12
The analysed elution sample of the protein A affinity chromatography run indicates significant purification of the monoclonal antibody. The percentage of aggregates and contaminants is considerably reduced. Fractionation by HPLC-SEC analysis shows two peaks (Figure 6). Based on the respective retention times, these peaks can be calculated as molecular sizes using the standard. The areas of the peaks obtained provide information on the percentages of aggregates and monomers in the mAb solutions (Table 4).
Summary
HPLC-SEC analysis is an important tool for determining the purity of therapeutic proteins and is used worldwide in the manufacturing process of monoclonal antibodies as a standard method to measure the aggregation content and the percentage of contaminants.
As the integrity of proteins – in this case, monoclonal antibodies – is at risk due to temperature changes, the effects of mechanical stress, pH fluctuations and exposure to UV light during production and throughout their entire lifecycle (see also [3]), it is of crucial importance to establish a reliable method for determining the purity of an mAb.
The method described in this paper enabled excellent determination of the aggregation states of monoclonal antibodies. In addition, it demonstrated excellent purification results. An essential prerequisite for performing this method is the purity of the substances and solutions used and, above all, that of water employed as the basic solvent for preparing the solutions. As shown in the preliminary trials (Figure 3), ultrapure water produced by arium®
ultrapure water is used, no ghost peaks [8] caused by an interaction between the mobile and stationary phases and no UV-active contaminants or peak shifts are detectable. Strong organic impurities, expressed in the form of high TOC values, might be manifested in peak shifts because the column matrix becomes more nonpolar as a run progresses. Heavy metal salts, or chelating agents, can shield charges in the column, which could lead to protein folding (increased formation of antibody aggregates). These assumptions must be clarified in further trials.
When arium® References
Figure 6. HPLC-SEC analysis of a monoclonal antibody after protein A affinity chromatography during downstream processing (conditions are described in Table 2).
Table 4. Percentages of aggregates of an mAb during purification mAb aggregates [%] mAb monomers [%]
After harvesting and initial clarification 10 After protein A affinity chromatography 3
90 97
SEC analysis of the percentages of the mAb aggregates and monomers after harvesting and initial clarification (Figure 5) show that 10% aggregates contaminating the mAb still remain (Table 4). The SEC analytical run after the additional purification step using protein A affinity chromatography indicates a decrease in the percentage of aggregates to 3% (Figure 6). No further contaminants are detected by SEC analysis. The results obtained clearly demonstrate that the method used is suitable.
1. Quaglia M., Machtejevas E., Hennessy T., Unger K.K.: Gelfiltration-Größenausschluss-Chromatographie von Biopolymeren-Optimierungsstrategien und Fehlersuche. Excerpted from “HPLC richtig optimiert. Ein Handbuch für Praktiker.” Herausgeber Stavros Kromidas [Publisher]. WILEY-VCH, Weinheim (2006).
2. Schmid Thomas: Vorlesung ETH Zürich. Analytische Chemie (für Biol./Pharm. Wiss.) Teil “Chromatographische und elektrophoretische Trennverfahren,“ p. 58, (2011).
3. Frieß W., Ruberg E.-M.: Proteinarzneistoffe - Sensibel und stressanfällig PHARM. ZTG., 156 JG., 50. AUSG. [50th issue], (2011).
4. Richter A., Jostameling M., Müller K., Hermann A. and Pitschke M.: Quality control of antibodies for human use. Excerpted from: “Antibodies Volume 1, Production and Purification,” edited by G. Subramanian, Kluwer Academic/Plenum Publishers, New York (2004).
5. Nitzki F., Herbig E.: In situ Hybridisierung – Die Bedeutung von Reinstwasser für RNA Technologien. GIT Labor-Fachzeitschrift 57. Jahrgang, 3, (2013) [also available in English: In Situ Hybridization: The Importance of Ultrapure Water for RNA Technologies].
6. Phenomenex Chromatography Product Guide, p. 311 (2015/16).
7. Fish Brendan: Concepts in development of manufacturing strategies for monoclonal antibodies. Excerpted from: “Antibodies Volume 1, Production and Purification,” edited by G. Subramanian, Kluwer Academic/ Plenum Publishers, New York (2004).
8. Gottwald, W.: RP-HPLC für Anwender. Reihe: Die Praxis der instrumentellen Analytik, Herausgeber [editor] Gruber, U., Klein, W., VCH Verlagsgesellschaft, Weinheim, pp. 7-8 (1993).
pro VF is suitable as a medium for dissolving buffer substances and salts to prepare the mobile phase used during SEC.
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LAB ASIA - NOVEMBER/DECEMBER 2016
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