Glycoform-resolved pharmacokinetic studies in a rat model employing glycoengineered variants of a therapeutic monoclonal antibody

mAbs, vol. 13, pp. 1865596

Abstract

Good pharmacokinetic (PK) behavior is a key prerequisite for sufficient efficacy of therapeutic monoclonal antibodies (mAbs). Fc glycosylation is a critical quality attribute (CQA) of mAbs, due to its impact on stability and effector functions. However, the effects of various IgG Fc glycoforms on antibody PK remain unclear. We used a combination of glycoengineering and glycoform-resolved PK measurements by mass spectrometry (MS) to assess glycoform effects on PK. Four differently glycoengineered mAbs, each still containing multiple glycoforms, were separately injected into rats. Rat models have been shown to be predictive of human PK. At different time points, blood was taken, from which the mAbs were purified and analyzed with a liquid chromatography-MS-based bottom-up glycoproteomics approach. This allowed us to follow changes in the glycosylation profiles of each glycoengineered mAb over time. Enzyme-linked immunosorbent assay measurements provided an absolute concentration in the form of a sum value for all glycoforms. Information from both readouts was then combined to calculate PK parameters per glycoform. Thereby, multiple glycoform kinetics were resolved within one mAb preparation. We confirmed increased clearance of high-mannose (Man5) and hybrid-type (Man5G0) glycoforms. Specifically, Man5 showed a 1.8 to 2.6-fold higher clearance than agalactosylated, complex glycans (G0F). Unexpectedly, clearance was even higher (4.7-fold) for the hybrid-type glycan Man5G0. In contrast, clearance of agalactosylated, monoantennary glycoforms (G0F-N) was only slightly increased over G0F (1.2 to 1.4-fold). Thus, monoantennary, hybrid-type and high-mannose glycoforms should be distinguished in CQA assessments. Strikingly, α2,3-linked sialylation did not affect clearance, contradicting the involvement of the asialoglycoprotein receptor in mAb clearance.

Authors 8

1. David Falck corresponding Aachen Center for Proteomics and Metabolomics

   Leiden University Medical Center

   Affiliation as printed

   Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands

2. Marco Thomann
   Affiliation as printed

   Pharma Technical Development Europe, Roche Diagnostics GmbH, Penzberg, Germany

3. Martin Lechmann

   Roche Pharma AG (Germany)

   Affiliation as printed

   Roche Pharma Research and Early Development, Pharmaceutical Sciences, Roche Innovation Center Munich, Penzberg, Germany

4. Carolien A. M. Koeleman Aachen Center for Proteomics and Metabolomics

   Leiden University Medical Center

   Affiliation as printed

   Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands

5. Sebastian Malik
   Affiliation as printed

   Pharma Technical Development Europe, Roche Diagnostics GmbH, Penzberg, Germany

6. Cordula Jany

   Roche Pharma AG (Germany)

   Affiliation as printed

   Roche Pharma Research and Early Development, Pharmaceutical Sciences, Roche Innovation Center Munich, Penzberg, Germany

7. Manfred Wuhrer Aachen Center for Proteomics and Metabolomics

   Leiden University Medical Center

   Affiliation as printed

   Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands

8. Dietmar Reusch
   Affiliation as printed

   Pharma Technical Development Europe, Roche Diagnostics GmbH, Penzberg, Germany