TY - JOUR

T1 - Charged aerosol detector HPLC as a characterization and quantification application of biopharmaceutically relevant polysialic acid from E. coli K1

AU - Boßmann, Daniel

AU - Bartling, Bastian

AU - de Vries, Ingo

AU - Winkler, Jonas

AU - Neumann, Harald

AU - Lammers, Frank

AU - Beutel, Sascha

AU - Scheper, Thomas

N1 - Funding information: This work was financially supported by the German Research Foundation [DFG, grant number: DFG-SCHE279/35-1 ] as well as the German Federal Ministry of Education and Research [BMBF, grant numbers: BMBF-03VP00271 and BMBF-03VP00273 ].

PY - 2019/8/16

Y1 - 2019/8/16

N2 - Polysialic acid (polySia) is widely investigated in various biopharmaceutical applications (e.g. treatment of inflammatory neurodegenerative diseases), whereby a certain polySia chain length with an average degree of polymerization 20 (polySia avDP20) shows most promising effects. In this study, a rapid analytical method using a HPLC and charged aerosol detector (CAD) for the direct chain length characterization of biopharmaceutically relevant polySia was developed. It was evaluated as a fast alternative to the commonly used 1,2-diamino-4,5-methylenedioxybenzene (DMB) HPLC application. In contrast to HPLC-FLD, the CAD-application provides the actual chain length of polySia within ∼3 h. The reliability of the HPLC-CAD was evaluated with a commercial reference sample of known chain length and biotechnologically produced LC polySia (long chain polySia with a DP ∼130). Moreover, HPLC-CAD was successfully applied in the direct detection of oligo- and polySia until DP ∼65 and can be used to monitor the thermal hydrolysis and subsequent chromatographic isolation of polySia avDP20 (average degree of polymerization 20) without DMB sample derivatization. In addition, CAD was successfully applied for polySia quantification using a modified elution gradient. It was tested as a fast alternative to commonly used thiobarbituric acid (TBA) assay. A differentiation between LC polySia and smaller, hydrolysed polySia chains was intended and possible. For LC polySia and polySia avDP20, a quadratic relation between polySia mass-concentration and CAD signal was observed. In case of LC polySia, a quadratic dependency with a determination coefficient of R2 = 0.99 in a broad concentration range between 0.025 and 15 mg mL−1 was determined. Quantification of polySia avDP20 was found to have quadratic dependency with a determination coefficient of R2 = 0.99 in a concentration range between 0.02 and 0.25 mg mL−1. The HPLC-CAD was tested for quantification with polySia references of known concentration and showed high accordance with a concentration deviation ≤6.7%. The CAD quantification method was also applied in the polySia avDP20 production process and was compared to the TBA assay. Results of a correlation plot showed a high determination coefficient of R2 = 0.98. Overall, HPLC-CAD analysis was successfully tested as a suitable characterization and quantification application in the biopharmaceutical production of polySia.

AB - Polysialic acid (polySia) is widely investigated in various biopharmaceutical applications (e.g. treatment of inflammatory neurodegenerative diseases), whereby a certain polySia chain length with an average degree of polymerization 20 (polySia avDP20) shows most promising effects. In this study, a rapid analytical method using a HPLC and charged aerosol detector (CAD) for the direct chain length characterization of biopharmaceutically relevant polySia was developed. It was evaluated as a fast alternative to the commonly used 1,2-diamino-4,5-methylenedioxybenzene (DMB) HPLC application. In contrast to HPLC-FLD, the CAD-application provides the actual chain length of polySia within ∼3 h. The reliability of the HPLC-CAD was evaluated with a commercial reference sample of known chain length and biotechnologically produced LC polySia (long chain polySia with a DP ∼130). Moreover, HPLC-CAD was successfully applied in the direct detection of oligo- and polySia until DP ∼65 and can be used to monitor the thermal hydrolysis and subsequent chromatographic isolation of polySia avDP20 (average degree of polymerization 20) without DMB sample derivatization. In addition, CAD was successfully applied for polySia quantification using a modified elution gradient. It was tested as a fast alternative to commonly used thiobarbituric acid (TBA) assay. A differentiation between LC polySia and smaller, hydrolysed polySia chains was intended and possible. For LC polySia and polySia avDP20, a quadratic relation between polySia mass-concentration and CAD signal was observed. In case of LC polySia, a quadratic dependency with a determination coefficient of R2 = 0.99 in a broad concentration range between 0.025 and 15 mg mL−1 was determined. Quantification of polySia avDP20 was found to have quadratic dependency with a determination coefficient of R2 = 0.99 in a concentration range between 0.02 and 0.25 mg mL−1. The HPLC-CAD was tested for quantification with polySia references of known concentration and showed high accordance with a concentration deviation ≤6.7%. The CAD quantification method was also applied in the polySia avDP20 production process and was compared to the TBA assay. Results of a correlation plot showed a high determination coefficient of R2 = 0.98. Overall, HPLC-CAD analysis was successfully tested as a suitable characterization and quantification application in the biopharmaceutical production of polySia.

KW - Calibration

KW - Chain length distribution

KW - Charged aerosol detector (CAD)

KW - Polymer analysis

KW - Polysialic acid (polySia)

KW - Quantification

UR - http://www.scopus.com/inward/record.url?scp=85063874123&partnerID=8YFLogxK

U2 - 10.1016/j.chroma.2019.03.069

DO - 10.1016/j.chroma.2019.03.069

M3 - Article

C2 - 30961966

AN - SCOPUS:85063874123

VL - 1599

SP - 85

EP - 94

JO - Journal of Chromatography A

JF - Journal of Chromatography A

SN - 0021-9673

ER -