Conjugation site heterogeneity causes variable electrostatic properties in Fc conjugates

Abstract

Immunoconjugates, including antibody-drug conjugates and Fc-conjugates, represent a rapidly growing class of therapeutics undergoing clinical development. Despite their growing popularity, the high intrinsic heterogeneity of immunoconjugates often complicates the development process and limits their widespread application. In particular, immunoconjugate charge variants exhibit markedly different colloidal stabilities, solubilities, pharmacokinetics, and tissue distributions. Charge variants arise spontaneously due to degradation and, depending on the type of drug, linker, and conjugation site, through drug conjugation. Electrostatic changes in naked antibodies often result in poor performance characteristics, and therefore, charge alterations due to degradation are critical to control. Charge properties are expected to be equally important to producing well-behaved ADCs. Charge-based methods of analysis, such as isoelectric focusing and ion exchange chromatography, are capable of probing the underlying complexities within immunoconjugate drug products. Despite the utility of these methods, there are only a few published reports of charge-based assays applied to immunoconjugates. In the present study, we sought to identify the effects of chemical conjugation on the electrostatic properties of Fc-conjugates. In order to minimize the effects of post-translational modifications (e.g., deamidation), a single Fc charge variant was isolated prior to conjugation of a fluorescent probe, Alexa Fluor 350, to the side chains of lysine residues. The resulting Fc-conjugates were assessed by a variety of analytical techniques, including isoelectric focusing and ion exchange chromatography, to determine their charge properties.

Figure 1

IEF analysis of IgG1 Fc and Fc-conjugates. Lanes: 1) Protein G affinity-purified IgG1 Fc, 2) SAX-purified IgG1 Fc, and Fc-conjugates 3) DAR 0, 4) DAR 1.5, 5) DAR 2.8, 6) DAR 4.9, and 7) DAR 7.8. The isoelectric point (pI) for the labeled bands were identified using SERVA IEF marker 3-10 from Invitrogen. 58×42mm (600 × 600 DPI)

Figure 2

UV spectra of Fc-conjugates prepared at various DARs, normalized to absorbance at 280 nm. Overlay shows a proportional increase in 346 nm absorbance as the amount of conjugated AF350 increases. 59×43mm (600 × 600 DPI)

Figure 3

Analysis of native Fc-conjugate DAR 7.8 via SE-UPLC with UV absorbance detection at 280 and 346 nm. Labeled peaks correspond to unconjugated AF350 and Fc-conjugates. 100×77mm (300 × 300 DPI)

Figure 4

Distribution of DAR species. Symbols represent the relative abundance of each individual DAR species as determine by LC-ESI-MS analysis of reduced and deglycosylated Fc-conjugates. The diamonds (red), squares (orange), triangles (green), and circles (blue) represent Fc-conjugates with average DAR 1.5, 2.8, 4.9 and 7.8, respectively. Solid lines in the same color represent the theoretical normal distribution for the corresponding average DAR. 60×46mm (300 × 300 DPI)

Figure 5

Analysis of Fc-conjugate DAR 1.5 via SAX-UPLC. Overlay of individual 280 and 346 nm absorbance traces (top) and fluorescence detected at 442 nm (bottom) are shown. Shaded areas represent unconjugated AF350 and Fc-conjugates with calculated dye-to-antibody ratio derived from measured absorbance at 346 nm (dye) and 280 nm (Fc). 100×60mm (300 × 300 DPI)