Stability analysis of an inline peptide-based conjugate for metal delivery: nickel(II)-claMP Tag epidermal growth factor as a model system

Abstract

Metals are a key component of many diagnostic imaging and biotechnology applications, and the majority of cancer patients receive a platinum-based drug as part of their treatment. Significant effort has been devoted to developing tight binding synthetic chelators to enable effective targeted delivery of metal-based conjugates, with most successes involving lanthanides rather than transition metals for diagnostic imaging. Chemical conjugation modifies the protein's properties and generates a heterogeneous mixture of products. Chelator attachment is typically carried out by converting the amino group on lysines to an amide, which can impact the stability and solubility of the targeting protein and these properties vary among the set of individual conjugate species. Site-specific attachment is sought to reduce complexity and control stability. Here, the metal abstraction peptide technology was applied to create the claMP Tag, an inline platform for generating site-specific conjugates involving transition metals. The claMP Tag was genetically encoded into epidermal growth factor (EGF) and loaded with nickel(II) as a model system to demonstrate that the tag within the homogeneous inline conjugate presents sufficient solution stability to enable biotechnology applications. The structure and disulfide network of the protein and chemical stability of the claMP Tag and EGF components were characterized.

Keywords: MAP; NMR; absorption spectroscopy; analytical biochemistry; chromatography; conjugation; metal abstraction peptide; protein structure; stability; targeted drug delivery.

Figure 1

Size exclusion chromatography assessment of the monomeric content of EGF-Ni-claMP prepared in either Tris-Cl (a) or KPi (b). At the initial time point, only one species is present in both samples. After three months, mostly monomeric protein remains with the emergence of a shoulder on the backside of the peak suggests the formation of a smaller molecular weight species.

Figure 2

UV-vis spectroscopy confirms Ni(II) is incorporated into the claMP Tag in both Tris-Cl (a) and KPi (b). Ni(II) remains incorporated in the claMP Tag regardless of buffer system chosen, as the intensity of the feature at 310 nm remains constant over the 12-week period with Tris-Cl (c) and KPi (d). At the 24-week time point, the intensity of the feature at 310 nm decreases, suggesting alterations occur in the Ni-claMP complex. The intensity of the feature at 280 nm remains constant, indicating the protein concentration remains the same over the time period investigated.

Figure 3

Trend analysis by anion exchange chromatography to assess retention of intact EGF-Ni-claMP. The initial chromatograms of the conjugate prepared in either Tris-Cl (a) or KPi (b) are very similar, indicating the buffer choice does not alter formation of conjugate. In the sample prepared in KPi buffer, a peak emerges at 50 minutes, indicating formation of a species with a less negative charge. c) The area of the peak at 74 minutes was plotted to reflect the amount of intact EGF-Ni-claMP remaining. At 12 weeks, the area of this peak decreases by 25% and 35% for the samples prepared in Tris-Cl and KPi, respectively. At 24 weeks, the area of this peak in samples prepared in Tris-Cl and KPi decreases by approximately 40% and 70%, respectively. d) The area of the peak at 35 minutes was plotted to reflect the amount of intact EGF in solution, and it remains constant over the 24-week time period. 20% variability was observed for both EGF and EGF-Ni-claMP.

Figure 4

Plot of NMR peak heights. The peak height corresponding to Cys residues confirms the protein structure is maintained and no loss of protein from solution occurs. The peak heights corresponding to the Gly from the claMP tag and C-terminal residues from EGF (L52, R53) decrease, indicating a change in structure in this region.

Figure 5

¹H-¹⁵N HSQC spectra confirm that buffer selection has no effect on the structure of EGF-Ni-claMP, as the peaks of the samples prepared in Tris-Cl (blue) and KPi (red) overlap completely.

Figure 6

¹H-¹⁵N HSQC spectra of EGF-Ni-claMP at T=0 (red), T=12 weeks (blue), and T=24 weeks (green). The tertiary structure of the protein is maintained, but specific differences are observed in peaks corresponding to the C-terminal region (boxed).