A relatively low clearance is one of the prominent favorable features of immunoglobulin G1-based therapeutic monoclonal antibodies (mAbs). Various studies have observed differential clearance of... Show moreA relatively low clearance is one of the prominent favorable features of immunoglobulin G1-based therapeutic monoclonal antibodies (mAbs). Various studies have observed differential clearance of mAb glycoforms, including oligomannose glycoforms, which are considered a critical quality attribute because they show higher clearance than complex type glycoforms. Glycoform clearance, however, has not previously been studied after subcutaneous injection or in a porcine model system. Here, we performed glycoform-resolved pharmacokinetic (PK) analysis of two mAbs in Gottingen minipigs. We found glycoform effects on clearance to be largely the same for subcutaneous and intravenous injection and in line with observations in other species. Oligomannose glycoforms were cleared up to 25% faster and monoantennary glycoforms up to 8% faster than agalactosylated complex glycoforms. Sialylated glycoforms were cleared at approximately the same rate as fully galactosylated glycoforms. Importantly, we report here an impact of galactosylation on the PK of a mAb for the first time. Whether increased galactosylation led to slower or faster clearance seemed to depend on the overall glycosylation profile. When clearance of galactosylated glycoforms was slower, the mAb showed higher galactosylation in serum at maximum concentration after subcutaneous injection compared to both intravenous injection and the injected material. Whether this higher galactosylation after subcutaneous injection has consequences for therapeutic efficacy remains to be investigated. In conclusion, preferential clearance of antibody glycoforms can be simulated in the minipig model with intravenous as well as subcutaneous injections. Furthermore, we observed a glycoform bias in the absorption from skin into circulation after subcutaneous injection based on galactosylation. Show less
Falck, D.; Thomann, M.; Lechmann, M.; Koeleman, C.A.M.; Malik, S.; Jany, C.; ... ; Reusch, D. 2021
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... Show moreGood 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, alpha 2,3-linked sialylation did not affect clearance, contradicting the involvement of the asialoglycoprotein receptor in mAb clearance. Show less
