Levi Collin Nelemans, Vinicio Alejandro Melo, Matej Buzgo, Edwin Bremer, He was Simaite
Abstract
About 30% of the FDA approved drugs in 2021 were protein-based therapeutics. However, therapeutic proteins can be unstable and rapidly eliminated from the blood, compared to conventional drugs. Furthermore, on-target but off-tumor protein binding can lead to off-tumor toxicity, lowering the maximum tolerated dose. Thus, for effective treatment therapeutic proteins often require continuous or frequent administration. To improve protein stability, delivery and release, proteins can be encapsulated inside drug delivery systems. These drug delivery systems protect the protein from degradation during (targeted) transport, prevent premature release and allow for long-term, sustained release. However, thus far achieving high protein loading in drug delivery systems remains challenging. Here, the use of protein desolvation with acetonitrile as an intermediate step to concentrate monoclonal antibodies for use in drug delivery systems is reported. Specifically, trastuzumab, daratumumab and atezolizumab were desolvated with high yield (∼90%) into protein nanoparticles below 100 nm with a low polydispersity index (<0.2).
Introduction
Around 30% of the FDA approved drugs in 2021 were therapeutic proteins and their approval for clinical use has, since 2014 till 2021, accounted for an average of 27% [1]. Currently, most commercial protein therapeutics are administered intravenously or subcutaneously and, due to low protein half-lives in vivo, often require frequent injections. Rapid protein clearance from the blood is caused by multiple factors such as physicochemical instability and enzymatic degradation [2,3]. Furthermore, most therapeutic protein targets are ubiquitously expressed, which can lead to off-target cell toxicity [4,5]. Thus, drug delivery systems (DDS) for proteins that preserve protein activity, provide sustained release, and passive or active targeting and, thus, toxicity reduction, are in demand [6–9]. Despite tremendous progress, DDS for therapeutic monoclonal antibodies (mAbs) are not yet clinically available. One of the challenges is that mAbs are administered in large doses (usually in doses above 300 mg), and any additional excipients needed for a DDS considerably increase the volume of the injection. This leads to an even greater burden to the patient when injected intravenously and the large volume often prevents subcutaneous injection all together [10].
Materials and methods
Materials
All monoclonal antibodies (atezolizumab (tecentriq), cetuximab (erbitux), daratumumab (darzalex), elotuzumab (empliciti), rituximab (truxima), trastuzumab (herzuma)) were kindly provided by the pharmacy of the University Medical Center in Groningen (UMCG, Groningen, the Netherlands). Goat anti-human IgG biotin (PAB10694) was obtained from Abnova (Taipei, Taiwan). Human HER2 with his-tag (10126-ER) and recombinant human Fc gamma RIIIA alexa fluor 647 protein (AFR4325-020) were purchased from R&D systems (Minneapolis, United States). Tween-20 (polysorbate-20) (P9416) from Sigma (Saint Louis, United States). 1-step Turbo TMB-ELISA (34022), High sensitivity streptavidin-HRP (21130) and nickel-coated 96 wells plates (15442) were obtained from ThermoFisher (Waltham, United States). Finally, 100 kDa Spectra-Por Float-A-Lyzer G2 (734–3576), PBS 10x (K813), Acetonitrile (HiPerSolv, 83640), NaCl (GRP RECTAPUR, 27800), water (GRP RECTAPUR, 83612), and Sodium dodecyl sulphate (442442F) were purchased from VWR (Radnor, United States). OVCAR-3, ES-2, U2932, MDA-MB-231 and SK-BR-3 were purchased from the American Type Culture Collection (ATCC, Manassas, United States).
Results
Six commercially available mAbs were precipitated; atezolizumab (ATZ), cetuximab (CTX), daratumumab (DARA), elotuzumab (ELO), rituximab (RTX), and trastuzumab (TRA), and the influence of the process parameters on size, protein particle yield, and activity of the proteins was investigated. The known physicochemical properties of the antibodies, as well as the composition of their commercial formulations, are summarized in Table 1.
Discussion
Mechanism behind protein desolvation
The results presented in this paper demonstrate that therapeutic antibodies can be precipitated into nanoparticles using the desolvation method, by first dialyzing the antibodies in water with 1 mg/mL polysorbate-20. Furthermore, their particle size can be controlled by the addition of low concentrations of NaCl (<2 mM), while retaining binding activity after being redissolved.
Conclusions
The commercial antibodies trastuzumab, daratumumab and atezolizumab could be desolvated into protein nanoparticles with high yield (>80%) after dialysis in water with 1 mg/mL polysorbate-20. Furthermore, the formed protein nanoparticles could be redissolved in aqueous solutions without affecting binding activity. Finally, low concentrations of NaCl (0.5 to 2 mM) were used to precisely control mAb particle size, without influencing mAb activity.
Acknowledgments
We would like to thank Prof. Leonid Gurevich from Aalborg University, Denmark for reviewing the first draft of this paper and Jimena Alvarez Freile, Lisa Jacob and Yuzhu Qi for performing the experiments for the revisions.
Citation: Nelemans LC, Melo VA, Buzgo M, Bremer E, Simaite A (2024) Antibody desolvation with sodium chloride and acetonitrile generates bioactive protein nanoparticles. PLoS ONE 19(3): e0300416. https://doi.org/10.1371/journal.pone.0300416
Publisher: AM Abd El-Aty, Cairo University, EGYPT
Received: September 27, 2023; Accepted: February 26, 2024; Published: March 14, 2024
Copyright: © 2024 Nelemans et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: Supported by a grant from the Marie Sklodowska-Curie European Union grant agreement 813871 I-DireCT – H2020-MSCAITN-2018. https://cordis.europa.eu/project/id/813871 The funders did not and will not have a role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
Competing interests: The authors have declared that no competing interests exist.
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0300416#abstract0
