Molecular characterization of multivalent bioconjugates by size-exclusion chromatography with multiangle laser light scattering

Abstract

The degree of substitution and valency of bioconjugate reaction products are often poorly judged or require multiple time- and product-consuming chemical characterization methods. These aspects become critical when analyzing and optimizing the potency of costly polyvalent bioactive conjugates. In this study, size-exclusion chromatography with multiangle laser light scattering was paired with refractive index detection and ultraviolet spectroscopy (SEC-MALS-RI-UV) to characterize the reaction efficiency, degree of substitution, and valency of the products of conjugation of either peptides or proteins to a biopolymer scaffold, i.e., hyaluronic acid (HyA). Molecular characterization was more complete compared to estimates from a protein quantification assay, and exploitation of this method led to more accurate deduction of the molecular structures of polymer bioconjugates. Information obtained using this technique can improve macromolecular engineering design principles and help to better understand multivalent macromolecular interactions in biological systems.

Figure 1

Hyaluronic acid-peptide conjugate chemical structure. A heterobifunctional linker was used to anchor peptide and protein via their terminal thiol-functional cysteine residues to the carboxylic acid groups of the linear hyaluronic acid polymer.

Figure 2

Representative chromatogram (a) with 90° light scattering (LS), refractive index (RI), and ultraviolet light absorbance (UV) signals and Debye plot (b) calculated from mulit-angle laser light scattering (MALS) data at slices along the size-exclusion chromatogram for HyA-bsp-RGD(15) (1:25, HyA:bsp-RGD(15) molar reaction ratio). A Zimm plot (c) created from injectons of varying total conjugate mass for HyA-bsp-RGD(15) (1:25) used to calculate weight-averaged molecular mass (M_w), z-averaged radius of gyration, (R_G,z,) and second virial coefficient, (A₂).

Figure 3

Detector signals (90° light scattering, LS, refractive index, RI, and ultraviolet light absorbance, UV) (a) collected during size-exclusion chromatography (SEC) combined with MALS data allowed calculation of hyaluronic acid and protein molecular weight distributions (b) of the HyA-Shh (1:40, HyA:Shh molar reaction ratio) conjugate.

Figure 4

Protein fraction (dark line) and 90° light scattering intensity (light line) of HyA-bsp-RGD(15) (1:25) (a), HyA-Shh (1:20) (b), and HyA-Shh (1:40) (c) following size-exclusion chromatography. Light scattering instensity depends on concentration and total molar mass, with higher molar masses eluted first. Conjugation of bsp-RGD was more efficient than Shh, with smaller input reaction ratios achieving a higher final protein fraction due to the small size and ease of accessibility to the bsp-RGD reaction site when compared to Shh.

Figure 5

Cumulative (a, c) and differential (b, d) mass fractions versus molar mass of 0.5 MDa HyA and HyA-bsp-RGD(15) (1:25) (a, b) along with 1.0 MDa HyA, HyA-Shh (1:20), and HyA-Shh (1:40) (c, d).

Figure 6

Weight-averaged molecular weights of hyaluronic acid in conjugates of varying reaction ratio. The error bars represent the standard deviation (N=3).

Figure 7

Input and output degrees of substitution (DS) values for HyA-Shh conjugates. The line represents correspondence of 1:1.

Figure 8

Comparison of input and output molar ratios as determined by SEC-MALS-RI-UV and BCA assay methods. Error bars represent the standard deviation (N=3).