Quantitative MALDI

The first step in biopharmaceutical development is the selection of a clone in a specific cell line. Whole-mass analysis, if possible, is a fairly simple and powerful tool at this stage to verify the successful expression and translation of the desired protein. VanAdrichem et al.65 described the use of MALDI MS to monitor protein expression in several mammalian cell lines like CHO DXB11, CHO SSF3, and hybridomas. Quantitative MALDI-TOF MS measurements of an IgG antibody and insulin during large-scale production in hybridoma cells were comparable to affinity chromatography results. 

Bungert, D., Bastian, S., Heckmarm-Pohl, D.M., Giffhom, E., Heinzle, E., Tholey, A. Screening of sugar converting enzymes using quantitative MALDI-ToE mass spectrometry. Biotechnol. Lett. 2004,... 

Fig. 4. a rac-Phenylethylamine (PEA) and 2-methoxy-J T-[(li )-l-phenylethyl]-acetamide (MET) are substrate and product in a lipase catalyzed transesterification, b Linear relationship between relative signal intensities in MALDI-MS and the relative concentrations of PEA/d5-labeled PEA. c Comparison of decrease in PEA during enzymatic conversion as measured by quantitative MALDI-MS and gas chromatography (GC)... 

NMR peaks at 6 3.1 and 3.6 ppm were observed corresponding to the dimeric product in addition to the peak at d 3.3 ppm for the monoaddition product. It was found that the results from quantitative NMR analysis correlated well with the quantitative MALDI-TOF MS data with respect to the amount of oligomerization present. Quantitative NMR analysis using the peaks for the model dimeric oligomer product was found to be less sensitive than MALDI-TOF MS and... 

Mass Analyzers Ionization Sources Tissue Preparation MALDI Matrix Quantitative MALDI-MS... 

Although MALDI—MS is an established method for qualitative analysis, quantitative analysis is more difficult because MALDI exhibits irreproducible analyte signals as a result of inhomogeneous erystal formation, inconsistent sample preparation, and laser shot-to-shot variability (OnnerQord et al., 1999). Indeed, relative standard deviations ean be higher than 50% (Cohen and Gusev, 2002 Sleno and Volmer, 2006). The addition of an internal standard can compensate for several of these experimental factors that seriously complicate quantitative MALDI—MS (Nicola et al., 1996 Ling et al., 1998 Hatsis et al., 2003 Cui et al., 2004 Sleno and Volmer, 2006). 

Reich RF, Cudzilo K, Levisky JA, Yost RA. Quantitative MALDI-MS" analysis of cocaine in the autopsied brain of a human cocaine user employing a wide isolation window and internal standards. J Am Soc Mass Spectrom 2010 21 564-571. 

The use of multicomponent matrices with accelerated sample drying along with a modified instrumental set-up was also considered when preparing samples for quantitative MALDI analysis. This method has been shown to improve the measured relative standard deviation (RSD) to better than 10% of analyte to internal standard (IS) intensity ratio [18]. Both, signal reproducibility and precision of the standard curve slope were improved by a factor of two when a DHB/fucose/5-methoxysaHcyUc acid multicomponent matrix was used compared to DHB alone. Similar results were observed when a feruhc acid/fucose multicomponent matrix was used compared to the use of ferulic add alone. 

The wide variety of both numerous and diverse applications described in this chapter clearly demonstrates the utility and future potential of MALDI for both quahtative and quantitative characterization of LMM compounds. Many new instrumentation and sample preparation techniques are now available for automated and painless MALDI characterization. Arguably, sample preparation from complex mUieu such as blood still requires considerable attention and effort The sample dean-up procedures prior to MALDI analysis need not be more compUcated or lengthy than those conducted for ESl-MS analysis. Realistically, problems such as shot-to-shot and sample-to-sample reproducibility still exist, but can be overcome by the techniques discussed earlier. Other criticisms of quantitative MALDI such as poor sensitivity, limited linear range and imprecision (higher RSD) are primarily compound-dependent and have been significantly improved by instramental advancements. 

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