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Design and evaluation of lyophilized fibroblast growth factor 21 and its protection against ischemia cerebral injury xuanxin yang, Qi Hui, Bingjie Yu, Zhen Huang, Peipei Zhou, Panfeng Wang, Zhitao Wang, Shucai Pang, Jinghang Li, Hanshi Wang, Li Lin, Xiaokun Li, and xiaojie wang Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00588 • Publication Date (Web): 26 Dec 2017 Downloaded from http://pubs.acs.org on December 27, 2017
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Bioconjugate Chemistry
Article type: Research article
Design and evaluation of lyophilized fibroblast growth factor 21 and its protection against ischemia cerebral injury
XuanxinYang1#, Qi Hui1#, BingjieYu1#, Zhen Huang1, PeiPei Zhou1, Panfeng Wang1, Zhitao Wang1, Shucai Pang1, Jinghang Li2, Hanshi Wang1, Li Lin1, Xiaokun Li1*, Xiaojie Wang1*
Author affiliations:
1
School of Pharmacy, Wenzhou Medical University, Chashan University Park, Wenzhou 325035,
China. 2
Swanson School of Engineering University of Pittsburgh Benedum Hall Pittsburgh, PA 15261
*Corresponding Authors: Xiaojie Wang,
[email protected]; Xiaokun Li,
[email protected] School of pharmacy, Wenzhou Medical University Chashan University Park, Wenzhou 325035, China. Tel: +86-577-86699523; Fax number: +86-577-86699523.
E-mail addresses:
[email protected] (X.J. Wang),
[email protected] (X.K. Li),
[email protected] (X.X. Yang),
[email protected] (Q. Hui),
[email protected] (B.J. Yu),
[email protected] (Zh. Huang),
[email protected](P.P. Zhou),
[email protected] (P.F. Wang),
[email protected] (Z.T. Wang),
[email protected] (S.C. Pang),
[email protected] (J.H. Li),
[email protected] (H.S. Wang)
[email protected] (L. Lin) # These authors contributed equally to this work.
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Abstract This study investigated the effect of the excipients, including glycine, mannitol, arginine, trehalose, sorbitol, and poloxamer188 on the stability of recombinant human fibroblast growth factor 21(FGF21) during the process of lyophilization and storage. The glass transition temperature (Tg), protein secondary structure, aggregation ratio and the bioactivity of lyophilized FGF21 were measured. We furthermore investigated the effect of FGF21 against ischemia cerebral injury using the middle cerebral artery occlusion (MCAO) model in rats. The ischemia cerebral injury of MCAO rats was analyzed via 2,3,5-triphenyltetrazolium chloride and Nissl-staining. Endoplasmic reticulum (ER) stress related proteins were detected via western blot. In this study, we found that aggregation was the primary mode of deterioration of lyophilized FGF21under accelerated storage conditions. Mannitol combined with trehalose and glycine formulations offers the most effective protein protection to reduce the aggregation. Administration of FGF21 protected cerebral ischemia and decreased ER stress related proteins in MCAO rats and PC12 cells.
Keywords: FGF21; formulation; lyophilization; protein stability; ischemia cerebral injury; ER stress
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Bioconjugate Chemistry
Introduction FGF21 is an important member of the fibroblast growth factor family, which is preferentially secreted by the liver and involved in both the glucose and lipid metabolisms.1-4 In combination with changes of FGF21 in the circadian rhythm, a recent study showed that FGF21 regulates the central nervous system and that intravenous injections of FGF21 can cross the blood-brain barrier, showing its potential as neuroprotective treatment.5 However, in contrast to other proteins, long-term research and clinical practice have shown FGF21 to have poor stability and short shelf-life, thus diminishing its therapeutic effect.6 Lyophilization is the most commonly used storage method for a therapeutic protein drug in the pharmaceutical industry to increase the long-term stability, and allow easy handling and storage of drug product.7-9 This process may be used to maintain their quality, physicochemical characteristics, performance, and low residual moisture content, as well as distribution and good stability of the drug product.10 Overall, the lyophilization process generally occurs in three sequential steps, freezing, primary drying and secondary drying. 11 Specifically, it consists in freezing the samples, and removing the water by sublimation and desorption under vacuum. The exact mechanism for the freezing during the lyophilization is not clear. But apparently during the freezing, ice crystals nucleate and grow, excluding the solutes in the formulation in which a phase separation or even crystallization may occur. The freezing step is also the most aggressive step in lyophilization and may possibly leads to protein aggregation. For instance, proteins suffer from a variety of unstable factors during lyophilization, such as cold shock, ice-water interfaces, pH changes, dehydration stress, easy aggregation, and chemical degradation during the process of lyophilization.8, 12-15 To stabilize the bioactivity of FGF21 during the process of preparation and storage, it is necessary to add protectants to the lyophilized formulation to
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protect them from freezing stresses.16,
17
The most common lyoprotectant have been considered,
including sugars/polyols, polymers, surfactants, amino acids, metal ions, and inorganic substances, 15, 18, 19
which influence the glass transition temperature of formulation to with low residual moisture content,
yielding better protein stability. Among these lyoprotectants, sugars (such as sucrose and trehalose) tend to produce an amorphous form, consequently enhancing protein stability during freeze-drying and storage.9,
20, 21
Polyols, such as mannitol, can not only be used as bulking agents, but also as
lyoprotectant in some formulations.19, 22, 23 The purpose of this work was to understand how various physicochemical properties affect the stability of lyophilized FGF21 as a model therapeutic protein, and assess the effect of freezing on structural stability of FGF21. We examined the protecting effect of various excipients on the deterioration behavior of lyophilized FGF21 at elevated storage temperature. Furthermore, the effect of FGF21 was investigated against ischemia cerebral injury, using the middle cerebral artery occlusion (MCAO) model in rats. Results and discussion Glass transition temperature Tg value is an important parameter of any lyophilized formulation. The Tgs of the five FGF21 formulations a to e were 51.36±0.75°C, 50.96±0.90°C, 51.56±0.09°C, 47.35±0.12°C, and 45.36±0.11°C, respectively. Tg values were higher for mannitol-containing formulations (formulation a, b and c) than of corresponding sorbitol-containing formulations (formulation d and e). The ingredients of five FGF21 formulations are shown in table 1. Protein secondary structure of lyophilized FGF21 formulations The secondary structure of lyophilized FGF21 in each formulation is shown in Figure 1. The range of α-helix contents of each of the formulations is: c (11.18±0.83%) > a (11.02±0.82%)> d
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Bioconjugate Chemistry
(10.86±1.18%) > b (10.38±0.83%) > e (10.29±1.10%) > control (9.97±1.10%), while the differences between each formulation and the control group was not significant (Figrue 1G, p>0.05, n=3). Moreover, the range of β-pleated sheet contents resulted in the following order: control sample (45.98±0.58%) > e (40.51±0.42%) > d (40.50±0.83%) > b (39.43±1.67%) > a (39.39±0.22%) > c (39.36±0.61%). There was a significant difference between control group and the five lyophilized FGF21 formulations (Figure 1H, p
