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N-glycan PK Profiling Using a High Sensitivity nanoLCMS Work-Flow with Heavy Stable Isotope Labeled Internal Standard and Application to a Preclinical Study of an IgG1 Biopharmaceutical.

Higel F, Seidl A, Demelbauer U, Viertlboeck-Schudy M, Koppenburg V, Kronthaler U, Sörgel F, Friess W - Pharm. Res. (2015)

Bottom Line: Possible effects on the pharmacokinetics caused by glyco-variants have been subject of several studies with in part contradictory results which can be related to differences in the set-up.It was concluded that M6 was converted by mannosidases in circulation to M5 which in turn was selectively cleared by mannose receptor binding which is in-line with previously published results.Therefore the developed technology delivers reliable results and can be applied for PK profiling of other mAbs and other types of biopharmaceuticals.

View Article: PubMed Central - PubMed

Affiliation: Analytical Characterization, Sandoz Biopharmaceuticals, HEXAL AG, Keltenring 1+3, 82041, Oberhaching, Germany. fabian.higel@sandoz.com.

ABSTRACT

Purpose: In this study an innovative, highly sensitive work-flow is presented that allows the analysis of a possible influence of individual glyco-variants on pharmacokinetics already during pre-clinical development. Possible effects on the pharmacokinetics caused by glyco-variants have been subject of several studies with in part contradictory results which can be related to differences in the set-up.

Methods: Using 96-well plate based affinity purification an IgG1 antibody was isolated from preclinical samples and glycans were analyzed individually by nanoLCMS. Prerequisite was a reference standard based on stable heavy isotope labeled glycans. The high sensitivity and low sample consumption enabled the integration into the preclinical development program.

Results: The data of an IgG1 biopharmaceutical from a preclinical rabbit study showed that some N-glycoforms have a different PK profile compared with the average of all molecule variants as determined by ELISA. IgG1 high mannose glycoforms M5 and M6 were removed from circulation at a higher rate.

Conclusion: The results of the preclinical study demonstrated the applicability of the developed innovative workflow. The PK profile of glyco-variants could be determined individually. It was concluded that M6 was converted by mannosidases in circulation to M5 which in turn was selectively cleared by mannose receptor binding which is in-line with previously published results. Therefore the developed technology delivers reliable results and can be applied for PK profiling of other mAbs and other types of biopharmaceuticals.

No MeSH data available.


Comparison of nanoLC-MS based glycan PK data (blue) and ELISA data (red) for G0F (a), G1F (b), G2F (c), M5G1F/M6G0F (d), M3 (e), M3G1F (f), M3G0F (g). Concentration is relative to the maximum of each curve to enable comparison. Mean profiles from 15 animals are shown.
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Fig5: Comparison of nanoLC-MS based glycan PK data (blue) and ELISA data (red) for G0F (a), G1F (b), G2F (c), M5G1F/M6G0F (d), M3 (e), M3G1F (f), M3G0F (g). Concentration is relative to the maximum of each curve to enable comparison. Mean profiles from 15 animals are shown.

Mentions: The determined glycan L/H ratios were plotted against the sampling time to obtain individual PK profiles for each N-glycan. Mean L/H ratios for each N-glycan were normalized to the maximum of each N-glycan L/H curve to obtain a relative concentration (Fig. 5). ELISA data were normalized in the same way. The PK profile of the most abundant complex type G0F was very similar to the ELISA profile (Fig. 5a). The maximum concentration (tmax) was reached after 72 h with congruency of the ELISA and the L/H glycan graph. Elimination occured at identical rates and the PK profiles were regarded as comparable. This finding was expected as the ELISA represents the average profile of all mAb1 glycoforms and G0F being the major N-glycan representing the majority of these glycoforms. The second most abundant complex type N-glycan G1F that accounted for approximately 16% of all mAb1 N-glycans also showed a PK profile which was very similar compared to the ELISA profile (Fig. 5b). Again tmax was reached at 72 h. Complex type G2F with a portion of 2% had the best match with the ELISA profile showing almost perfect congruency (Fig. 5c). The profile of the M6G0F/M5G1F hybrid type glycan represented two isomers that could not be differentiated with the nanoLC-MS approach and which had a relative content of only 0.1%. The profile and tmax were similar to the ELISA profile as well (Fig. 5d). The low abundance brought the analysis close to the LLOQ which resulted in lower precision and higher variation. The glycan PK profiles of M3, M3G0F and M3G1F were also highly similar to the ELISA profile (Fig. 5e, f and g). These results demonstrated that PK profiles could be obtained for each N-glycan individually. The complex glycan PK profiles were highly similar to the ELISA for the most abundant glycans. For N-glycans with a portion smaller than 0.5% the graphs showed higher variation. Considering all the glycan forms analyzed, only the high mannose type N-glycans M5 and M6 with 9.5 and 2% relative abundance respectively showed a clear discrepancy from the ELISA profile (Fig. 6) The maximum concentration of M6 was reached after 24 h followed by either a conversion to M5 or an increased elimination rate that led to a faster clearance to a level below the LLOD or a complete removal from circulation at the 168 h time point. The high mannose glycan M5 profile also differed from the overall mAb ELISA PK profile (Fig. 6b). The tmax was reached 24 h earlier already after 48 h and these molecules were cleared faster between 48 and 168 h. PK profiles for G0 could not be obtained due to co-elution of a contaminant with the same m/z value. The tmax values are listed in Table II.Fig. 5


N-glycan PK Profiling Using a High Sensitivity nanoLCMS Work-Flow with Heavy Stable Isotope Labeled Internal Standard and Application to a Preclinical Study of an IgG1 Biopharmaceutical.

Higel F, Seidl A, Demelbauer U, Viertlboeck-Schudy M, Koppenburg V, Kronthaler U, Sörgel F, Friess W - Pharm. Res. (2015)

Comparison of nanoLC-MS based glycan PK data (blue) and ELISA data (red) for G0F (a), G1F (b), G2F (c), M5G1F/M6G0F (d), M3 (e), M3G1F (f), M3G0F (g). Concentration is relative to the maximum of each curve to enable comparison. Mean profiles from 15 animals are shown.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig5: Comparison of nanoLC-MS based glycan PK data (blue) and ELISA data (red) for G0F (a), G1F (b), G2F (c), M5G1F/M6G0F (d), M3 (e), M3G1F (f), M3G0F (g). Concentration is relative to the maximum of each curve to enable comparison. Mean profiles from 15 animals are shown.
Mentions: The determined glycan L/H ratios were plotted against the sampling time to obtain individual PK profiles for each N-glycan. Mean L/H ratios for each N-glycan were normalized to the maximum of each N-glycan L/H curve to obtain a relative concentration (Fig. 5). ELISA data were normalized in the same way. The PK profile of the most abundant complex type G0F was very similar to the ELISA profile (Fig. 5a). The maximum concentration (tmax) was reached after 72 h with congruency of the ELISA and the L/H glycan graph. Elimination occured at identical rates and the PK profiles were regarded as comparable. This finding was expected as the ELISA represents the average profile of all mAb1 glycoforms and G0F being the major N-glycan representing the majority of these glycoforms. The second most abundant complex type N-glycan G1F that accounted for approximately 16% of all mAb1 N-glycans also showed a PK profile which was very similar compared to the ELISA profile (Fig. 5b). Again tmax was reached at 72 h. Complex type G2F with a portion of 2% had the best match with the ELISA profile showing almost perfect congruency (Fig. 5c). The profile of the M6G0F/M5G1F hybrid type glycan represented two isomers that could not be differentiated with the nanoLC-MS approach and which had a relative content of only 0.1%. The profile and tmax were similar to the ELISA profile as well (Fig. 5d). The low abundance brought the analysis close to the LLOQ which resulted in lower precision and higher variation. The glycan PK profiles of M3, M3G0F and M3G1F were also highly similar to the ELISA profile (Fig. 5e, f and g). These results demonstrated that PK profiles could be obtained for each N-glycan individually. The complex glycan PK profiles were highly similar to the ELISA for the most abundant glycans. For N-glycans with a portion smaller than 0.5% the graphs showed higher variation. Considering all the glycan forms analyzed, only the high mannose type N-glycans M5 and M6 with 9.5 and 2% relative abundance respectively showed a clear discrepancy from the ELISA profile (Fig. 6) The maximum concentration of M6 was reached after 24 h followed by either a conversion to M5 or an increased elimination rate that led to a faster clearance to a level below the LLOD or a complete removal from circulation at the 168 h time point. The high mannose glycan M5 profile also differed from the overall mAb ELISA PK profile (Fig. 6b). The tmax was reached 24 h earlier already after 48 h and these molecules were cleared faster between 48 and 168 h. PK profiles for G0 could not be obtained due to co-elution of a contaminant with the same m/z value. The tmax values are listed in Table II.Fig. 5

Bottom Line: Possible effects on the pharmacokinetics caused by glyco-variants have been subject of several studies with in part contradictory results which can be related to differences in the set-up.It was concluded that M6 was converted by mannosidases in circulation to M5 which in turn was selectively cleared by mannose receptor binding which is in-line with previously published results.Therefore the developed technology delivers reliable results and can be applied for PK profiling of other mAbs and other types of biopharmaceuticals.

View Article: PubMed Central - PubMed

Affiliation: Analytical Characterization, Sandoz Biopharmaceuticals, HEXAL AG, Keltenring 1+3, 82041, Oberhaching, Germany. fabian.higel@sandoz.com.

ABSTRACT

Purpose: In this study an innovative, highly sensitive work-flow is presented that allows the analysis of a possible influence of individual glyco-variants on pharmacokinetics already during pre-clinical development. Possible effects on the pharmacokinetics caused by glyco-variants have been subject of several studies with in part contradictory results which can be related to differences in the set-up.

Methods: Using 96-well plate based affinity purification an IgG1 antibody was isolated from preclinical samples and glycans were analyzed individually by nanoLCMS. Prerequisite was a reference standard based on stable heavy isotope labeled glycans. The high sensitivity and low sample consumption enabled the integration into the preclinical development program.

Results: The data of an IgG1 biopharmaceutical from a preclinical rabbit study showed that some N-glycoforms have a different PK profile compared with the average of all molecule variants as determined by ELISA. IgG1 high mannose glycoforms M5 and M6 were removed from circulation at a higher rate.

Conclusion: The results of the preclinical study demonstrated the applicability of the developed innovative workflow. The PK profile of glyco-variants could be determined individually. It was concluded that M6 was converted by mannosidases in circulation to M5 which in turn was selectively cleared by mannose receptor binding which is in-line with previously published results. Therefore the developed technology delivers reliable results and can be applied for PK profiling of other mAbs and other types of biopharmaceuticals.

No MeSH data available.