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Micro simulated moving bed chromatography-mass spectrometry as a continuous on-line process analytical tool

Diehm, Juliane, Witting, Lennart, Kirschhöfer, Frank, Brenner-Weiß, Gerald, Franzreb, Matthias·2024·Analytical and Bioanalytical Chemistry
DOI 10.1007/s00216-023-05023-9PMC PMC10761468
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Abstract

Continuous manufacturing is becoming increasingly important in the (bio-)pharmaceutical industry, as more product can be produced in less time and at lower costs. In this context, there is a need for powerful continuous analytical tools. Many established off-line analytical methods, such as mass spectrometry (MS), are hardly considered for process analytical technology (PAT) applications in biopharmaceutical processes, as they are limited to at-line analysis due to the required sample preparation and the associated complexity, although they would provide a suitable technique for the assessment of a wide range of quality attributes. In this study, we investigated the applicability of a recently developed micro simulated moving bed chromatography system (µSMB) for continuous on-line sample preparation for MS. As a test case, we demonstrate the continuous on-line MS measurement of a protein solution (myoglobin) containing Tris buffer, which interferes with ESI-MS measurements, by continuously exchanging this buffer with a volatile ammonium acetate buffer suitable for MS measurements. The integration of the µSMB significantly increases MS sensitivity by removing over 98% of the buffer substances. Thus, this study demonstrates the feasibility of on-line µSMB-MS, providing a versatile PAT tool by combining the detection power of MS for various product attributes with all the advantages of continuous on-line analytics. Graphical abstract

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Figures

Fig. 1 Instrumentation and piping diagram of the used µSMB setup. The conductivity sensor in the raffinate stream is only used when the µSMB is not coupled to the MS
Fig. 1 Instrumentation and piping diagram of the used µSMB setup. The conductivity sensor in the raffinate stream is only used when the µSMB is not coupled to the MSCC BY · PubMed Central
Fig. 2 Measurement of Mb in native and non-native state. (a) background spectrum of 10 mM NH4OAc buffer and spectra of Mb under native and non-native measurement conditions. (b) Protein reconstruction of the Mb spectra from (a). (c) Close up of the Mb main peak with isotopic peaks and sodium adducts. In (a) and (c), charge states are assigned in parenthesis after m/z labels. In (a) and (b), peaks of holoMb are marked with white squares, of apoMb with black circles, and of heme with white circles
Fig. 2 Measurement of Mb in native and non-native state. (a) background spectrum of 10 mM NH4OAc buffer and spectra of Mb under native and non-native measurement conditions. (b) Protein reconstruction of the Mb spectra from (a). (c) Close up of the Mb main peak with isotopic peaks and sodium adducts. In (a) and (c), charge states are assigned in parenthesis after m/z labels. In (a) and (b), peaks of holoMb are marked with white squares, of apoMb with black circles, and of heme with white circlesCC BY · PubMed Central
Fig. 3 Influence of Tris buffer on MS detection of Mb. (a) Mass spectra of solutions containing 10 µg/mL Mb with increasing Tris concentrations. Charge states are assigned in parenthesis after m/z labels. (b) Normalized Tris signal (m/z 593.23) and amount of detected holoMb in dependence of Tris concentration. (c) Influence of Tris concentration on detected percentage of apoMb and heme group. The detected amount of apo/holoMb was calculated by integration of the respective peaks in the protein reconstruction chromatogram
Fig. 3 Influence of Tris buffer on MS detection of Mb. (a) Mass spectra of solutions containing 10 µg/mL Mb with increasing Tris concentrations. Charge states are assigned in parenthesis after m/z labels. (b) Normalized Tris signal (m/z 593.23) and amount of detected holoMb in dependence of Tris concentration. (c) Influence of Tris concentration on detected percentage of apoMb and heme group. The detected amount of apo/holoMb was calculated by integration of the respective peaks in the protein reconstruction chromatogramCC BY · PubMed Central
Fig. 4 Tris concentrations in the raffinate and extract streams during a µSMB experiment with 100 mM Tris in the feed solution
Fig. 4 Tris concentrations in the raffinate and extract streams during a µSMB experiment with 100 mM Tris in the feed solutionCC BY · PubMed Central
Fig. 5 Comparison of the different in- and outlet flow rates without (a) and with (b) direct coupling of the raffinate stream to the ESI interface of the MS system
Fig. 5 Comparison of the different in- and outlet flow rates without (a) and with (b) direct coupling of the raffinate stream to the ESI interface of the MS systemCC BY · PubMed Central
Fig. 6 Normalized signals of Mb, heme group, and Tris (a) during the entire first µSMB-MS experiment (run 1); (b) for the third process cycle at every Tris concentration (marked with black boxes in (a))
Fig. 6 Normalized signals of Mb, heme group, and Tris (a) during the entire first µSMB-MS experiment (run 1); (b) for the third process cycle at every Tris concentration (marked with black boxes in (a))CC BY · PubMed Central
Fig. 7 (a) Normalized Tris signal as a function of the Tris concentration in the feed of the µSMB experiments. Depicted values are the mean values of the third cycle of each concentration. The Tris concentrations of the calibrations were multiplied with the expected desalting level of 98% in order to match the x-axis. (b) Detected apoMb percentage and detected amount of Mb as a function of the feed Tris concentration during the experiments. The values are mean values of the third cycle of the µSMB process for each concentration. (c) Detected apoMb percentage and Tris signal level for three consecutive cycles (4–6) of µSMB run 2 with a constant Tris concentration of 10 mM in the feed stream. The apoMb level was calculated for each switching interval, as indicated by the dotted vertical lines
Fig. 7 (a) Normalized Tris signal as a function of the Tris concentration in the feed of the µSMB experiments. Depicted values are the mean values of the third cycle of each concentration. The Tris concentrations of the calibrations were multiplied with the expected desalting level of 98% in order to match the x-axis. (b) Detected apoMb percentage and detected amount of Mb as a function of the feed Tris concentration during the experiments. The values are mean values of the third cycle of the µSMB process for each concentration. (c) Detected apoMb percentage and Tris signal level for three consecutive cycles (4–6) of µSMB run 2 with a constant Tris concentration of 10 mM in the feed stream. The apoMb level was calculated for each switching interval, as indicated by the dotted vertical linesCC BY · PubMed Central

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Citations

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Metadata

Authors
Diehm, J., Witting, L., Kirschhöfer, F., Brenner-Weiß, G., & Franzreb, M.
ORCID
Venue
Analytical and Bioanalytical Chemistry
Publisher
Springer Science and Business Media LLC
Published
2023-11-10
Volume
416
Issue
2
Pages
373-386
ISSN
  • 1618-2642
  • 1618-2650
DOI
10.1007/s00216-023-05023-9
PMC
PMC10761468
Cited by
2
References
1
Language
en
Licence
Funders
  • Stiftung Begabtenförderung Cusanuswerk
  • Karlsruher Institut für Technologie (KIT)

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