Influence of Molecular size on the clearance of antibody fragments

Abstract

Purpose: To establish a continuous relationship between the size of various antibody fragments and their systemic clearance (CL) in mice.

Methods: Two different orthogonal approaches have been used to establish the relationship. First approach uses CL values estimated by non-compartmental analysis (NCA) to establish a correlation with protein size. The second approach simultaneously characterizes the PK data for all the proteins using a 2-compartment model to establish a relationship between protein size and pharmacokinetic (PK) parameters.

Results: Simple mathematical functions (e.g. sigmoidal, power law) were able to characterize the CL vs. protein size relationship generated using the investigated proteins. The relationship established in mouse was used to predict rat, rabbit, monkey, and human relationships using allometric scaling. The predicted relationships were found to capture the available spares data from each species reasonably well.

Conclusions: The CL vs. protein size relationship is important for establishing a robust quantitative structure-PK relationship (QSPKR) for protein therapeutics. The relationship presented here can help in a priori predicting plasma exposure of therapeutic proteins, and together with our previously established relationship between plasma and tissue concentrations of proteins, it can predict the tissue exposure of non-binding proteins simply based on molecular weight/radius and dose.

Keywords: Antibody Fragments; Compartmental Modeling; Monoclonal Antibody (mAb); QSPKR; Systemic Clearance.

Figure 1

Digitized plasma PK data for nanobody (13 kDa, n=3), scFv (27 kDa, n=13), diabody (50 kDa, n=2), Fab (50 kDa, n=6), scFv₂ (55 kDa, n=2), minibody (80 kDa, n=3), F(ab)₂ (100 kDa, n=6), scFv-Fc (105 kDa, n=2), and IgG in FcRn KO mice (150 kDa, n=1).

Figure 2

(A) Clearance vs. molecular weight, and (B) clearance vs. radius relationships. Solid circles represent CL values calculated using NCA for all proteins, except Fab, scFv-Fc, and IgG in wild type mice. Solid diamond represents Fab, and solid triangles represent scFv-Fc and IgG. Solid line represents fitted relationships, and the gray area represent the two-fold error envelop.

Figure 3

Observed and model predicted PK profiles for nanobody (13 kDa), scFv (27 kDa,), diabody (50 kDa), Fab (50 kDa), scFv₂ (55 kDa), minibody (80 kDa), F(ab)₂ (100 kDa), and IgG in FcRn KO mice (150 kDa). Solid circles represent digitized raw PK data, and solid lines represent 2-compartment model fitted PK profiles. The gray area represent the zone obtained by changing the systemic clearance two-fold.

Figure 4

Simulated, (A) V2 vs. MW, (B) CLD vs. MW, (C) CL vs. MW, and (D) CL vs. a_e profiles, generated using parameters estimated by simultaneously fitting all the protein PK data using a 2-compartment model.

Figure 5

(A) CL vs. MW, and (B) CL vs. radius relationships generated by the two different approaches superimposed over each other. Solid circles represent NCA calculated CL values for all proteins except Fab, scFv-Fc, and IgG in wild type mice. Solid diamond represents NCA calculated CL for Fab, and solid triangles represent NCA calculated CL for scFv-Fc and IgG in wild type mice. Solid line represents continuous relationship between CL and MW/radius obtained using compartmental modeling fitting approach. Dashed line represents continuous relationship between CL and MW/radius obtained using NCA approach.

Figure 6

CL vs. molecular weight relationships for antibody and antibody fragments. The lines represent the relationships generated using equations and the symbols represent the observed clearance values. The proteins at 150 kDa are monoclonal antibodies. Panels A, B, C, D, and E represent human, monkey, rabbit, rat, and mouse. The gray area in those panels represent twofold error envelop. Panel F superimposes the CL vs. molecular weight relationships for all the animal species.

Figure 7

This figure compares various CL vs. MW relationships. Solid line represents fitted CL vs. MW relationship established using NCA based CL values calculated in the current study. Dashed line represents calculated renal CL vs. MW relationship in mice, obtained using sieving coefficient vs. MW relationship reported by Norden et al. (16) Renal clearance was obtained by simply multiplying the sieving coefficients with glomerular filtration rate (GFR). Close squares represent NCA calculated CL for proteins from the current study, except for Fab, scFv-Fc, and IgG in wild type mice, which are shown as open squares. Open circles represent CL values for various proteins that are derived from k_clear values reported by Schmidt and Wittrup.(14)