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. 2023 Jan 30;21(1):e134282.
doi: 10.5812/ijpr-134282. eCollection 2022 Dec.

Application of SPRA Technology for Delivery of Erythropoietin: Stability Evaluation of Conjugated Erythropoietin with Adamantane and in SPRA Inclusion Complex

Bahareh Alizadeh  1 , Afshin Zarghi  2 , Arash Mahboubi  1   3 , Reza Aboofazeli  1   4
Affiliations

Application of SPRA Technology for Delivery of Erythropoietin: Stability Evaluation of Conjugated Erythropoietin with Adamantane and in SPRA Inclusion Complex

Bahareh Alizadeh et al. Iran J Pharm Res. .

Abstract

Background: As a widely used therapeutic protein, recombinant human erythropoietin (rhEPO) is currently one of the most effective biopharmaceuticals on the market for the treatment of anemia in patients with chronic renal disease. Increasing in vivo rhEPO half-life and its bioactivity is a significant challenge. It was hypothesized that the application of self-assembly PEGylation retaining activity, named supramolecular (SPRA) technology, could prolong the protein half-life without a significant loss of bioactivity.

Objectives: This study aimed to assess the stability of rhEPO during synthetic reactions, including the conjugation with adamantane and the formation of the SPRA complex. To do this, the secondary structure of the protein was also evaluated.

Methods: FTIR, ATR-FTIR, Far-UV-CD, and SDS-PAGE methods were employed. Thermal stability studies of SPRA-rhEPO complex and rhEPO were investigated at 37°C for ten days using a nanodrop spectrophotometer.

Results: The secondary structure of lyophilized rhEPO, AD-rhEPO, and rhEPO (pH 8) was compared to rhEPO. Results showed that the secondary structure of the protein was unaffected by lyophilization, pH change, and the formation of covalent bonds in conjugation reaction. SPRA-rhEPO complex was also stable for seven days in phosphate buffer (pH 7.4) at 37°C.

Conclusions: It was concluded that the stability of rhEPO could increase by complexation using SPRA technology.

Keywords: Erythropoietin; Host-guest Interaction; Poly (Ethylene Glycol); SPRA Chemistry; Stability; Supramolecular; β-Cyclodextrin.

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Conflict of interest statement

Conflict of Interests: The authors declared that there is no conflict of interest.

Figures

Figure 1.
Figure 1.. Pathways for the synthesis of (A) activated AD, (B) AD-rhEPO conjugate, (C) mPEG-β-CD, and (D) SPRA-rhEPO inclusion complex (mPEG-β-CD-AD-rhEPO).
Figure 2.
Figure 2.. SDS-PAGE results of (A) rhEPO; (B) rhEPO at pH 8, and AD-rhEPO.
Figure 3.
Figure 3.. ATR-FTIR spectra of (A) rhEPO and (B) lyophilized rhEPO.
Figure 4.
Figure 4.. FTIR spectra of the activated AD, rhEPO, and AD-rhEPO conjugate.
Figure 5.
Figure 5.. Secondary structure changes of rhEPO and AD-rhEPO. CD spectra were obtained at room temperature and a protein concentration of 0.2 mg/mL.
Figure 6.
Figure 6.. Thermal stability of rhEPO, AD-rhEPO, and SPRA-rhEPO complex in phosphate buffer (pH 7.4) at 37°C for 7 days (Mean ± SD, n = 3. SD value are not presented).
See this image and copyright information in PMC

References

    1. Higashi T, Iohara D, Motoyama K, Arima H. Supramolecular pharmaceutical sciences: A novel concept combining pharmaceutical sciences and supramolecular chemistry with a focus on cyclodextrin-based supermolecules. Chem Pharm Bull (Tokyo). 2018;66(3):207–16. doi: 10.1248/cpb.c17-00765. - DOI - PubMed
    1. Khameneh B, Saberi MR, Hassanzadeh-Khayyat M, Mohammadpanah H, Ghandadi M, Iranshahi M, et al. Evaluation of physicochemical and stability properties of human growth hormone upon enzymatic PEGylation. J Appl Biomed. 2016;14(4):257–64. doi: 10.1016/j.jab.2016.06.002. - DOI
    1. Moncalvo F, Martinez Espinoza MI, Cellesi F. Nanosized delivery systems for therapeutic proteins: Clinically validated technologies and advanced development strategies. Front Bioeng Biotechnol. 2020;8:89. doi: 10.3389/fbioe.2020.00089. - DOI - PMC - PubMed
    1. Yin L, Chen X, Vicini P, Rup B, Hickling TP. Therapeutic outcomes, assessments, risk factors and mitigation efforts of immunogenicity of therapeutic protein products. Cell Immunol. 2015;295(2):118–26. doi: 10.1016/j.cellimm.2015.03.002. - DOI - PubMed
    1. Duncan R. The dawning era of polymer therapeutics. Nat Rev Drug Discov. 2003;2(5):347–60. doi: 10.1038/nrd1088. - DOI - PubMed

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