Heparin chromatography as an in vitro predictor for antibody clearance rate through pinocytosis

Abstract

The pharmacokinetic (PK) properties of therapeutic antibodies directly affect efficacy, dose and dose intervals, application route and tissue penetration. In indications where health-care providers and patients can choose between several efficacious and safe therapeutic options, convenience (determined by dosing interval or route of application), which is mainly driven by PK properties, can affect drug selection. Therapeutic antibodies can have greatly different PK even if they have identical Fc domains and show no target-mediated drug disposition. Biophysical properties like surface charge or hydrophobicity, and binding to surrogates for high abundant off-targets (e.g., baculovirus particles, Chinese hamster ovary cell membrane proteins) were proposed to be responsible for these differences. Here, we used heparin chromatography to separate a polyclonal mix of endogenous human IgGs (IVIG) into fractions that differ in their PK properties. Heparin was chosen as a surrogate for highly negatively charged glycocalyx components on endothelial cells, which are among the main contributors to nonspecific clearance. By directly correlating heparin retention time with clearance, we identified heparin chromatography as a tool to assess differences in unspecific cell-surface interaction and the likelihood for increased pinocytotic uptake and degradation. Building on these results, we combined predictors for FcRn-mediated recycling and cell-surface interaction. The combination of heparin and FcRn chromatography allow identification of antibodies with abnormal PK by mimicking the major root causes for fast, non-target-mediated, clearance of therapeutic, Fc-containing proteins.

Keywords: FcRn; Pharmacokinetics; clearance; heparin; neonatal Fc receptor; pinocytosis; prediction.

Figure 1.

IVIG fractionation on a preparative heparin column. The blue trace shows the UV absorption at 280 nm; the red trace indicates conductivity. Gray areas indicate where fractions 1, 2 and 3, respectively, were collected.

Figure 2.

(A) Retention of IVIG fractions and control samples on hFcRn column; peaks 1 and 2 of the retention time standard, which are used for the calculation of the relative retention time, are indicated. Briakinumab and ustekinumab are shown as examples for unusually strong and “typical” examples, respectively. (B) Re-chromatography of IVIG fractions by analytical heparin HPLC.

Figure 3.

Serum concentration vs. time profiles for fractions 1, 2 and 3. (A) FcRn ko mice. (B) hFcRn Tg32 homozygous mice. (C) wild-type (C57BL/6) mice. (D) Clearance of IVIG fractions in the different mouse models. Clearance values represent the mean ± S.D. of five mice per experiment. The red-dotted line marks the assay detection limit. Asterisks denote statistically significant differences (** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001) as determine by one-way ANOVA plus a posteriori Holm-Sidak test.

Figure 4.

(A) Correlation of heparin and FcRn column retention for Roche clinical candidates colored according to qualitative clearance in cynomolgus monkeys (dark green, very slow, <2.5 mL/kg/day; green, slow, <8 ml/kg/day; yellow, intermediate >8 and <12 ml/kg/day; red >12 ml/kg/day). (B) Heparin and FcRn column retention of antibodies from to Jain et al.³⁵ Heparin and FcRn binding data were generated as part of this study. The color code reflects the year in which the INN was first assigned. Uste* and Bria* denotes the variable domains of briakinumab and ustekinumab on a common IgG1 constant region. Cutoffs were chosen manually to separate mAbs with fast and normal clearance. IVIG fractions F1, F2, and F3 were added for comparison.