Effects of Excipients on the Structure and Dynamics of Filgrastim Monitored by Thermal Unfolding Studies by CD and NMR Spectroscopy

Abstract

Product excipients are used to confer a number of desirable properties on the drug substance to maintain or improve stability and facilitate drug delivery. This is especially important for products where the active pharmaceutical ingredient (API) is a recombinant protein. In this study, we aimed to determine if excipients and formulation conditions affect the structure and/or modulate the dynamics of the protein API of filgrastim products. Samples of uniformly labeled 15N-Met-granulocyte-colony stimulating factor (GCSF) were prepared at 100 μM (near formulation concentration) with various concentrations of individual components (polysorbate-20 and -80, sorbitol) and three pH values. Nuclear magnetic resonance (NMR) spectroscopy techniques were applied to measure chemical shift perturbation (CSP) to detect structural changes, and relaxation parameters (T 1, T 2, and heteronuclear Overhauser effect) were measured to probe the effects on protein backbone motions. In parallel, the same solution conditions were subjected to protein thermal unfolding studies monitored by circular dichroism spectropolarimetry (CD). Detergents (polysorbate-20 and 80) do not induce any observable changes on the protein structure and do not modify its dynamics at formulation concentration. Lowering pH to 4.0, a condition known to stabilize the conformation of filgrastim, as well as the addition of sorbitol produced changes of the fast motion dynamics in the nanosecond and picosecond timescale. NMR-derived order parameters, which measure the local conformational entropy of the protein backbone, show that lowering pH leads to a compaction of the four-helix bundle while the addition of sorbitol relaxes helices B and C, thereby reducing the mobility of loop CD. CSPs and measurements of protein dynamics via NMR-derived order parameters provide a description in structural and motional terms at an atomic resolution on how formulation components contribute to the stabilization of filgrastim products.

Investigation of the pH-dependent aggregation mechanisms of GCSF using low resolution protein characterization techniques and advanced molecular dynamics simulations

“…Based on chemical shift analysis, the authors could show that changes in Trp-His interactions affect the conformational stability of GCSF [20] . In addition, Ghasriani et al have determined the relaxation parameters of GCSF using NMR spectroscopy and assessed the protein flexibility from the calculated order parameters [41] . The authors found that the main difference between pH 4.0 and 6.0 was due to the change in the loop and helical flexibility.…”

“…The well-tempered metadynamics scheme [40] was used to ensure a smooth convergence of the free energy landscape. The collective variables (CVs) were chosen based on the experimental observations [18] , [20] , [41] , [19] , and included the center of the mass distance (COM) between Trp and His residues to monitor the interactions between, Trp59-His157 ( d1 ) and His80-Trp119 ( d2 ), and the α-helical content ( α ) ( Table 1 ).…”

Backbone and side-chain resonance assignments of the NISTmAb-scFv and antigen-binding study

“…harboring the pET15b10-NISTmAb-scFv plasmid in minimal media (M9) using 13 C-glucose and 15 Nammonium chloride as sole source of carbon and nitrogen at 37°C. Protein expression was induced by the addition of isopropyl thio-D-galactopyranoside (IPTG) at an OD 600 of 0.8.…”

“…Protein concentration was determined by using UV spectroscopy with the theoretical extinction coe cient 18150 M − 1 cm − 1 (Swissprot). NMR samples contained 0.25 mM of the uniformly isotope-labeled 13 C-15 N-or 15 N-NISTmAb-scFv in 20 mM phosphate buffer at pH 6.0, and 5% 2 H 2 O was used for eld frequency lock. The sample temperature was kept at 313 K (40°C).…”

“…NMRFAM-Sparky(Goddard and Kneller; Lee et al, 2015) was employed for spectral visualization and spectral analysis. Automatic assignment routine PINE-Sparky (Lee et al, 2019) was invoked for probabilistic assignment of the backbone amide 1 H, 15 N, and backbone carbonyl 13 C chemical shifts. Assignment of side chain resonances was done through a semi-automatic approach by initial engagement of PINE-Sparky, followed by implementation of an "inspection-veri cation" strategy, where PINE result for each assignment was either accepted or rejected based on a holistic approach that included inspection of complementary NOESY data.…”