Rapid Commun. Mass Spectrom. 1996 , 10, 883. (4) Holland, R. D.; Wilkes, J. G.; Rafii, F.; Sutherland, J. B.; Persons, C. C.; Voorhees, K. J.; Lay, J. O., Jr. Rapid Commun. Mass Spectrom. 1996 , 10, 1227. (5) Cain, T. C.; Lubman, D. M.; Weber, W. J., Jr. Rapid Commun. Mass Spectrom. 1994 , 8, 1026. (6) Claydon, M. A.; Davey, S. N.; Edwards-Jones, V.; Gordon, D. B. Nat. Biotechnol. 1996 , 14, 1584. (7) Mendez, E.; Camafeita, E.; Sebastian, J. S.; Valle, I.; Solis, J.; Mayer-Posner, F. J.; Suckau, D.; Marfisi, C.; Soriano, F. Rapid Commun. Mass Spectrom. 1995 , 9, 123. (8) Leist, C. H.; Meyer, H. P.; Fiechter, A. J. Biotechnol. 1990 , 15,1-46. (9) Weinberger, S. R.; Bo ¨ rnsen, K. O.; Finnchy, J. W.; Robertson, V.; Musselman, B. D. Proceeedings of the 41st Annual Conference on Mass Spectrometry and Allied Topics, San Francisco, CA, ASMS: Santa Fe, NM, 1993; pp 775a- 775b. Anal. Chem. 1998, 70, 923-930 S0003-2700(97)00977-3 CCC: $15.00 © 1998 American Chemical Society Analytical Chemistry, Vol. 70, No. 5, March 1, 1998 923 Published on Web 01/27/1998