Molecular engineering of cyclic azobenzene-peptide hybrid ligands for the purification of human blood Factor VIII via photo-affinity chromatography

Abstract

The use of benign stimuli to control the binding and release of labile biologics for their isolation from complex feedstocks is a key goal of modern biopharmaceutical technology. This study introduces cyclic azobenzene-peptide (CAP) hybrid ligands for the rapid and discrete photo-responsive capture and release of blood coagulation Factor VIII (FVIII). A predictive method - based on amino acid sequence and molecular architecture of CAPs - was developed to correlate the conformation of cis/trans CAP photo-isomers to FVIII binding and release. The combined in silico and in vitro analysis of FVIII:peptide interactions guided the design of a rational approach to optimize isomerization kinetics and biorecognition of CAPs. A photoaffinity adsorbent, prepared by conjugating selected CAP G-cyclo_AZOB[Lys-YYKHLYN-Lys]-G on translucent chromatographic beads, featured high binding capacity (> 6 mg of FVIII per mL of resin) and rapid photo-isomerization kinetics (τ < 30s) when exposed to 420-450 nm light at the intensity of 0.1 W·cm^-2. The adsorbent purified FVIII from a recombinant harvest using a single mobile phase, affording high product yield (>90%), purity (>95%), and blood clotting activity. The CAPs introduced in this report demonstrate a novel route integrating gentle operational conditions in a rapid and efficient bioprocess for the purification of life-saving biotherapeutics.

Keywords: Factor VIII; affinity ligands; azobenzene-peptide hybrids; biomolecular recognition; photo-affinity chromatography.

Figure 1.. Photo-affinity chromatography of labile therapeutics.

The target recombinant human blood coagulation Factor VIII (rVIII) – a labile therapeutic protein – is captured by the CAPs displayed on translucent ChemMatrix beads, while the impurities are cleared, and subsequently released at high purity under benign conditions (< 10 mW·cm⁻²). Specifically, (i) the pore surface of the beads is functionalized with cyclic azobenzene-peptides (CAPs), each comprising a peptide segment, which interacts with the target rFVIII, and a photo-responsive azobenzene linker, which switches reversibly the peptide segment between a rFVIII-binding (cis) and a rFVIII-releasing (trans) conformation; (ii) when rFVIII in solution contacts the beads, it diffuses into their pores, and reaches the inner surface of the CAP-functionalized beads, where it is captured by the affinity binding of cis-CAPs ^§ (note: the CAPs developed in this study mainly target the A1 domain of rFVIII); conversely, the other species present in the feedstock do not interact with the CAPs and are therefore removed by washing; (iii) following the adsorption of rFVIII, the beads are washed and exposed to blue light (440 nm) to isomerize cis-CAPs into trans-CAPs, thus triggering the dissociation of the rFVIII:CAP complex and the release of rFVIII from the resin beads; finally, (iv) the beads can subsequently be exposed to UV light (330 nm), which switches the trans-CAPs back into cis-CAPs, thus preparing the beads for a subsequent round of photo-affinity purification of rFVIII.

Figure 2.. Secondary screening of candidate and variant rFVIII-targeting photo-responsive CAP ligands.

(A) The values of rFVIII binding strength were calculated as the ratio between the corrected intensity of red fluorescence (exc/em: 590/617 nm) of a cis-CAP-ChemMatrix bead and the corrected intensity of a control ChemMatrix bead (i.e. functionalized with anti-rFVIII antibodies) following AF594-rFVIII adsorption. The values of rFVIII binding selectivity were calculated as the ratio of the corrected intensity of red fluorescence of a cis-CAP-ChemMatrix bead after AF594-rFVIII adsorption against the total red and green (exc/em: 490/525 nm) fluorescence of a cis-CAP-ChemMatrix bead after AF594-rFVIII and AF488-HCPs adsorption. The values of rFVIII elution efficiency were calculated as the ratios of the corrected red fluorescence intensity of a cis-CAP-ChemMatrix bead after AF594-rFVIII adsorption and the corrected red fluorescence intensity of the same trans-CAP-ChemMatrix bead after AF594-rFVIII desorption upon exposure to visible light (λ_ex = 420-450 nm) at ~220 mW·cm⁻² for 2 mins at room temperature (25°C). Scale bar = 200 μm. (B) Sequences selected for in vitro analysis of rFVIII binding strength (dark red), elution efficiency (light red), and selectivity (blue diamonds), (C) their structural variants, and (C) performance of at different values of ionic strength of the mobile phase – namely, X M NaCl (X: 0.1, 0.25, 0.5, 0.75, and 1) added to 0.1 M HEPES buffer, 5 mM CaCl₂, and 0.01% v/v Tween20 at pH 7.4. Error bars represent one standard deviation of 30 beads. The raw values of average fluorescence and are in Figure S4.

Figure 3.. In silico evaluation of binding interaction between candidate CAPs and FVIII.

Structures of the complexes formed by human FVIII and the trans and cis isomers of (A) CAP.14” (G-cyclo_AZOB[Lys-KYYGSYY-Lys]-G) and (B) CAP.16” (G-cyclo_AZOB[Lys-YYKHLYN-Lys]-G); the trans-CAP isomers are in blue, the cis-CAP isomers are in magenta, the A1 domain of human FVIII (PDB ID: 3CDZ) is in pink, while all other FVIII domains are in grey. Paired interactions between FVIII and (C) cis-CAP.14” and (D) cis-CAP.16”; hydrogen bonds and polar contacts are denoted in green; hydrophobic and π-π interactions are denoted in yellow; electrostatic interactions are denoted in purple; the color intensity denotes the contribution of the paired interaction to the ΔG_b.

Figure 4.. Photo-affinity chromatographic purification of rFVIII from using CAP.16”-ChemMatrix beads and blood clotting activity of purified rFVIII.

(A) Values of concentration of rFVIII (■) and CHO HCPs (◆);rFVIII purity (▲) and recovery (●) vs. volume of effluent collected from the measurement cell; the chromatographic operation was entirely conducted at room temperature (25°C) and under constant flow of 0.1 M HEPES buffer added with 5 mM CaCl₂ and 0.01% v/v Tween20 at pH 7.4. (B) Clotting images generated by mixing platelet-poor plasma isolated from two Hemophilia A patients with aqueous 500 mM CaCl₂, a solution of AF488-Fibrinogen at 50 μg·mL⁻¹ in Binding Buffer, and either no FVIII or native FVIII, or FVIII purified via photo-affinity chromatography (fractions collected between 0.6 – 0.9 mL); clotting image generated by mixing healthy plasma with aqueous 500 mM CaCl₂ and a solution of AF488-Fibrinogen at 50 μg·mL⁻¹ in Binding Buffer. The images were generated from 5 μm z-stacks via confocal microscopy and the pixel density was measured in ImageJ. (C) Values of relative fiber density, calculated as the ratio of fiber pixels to background pixels after binarization of the images in panel (B). (D) Values of intersection density, calculated as the ratio of skeleton intersections post-erosion to the total number of fiber pixels pre-erosion of the images in panel (B). Plots show mean ± standard deviation. (**** p < 0.0001; two-way ANOVA).

Figure 5.

(A) Correlation of CAP photo-isomerization kinetics (κ/I) and energetics (ΔE_cis-trans). The values of κ/I ratio of ChemMatrix-bound CAPs are plotted against the normalized values of ΔE_cis-trans derived in silico via MD modeling of the corresponding CAPs; the dotted line data are reported from prior work on CAPs. (B) Structures of the complexes formed by human FVIII and the cis isomers of CAP.14^mY and CAP.16^mY; the cis-CAP isomers are in magenta, the A1 domain of human FVIII (PDB ID: 3CDZ) is in pink, while all other FVIII domains are in grey. (C) Values of rFVIII binding strength (dark red), elution efficiency (light red), and selectivity (blue diamonds) of sequence-based variants of CAP.14 and CAP.16 conjugated on ChemMatrix beads at different values of ionic strength of the mobile phase - namely X M NaCl (X: 0.1, 0.25, 0.5, 0.75, and 1) added to 0.1 M HEPES buffer, 5 mM CaCl₂, and 0.01% v/v Tween20 at pH 7.4. The values of rFVIII binding strength, selectivity, and elution, and error bars were calculated as described in Figure 2.

Figure 6.. Photo-affinity chromatographic purification of rFVIII from using (A) CAP.14^mY- and (B) CAP.16^mY-ChemMatrix beads.

Values of concentration of rFVIII (■) and CHO HCPs (◆); rFVIII purity (▲) and cumulative yield (●) vs. volume of effluent collected from the measurement cell. the chromatographic operation was entirely conducted at room temperature (25°C) and under constant flow of 0.1 M HEPES buffer added with 5 mM CaCl₂ and 0.01% v/v Tween20 at pH 7.4.