Review article (accepted manuscript): Ionic liquids and deep eutectic solvents for the stabilization of biopharmaceuticals: A review This is the accepted manuscript (after peer-review) version of the following review article: Nathalia Vieira Porphirio Veríssimo, Cassamo Usemane Mussagy, Heitor Buzetti Simões Bento, Jorge Fernando Brandão Pereira, Valéria de Carvalho Santos-Ebinuma, Ionic liquids and deep eutectic solvents for the stabilization of biopharmaceuticals: A review, Biotechnology Advances 71, 2024, 108316, DOI: j.biotechadv.2024.108316, which has been published in final form at https://doi.org/10.1016/j.biotechadv.2024.108316. This article may be used for non-commercial purposes in accordance with Elsevier policies for use of self-archived versions. [https://www.elsevier.com/about/policies/sharing]. © 2024 Elsevier B.V. All rights reserved. © <2024>. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ 2 Review Ionic liquids and deep eutectic solvents for the stabilization of biopharmaceuticals: A review Nathalia Vieira Porphirio Veríssimo 1,2*, Cassamo Usemane Mussagy 3, Heitor Buzetti Simões Bento 1, Jorge Fernando Brandão Pereira 4, and Valéria de Carvalho Santos-Ebinuma 1,* 1Department of Bioprocess Engineering and Biotechnology, School of Pharmaceutical Sciences, São Paulo State University, CEP: 14801-902, Araraquara SP, Brazil. NVPV: nathalia.v.santos@unesp.br; HBSB: heitor.bento@unesp.br; VCSE: valeria.ebinuma@unesp.br 2Department of Pharmaceutical Sciences, School of Pharmaceutical Sciences, São Paulo University, CEP: 14040-020, Ribeirão Preto, SP, Brazil. 3Escuela de Agronomía, Facultad de Ciencias Agronómicas y de los Alimentos, Pontificia Universidad Católica de Valparaíso, Quillota 2260000, Chile. cassamo.mussagy@pucv.cl 4Univ Coimbra, CIEPQPF, Department of Chemical Engineering, Pólo II – Pinhal de Marrocos, 3030-790 Coimbra, Portugal. jfbpereira@eq.uc.pt *Shared correspondence. Correspondence and requests for materials should be addressed to Nathalia Vieira Porphírio Veríssimo – E- mail: nathalia.v.santos@unesp.br or Valéria de Carvalho Santos-Ebinuma – E-mail: valeria.ebinuma@unesp.br. Universidade Estadual Paulista (UNESP), School of Pharmaceutical Sciences, Campus (Araraquara), Department of Engineering Bioprocess and Biotechnology, Rodovia Araraquara-Jaú/ Km 01, Campos Ville - Araraquara/SP 14800-903 - Araraquara - SP/Brazil. Phone: Phone: 55-16-3301- 4647. 3 Abstract Biopharmaceuticals have allowed the control of previously untreatable diseases. However, their low solubility and stability still hinder their application, transport, and storage. Hence, researchers have applied different compounds to preserve and enhance the delivery of biopharmaceuticals, such as ionic liquids (ILs) and deep eutectic solvents (DESs). Although the biopharmaceutical industry can employ various substances for enhancing formulations, their effect will change depending on the properties of the target biomolecule and environmental conditions. Hence, this review organized the current state-of-the-art on the application of ILs and DESs to stabilize biopharmaceuticals, considering the properties of the biomolecules, ILs, and DESs classes, concentration range, types of stability, and effect. We also provided a critical discussion regarding the potential utilization of ILs and DESs in pharmaceutical formulations, considering the restrictions in this field, as well as the advantages and drawbacks of these substances for medical applications. Overall, the most applied IL and DES classes for stabilizing biopharmaceuticals were cholinium-, imidazolium-, and ammonium-based, with cholinium ILs also employed to improve their delivery. Interestingly, dilute and concentrated ILs and DESs solutions presented similar results regarding the stabilization of biopharmaceuticals. With additional investigation, ILs and DESs have the potential to overcome current challenges in biopharmaceutical formulation. Keywords: proteins, nucleic acid, neoteric solvents, stability of biomolecules, pharmaceutical formulations, preservatives, biopharmaceuticals, ILs, DESs, protein stability 4 Abbreviations Ionic liquids (ILs) and Deep Eutectic Solvents (DESs) Ammonium-based ILs tetrabutylammonium bromide ([N₄,₄,₄,₄]Br) triethylammonium phosphate ([N0,2,2,2]PO₄) triethylammonium sulfate ([N0,2,2,2]SO₄) trimethylammonium acetate ([N0,1,1,1][CH₃COO]) trimethylammonium dihydrogen phosphate ([N0,1,1,1]H₂PO₄) trimethylammonium hydrogen sulfate ([N0,1,1,1]HSO₄) Ammonium IL-Robed siRNA benzyldimethyloctylammonium robed-siRNA1 ([N1,1,(Bz),8]–siRNA1) benzyldimethylstearylammonium robed-siRNA1 ([N1,1,(Bz),18]–siRNA1)] benzyldimethyltetradecylammonium robed-siRNA1 ([N1,1,(Bz),14]–siRNA1) Cholinium-based DESs choline chloride and ethylene glycol ([Ch]Cl–[(CH₂OH)₂]) choline chloride and glycerol ([Ch]Cl–[C₃H₅(OH)₃]) choline chloride and carbamide ([Ch]Cl–[CO(NH₂)₂]) cholinium geranate 1:2 ([Ch][C₉H₁₅COO]–[C₉H₁₅COOH]) cholinium geranate 1:4 ([Ch][C₉H₁₅COO]–[C₉H₁₅COOH]₃) cholinium hydroxyacetic acid 1:2 ([Ch][HOCH₂COO]–[HOCH₂COOH]) cholinium hydroxyacetic acid 2:1 ([Ch][HOCH₂COO]–[Ch]) Cholinium-based ILs cholinium acetate ([Ch][CH₃COO]) cholinium butanoate ([Ch][C₃H₇COO]) cholinium citrate ([Ch][C₅H₇O₅COO]) cholinium dihydrogen phosphate ([Ch]H₂PO₄) cholinium geranate ([Ch][C₉H₁₅COO]) cholinium hydroxyacetate ([Ch][HOCH₂COO]) cholinium hexanoate ([Ch][C₅H₁₁COO]) cholinium indole-3-acetate ([Ch][C₉H₈NCOO]) cholinium L-argininate ([Ch][Arg]) cholinium L-asparaginate ([Ch][Asn]) cholinium dodecanoate ([Ch][C₁₂:0]) cholinium L-glutaminate ([Ch][Gln]) 5 cholinium L-lysinate ([Ch][Lys]) cholinium cis-9-octadecenoate ([Ch][C₁₈:₁]) cholinium phenylpropanoate ([Ch][PhC₂H₅COO]) cholinium propanoate ([Ch][C₂H₅COO]) cholinium 2-oxopropanoate ([Ch][CH₃COCOO]) dicholinium L-asparaginate ([Ch]₂[Asn]) dicholinium L-glutaminate ([Ch]₂[Gln]) Phosphonium-based ILs tetrabutylphosphonium bromide ([P₄,₄,₄,₄]Br) Imidazolium-based ILs 1-butyl-3-methylimidazolium acetate ([C₄MIm][CH₃COO]) 1-butyl-3-methylimidazolium bromide ([C₄MIm]Br) 1-butyl-3-methylimidazolium chloride ([C₄MIm]Cl) 1-butyl-3-methylimidazolium dicyanamide ([C₄MIm][N(CN)₂]) 1-butyl-3-methylimidazolium hydrogen sulfate ([C₄MIm]HSO₄) 1-butyl-3-methylimidazolium iodine ([C₄MIm]I) 1-butyl-3-methylimidazolium methanesulfonate ([C₄MIm[CH₃SO₃]) 1-butyl-3-methylimidazolium nitrate ([C₄MIm]NO₃) 1-butyl-3-methylimidazolium thiocyanate ([C₄MIm][SCN]) 1-butyl-3-methylimidazolium tosylate ([C₄MIm][CH₃BzSO₃]) 1-butyl-3-methylimidazolium tricyanomethanide ([C₄MIm][C(CN)₃]) 1-butyl-3-methylimidazolium trifluoroacetate ([C₄MIm][CF₃COO]) 1-decyl-3-methylimidazolium acetate [C₁₀MIm][CH₃COO] 1-dodecyl-3-methylimidazolium acetate ([C₁₂MIm][CH₃COO]) 1-ethyl-3-methylimidazolium bromide ([C₂MIm]Br) 1-ethyl-3-methylimidazolium acetate ([C₂MIm][CH₃COO]) 1-hexyl-3-methylimidazolium acetate ([C₆MIm][CH₃COO]) 1-octyl-3-methylimidazolium acetate ([C₈MIm][CH₃COO]) Lidocainum-based IL lidocainum etodolac IL, Ionic Liquid Transdermal System from MEDRx (ILTS®) Poly(vinyl pyrrolidone)-based DES poly(vinyl pyrrolidone) and propanedioic acid 1:1 ([(C6H9NO)n]–[CH₂(COOH)₂]) Other active pharmaceutical ingredients (APIs) 6 adenine (A) analytical ultracentrifugation (AUC) entropy change (ΔS) Gibbs Free Energy change (ΔG) circular dichroism (CD) compound annual growth rate (CAGR) cryogenic electron microscopy (Cryo-EM) cytosine (C) deoxyribonucleic acid (DNA) differential scanning calorimetry (DSC) differential scanning fluorimetry (DSF) European Union (EU) Fourier transform infrared (FTIR) generally recognized as safe (GRAS) glucagon-like peptide 1 (GLP-1) grand average hydrophobicity index (GRAVY) guanine (G) half-life (t1/2) hydrogen bond acceptor (HBA) hydrogen bond donor (HBD) immunoglobulin G1 (IgG1) instability index (II) interleukin-2 (IL-2) ionic liquids (ILs) isothermal titration calorimetry (ITC) L-Asparaginase (L-ASNase) lysozyme (Lys) nuclear resonance spectroscopy (NMR) melting enthalpy (∆Hm) melting temperature (Tm) molecular dynamics (MD) ovalbumin epitope (OVA) protein-protein interactions (PPIs) ribonucleic acid (RNA) 7 SHAPE (Selective 2'-Hydroxyl Acylation analyzed by Primer Extension) small interfering RNA (siRNA) molecular weight (MW) thermal shift assay (TSA) thymine (T) ultraviolet (UV) uracil (U) α-chymotrypsin (CT) 8 1. Introduction Biopharmaceuticals have allowed the successful treatment of illnesses with low recovery rates, such as cancers, autoimmune diseases, and metabolic disorders (Rasmussen et al., 2021). These compounds can include a wide variety of biomolecules based on amino acids and nucleic acids (Walsh, 2013), such as peptides, proteins, DNA, and RNA derivates. Their effectiveness has led to a growing demand, confirmed by a compound annual growth rate (CAGR) of 9.3% between 2016 and 2024, with an expected value of USD 405 billion by 2024 (Kim et al., 2022). However, due to their high prices and low stability outside of cold chains, most biopharmaceuticals are still inaccessible to low-income countries and communities (Ferrari, 2022). For example, many bioactive macromolecules are degraded or have poor absorption and bioavailability in vivo even when presenting pharmacological action in vitro (Manning et al., 2010). The inherent instability of biopharmaceuticals when outside their natural environment may cause limitations not only in their medical application, but also in their production, storage, and transportation. Therefore, the development of new formulations to stabilize and enhance the delivery of biopharmaceuticals can potentially expand their applications and improve their access in marginalized communities. Several strategies have been applied to enhance the stability of biopharmaceuticals, such as the use of neoteric “green” solvents as additives in their formulations. For example, different classes of ionic liquids (ILs) and deep eutectic solvents (DESs) can be employed to improve the stability and delivery of biomolecules such as proteins and nucleic acids (Egorova et al., 2021; Veríssimo et al., 2022). Thus, the development of novel green solvent formulations for biopharmaceuticals may lead to more efficient, sustainable, and environmentally friendly production and application of biologics (Dhiman et al., 2023). To elucidate the advances in this field, this review will organize the state-of-the-art on the use of ILs and DESs for improving the stability and delivery of biopharmaceuticals. We will demonstrate the trends and knowledge gaps in this area and provide a perspective on the use of biocompatible ILs and DESs as additives in biopharmaceutical formulations. Firstly, we will present the main classes of biopharmaceuticals, their types of stability, and how to assess them. Then, we will discuss the properties of ILs and DESs, their biocompatibility, and their potential for application in biological systems. We will then demonstrate the effects of ILs and DESs on protein and nucleic acid biopharmaceuticals by examining their effects on individual biopharmaceuticals, followed by a debate regarding their overall use to stabilize and enhance the delivery of biotechnological medicines. As concluding remarks, we will provide our expert opinion concerning the trends and opportunities in the field, along with limitations and challenges to overcome. 2. Biopharmaceuticals Biopharmaceuticals are generally defined as medicines based on amino acid and nucleic acid and produced by biotechnological processes (Walsh, 2013). Therefore, small organic molecules like penicillin- 9 based antibiotics (e.g., benzylpenicillin) are not considered biopharmaceuticals even if obtained through biotechnological means, being classified as pharmaceuticals. Additionally, biological products such as donated blood and organs are not considered biopharmaceuticals as they are not obtained through biotechnological methods. However, there is still no unified definition for biopharmaceuticals by regulatory agencies such as the U.S. Food and Drug Administration (FDA) or the European Medicines Agency (EMA), which regulates them at times in the broader category of biological products and others with traditional pharmaceuticals (EMA, 2023; FDA, 2017). Regarding their constitution, amino acid-based biopharmaceuticals include peptides, proteins, and glycosylated peptides and proteins, while nucleic-based are mostly comprised of DNA, RNA (e.g., siRNA, mRNA), viral vectors, antisense oligonucleotides, aptamers, and ribozymes. As for their classes of application, the most relevant include vaccines, anticancer biopharmaceuticals, gene therapies, genome editing biopharmaceuticals, antisense therapy, enzymes, hormones, monoclonal antibodies, blood product derivatives, cytokines and interferons, growth factors, and fusion proteins (Ho, 2013). Historically, the origins of biopharmaceuticals can be traced back to the therapeutic use of insulin sourced from animal pancreas in the 1920s (Alyas et al., 2021). While this naturally-derived insulin served as a precursor to modern biopharmaceuticals, the true era of biotechnological medicines began with the development of recombinant DNA technology. The first biopharmaceutical, a recombinant human insulin developed using Genentech's techniques and then commercialized as 'Humulin' by Eli Lilly, was approved by the FDA in 1982 (Nielsen, 2013). As biotechnological techniques matured, the late 20th and early 21st centuries saw the rise of monoclonal antibodies, effectively targeting specific disease agents and mechanisms. The evolution of biopharmaceuticals reached a new frontier with the advent of nucleic acid- based therapies, with the first nucleic acid-based drug (“Glybera”, from uniQure) approved for use in the European Union (EU) in 2012 (Kesik‐Brodacka, 2018; Moran, 2012). However, despite the success of biopharmaceuticals in treating complex diseases, their unstable nature and vulnerability to denaturation still limit their widespread therapeutical application (Lin et al., 2021). Biopharmaceuticals have significantly greater molecular weight (MW) than traditional pharmaceuticals based on small organic compounds (e.g., MW of 5,734 g/mol for human insulin and 334 g/mol for benzylpenicillin, respectively), with more complex intra and intermolecular interactions required to maintain their structural integrity. Although their intricate nature can improve their therapeutical effectiveness and specificity, this characteristic can also lead to higher sensitivity to adverse environmental conditions and 10 increased inactivation or degradation during their clinical use. Moreover, these substances can exhibit complex mechanisms of action and are potentially immunogenic, which hinders their study and application (Kesik‐Brodacka, 2018; Molowa and Mazanet, 2003). In this sense, research regarding the use of ILs and DESs as greener and biocompatible stabilizer additives in the composition of biopharmaceuticals has demonstrated that they can be an alternative to the limitations of biomolecules. For example, ILs and DESs can enhance drug delivery, increase stability, and confer protection to bioactive compounds, which are valuable combinations in pharmaceutical formulations (Wang et al., 2022). Before discussing in detail the application of ILs and DESs in biopharmaceuticals, we will explore the relevant parameters and methods associated with the stability of macromolecules and the conventional strategies applied by the pharmaceutical industry to stabilize complex macromolecules. 3. Stability of biopharmaceuticals To comprehend the stability of biopharmaceuticals, it is necessary to understand their composition, structure, and the different parameters and methods that can be employed to determine their preservation. Proteins, peptides, and nucleic acids are intricate and diverse systems (Nelson and Cox, 2012). The structure of proteins and peptides is comprised of a chain of amino acids while nucleic acids are composed of a nucleotide sequence. Both proteins and nucleic acids will arrange their chains in complex three-dimensional arrangements that can be divided into four organizational levels. It should be noted that not all proteins, peptides, and nucleic acids present all four organizational levels, as their complexity varies depending on their size and function. To demonstrate these different levels, Figure 1 presents a summary of protein and nucleic acid structures. 11 Figure 1. Summary of protein, peptide, and nucleic acid structures (primary, secondary, tertiary, and quaternary). Sources: Protein Data Bank (PDB) IDs 1GFL, 1MKO, and 7VDV. Images of the proteins were produced with the PDB structures using UCSF Chimera 1.14 (Berman et al., 2002; Pettersen et al., 2004). Secondary and tertiary structures of nucleic acids from Thomas Shafee (Creative Commons 4.0) (Shafee, 2017; Thomas Shafee, 2016). As presented in Figure 1, proteins are composed of amino acids, which are organic molecules with amino and carboxylic acid functional groups. The primary structure of proteins comprises a polypeptide chain formed by amino acids linked by peptide bonds (Nelson and Cox, 2012). Its secondary structure includes the interactions of the polypeptides in motifs such as α-helix, β-sheets, and coils. As for the tertiary structure, it comprehends the three-dimensional folding of the protein structure, while the quaternary corresponds to the packing of distinct subunits of the protein (Voet and Voet, 2021). The structural levels of amino acids follow the same logic as the proteins but with differences in their composition and arrangement. Nucleic acids are composed of nucleotides, a base containing nitrogen [i.e., adenine (A), guanine (G), cytosine (C) in ribonucleic acid (RNA) and deoxyribonucleic acid (DNA); uracil 12 (U) in RNA; thymine (T) in DNA], a sugar (i.e., ribose in RNA; deoxyribose in DNA), and one phosphate (Neidle and Sanderson, 2021). Their primary structure is formed by the connection of nucleotides by phosphodiester bonds linking the oxygens between different nucleotides in carbons 5’ to 3’. The secondary structure of nucleic acids is formed by the specific interaction between the purine (i.e., A, G) and pyrimidine bases (i.e., C, U, T). These base pairings will lead to the formation of structures such as the helices (contiguous base pairs), stem-loops, and pseudoknots (unpaired nucleotides surrounded by helices). While the interactions of bases will form the secondary structure of DNA and RNA, their three-dimensional arrangement will determine its tertiary structure. Especially for DNA, their secondary and tertiary structures are closely related. DNA can take double-helical forms such as A-DNA, B-DNA, and Z-DNA, that differ based on the number of base pairs per turn, right- or left-handed helix, length of the helix, and size between minor and major grooves. As for RNA, it can form double helices, major and minor groove triplexes, quadruplexes, helical stacking, and other arrangements. Finally, their quaternary structure involves the interaction of different nucleic acids (e.g., the RNA enzyme Varkud satellite ribozyme) or between nucleic acid and protein (e.g., DNA and histones to form nucleosomes). Considering their complex nature, the structure of proteins and nucleic acids is not static, and environmental conditions will alter their conformation and can even denature them (i.e., irreversibly alter their structural arrangements). The conditions include changes in temperature, pH, ionic strength, pressure, enzymes, and chemical and organic substances (Manning et al., 2010; Neidle and Sanderson, 2021). Because the activity and behavior of biopharmaceuticals are directly related to the integrity of their macromolecular arrangements, it is necessary to characterize and monitor their structure and interactions to assure their security and effective application. However, multiple parameters can be evaluated to determine protein stability. Table 1 compiles the main parameters to evaluate the stability of protein, peptides, and nucleic acids, namely the four levels of structure of proteins, thermodynamic parameters, and activity. It also includes the most common techniques used to determine each type of stability and the metrics evaluated for each parameter. Table 1. Level of stability, composition, and recurrent evaluation methods for the different levels of proteins, peptides, and 1 nucleic acids structure. 2 Stability type Overall stability Composition Evaluation methods Proteins and peptides Primary structure Very high Amino acid chain. Amino acid sequencing (e.g., mass spectrometry, Edman degradation), electrophoresis, western blotting, chromatography, protein half-life. Secondary structure High to intermediary Local interactions of the polypeptide backbone (e.g., α- helix, β-sheet, random coil). CD, infrared spectroscopy. Tertiary structure Intermediary to low Three-dimensional folding of the protein structure. Direct: X-ray crystallography, neutron and X-ray scatterings, NMR, dual polarization interferometry, Cryo-EM. Indirect: fluorescence and absorbance (e.g., intrinsic UV-absorbance and fluorescence of proteins, proteins with chromophores and fluorophores, fluorescence labeling and probes), dynamic and static light scattering and AUC (protein aggregation and oligomeric state), Raman spectroscopy, computational modeling. Quaternary structure Low Packing of different subunits of the protein. Thermal Variable Thermodynamic parameters (e.g., Tm, ∆Hm, ΔS, ΔG). DSF, ITC, TSA, and DSC. CD and infrared spectroscopies associated with controlled heating. Activity Variable Varies depending on its intended application. E.g., enzymes: kinetic parameters; biosensors: absorbances and fluorescence; vaccines: potency, immunogenicity, and immunomodulation; monoclonal antibodies: binding. Nucleic acids Primary High Nucleotide sequence. DNA and RNA sequencing, microarrays, southern blots, and in situ hybridization, fluorescence-based assays with probes. Secondary Intermediary Interactions between bases (e.g., DNA and RNA: double helix; RNA: stem-loops, pseudoknots). CD, infrared spectroscopy, SHAPE assay. Tertiary Intermediary to low 3D folding of the nucleic acid chains. DNA: A-form, B-form, Z-form; RNA: e.g., helical duplexes, triple-stranded structures. Direct: X-ray crystallography, neutron and X-ray scatterings, NMR, Cryo-EM. Indirect: electrophoresis and chromatography (size determination), fluorescence (probes and labeling), UV absorbance (usually, λ 260 nm), computational modeling. Quaternary Low Higher level of organization of nucleic acids (e.g., DNA: chromatin; RNA: interaction of RNA units in the ribosome). Thermal Easily variable Thermodynamic parameters (e.g., Tm, ∆Hm, ΔS, ΔG). ITC, DSF, DSC. CD, UV, and infrared spectroscopies associated with controlled heating. Activity Easily variable Varies depending on its intended application. E.g., viral vectors: transduction efficiency; vaccines: potency, immunogenicity, and immunomodulation. 14 For proteins and peptides, their primary structure is highly resistant to stress, with 3 only extreme conditions or enzymes breaking its peptide bonds (Bischof and He, 2006). 4 Its evaluation consists of the direct sequence of their amino acid chain (e.g., mass 5 spectroscopy alone or combined with chromatography (Callahan et al., 2020), Edman 6 degradation (Zhou et al., 2012) or indirectly estimating alterations by assessing its size 7 with electrophoresis, western blotting, and chromatography or verifying its half-life 8 (Deller et al., 2016). 9 The secondary structure of proteins and peptides is still considerably resistant to 10 alterations but to a lesser degree than the primary. Conventional methods to evaluate it 11 include circular dichroism (CD) and infrared spectroscopies, such as Fourier transform 12 infrared (FTIR) and 2D-infrared (Greenfield, 2006; Kong and Yu, 2007), which will 13 estimate the proportions of secondary protein motifs chains (e.g., α-helix, β-sheet, 14 random coil). It should be noted that certain methods to evaluate the tertiary structure of 15 proteins can also provide information on their secondary forms, as we are listing the 16 most prominent methods for each case. 17 The tertiary and quaternary protein structures are more susceptible to alterations 18 due to their environment. The techniques for the tertiary structure need to demonstrate 19 the folding of the protein or suggest alterations to its 3D structure. As for the quaternary 20 structure, they should indicate changes in the protein oligomeric state. Overall, the 21 techniques to determine the tertiary and quaternary structure of proteins overlap. Direct 22 methods include nuclear magnetic resonance spectroscopy (NMR), X-ray 23 crystallography, dual polarisation interferometry, cryogenic electron microscopy (Cryo-24 EM), and neutron and X-ray scatterings (Alberts et al., 2002; Ilari and Savino, 2008; 25 Kikhney and Svergun, 2015; Milne et al., 2013; Petoukhov and Svergun, 2007; Swann 26 et al., 2004). To indirectly verify the tertiary structure of proteins, it is possible to assess 27 alterations in the absorbances and fluorescence of proteins in the case of proteins with 28 chromophores in their structure, such as fluorescent proteins or proteins with 29 fluorescent amino acid residues, such as tyrosine, tryptophan, and phenylalanine (dos 30 Santos et al., 2020, 2019). Even if the protein has no intrinsic fluorescence, conjugates 31 like fluorescein can be added to recombinant proteins or it is possible to evaluate the 32 interaction of the protein with fluorescence probes (Toseland, 2013). It is also possible 33 to monitor alterations to the oligomeric quaternary state of the protein or verify 34 aggregations by size exclusion chromatography, electrophoresis, dynamic and static 35 light scattering, and analytical ultracentrifugation (AUC) (Ahrer et al., 2003; Deller et 36 15 al., 2016; Liu et al., 2006). There are also in silico models such as molecular dynamics 37 (MD) and molecular docking, homology modeling, and protein-protein interactions 38 (PPIs) targets that allow the prediction of protein structure, stability, and interactions 39 with other molecules (Lee et al., 2017; Watson et al., 2005; Xiang, 2006; Xu et al., 40 2008). 41 The thermal stability of proteins is a property that can easily vary depending on the 42 environment of the protein. To assess it, the main indicator is the melting temperature 43 (Tm) i.e., the temperature of denaturation; however, there are other thermodynamic and 44 kinetic parameters such as activation energy, melting enthalpy (∆Hm), change in entropy 45 (ΔS), change in Gibbs free energy (ΔG) and also half-life (t1/2). The Tm of proteins can 46 be directly determined by differential scanning calorimetry (DSC), isothermal titration 47 calorimetry (ITC), and differential scanning fluorimetry (DSF, also called thermal shift 48 assay - TSA) (Bischof and He, 2006; Deller et al., 2016). However, by monitoring the 49 alterations of the secondary structure of proteins under controlled heat, it is also 50 possible to discover Tm. From the curves obtained by different techniques and related to 51 the concentration or amount of proteins as a function of the independent variable such 52 as temperature for thermal denaturation curves the other thermodynamic parameters can 53 be determined (Bischof and He, 2006; Schellman, 1987). Moreover, different models 54 can be used to estimate the thermodynamic parameters. As for the determination of the 55 biological activity of proteins, the methods will vary according to their intended 56 applications. For example, for protein vaccines, it is possible to measure their potency, 57 immunogenicity, and immunomodulation, for biosensors their absorbance and 58 fluorescence can be acquired, for monoclonal antibodies their binding can be tested, and 59 the kinetic parameters can be used to verify the activity of enzymes (dos Santos et al., 60 2020; Iyer and Ananthanarayan, 2008; Schofield, 2009; Veríssimo et al., 2021). 61 For nucleic acids, their primary structure also presents high stability. Their 62 integrity can be determined by gene sequencing or other methods to evaluate specific 63 nucleic acid sequences, such as microarrays, southern blots, and in situ hybridization 64 (Brown, 1993; Dorado et al., 2021; Jensen, 2014; Stoughton, 2005). The evaluation of 65 the secondary structure of nucleic acids will quantify their structural motifs, such as 66 double helices for DNA and RNA, and stem-loops and pseudoknots for RNA. Similar to 67 proteins, the methods employed will be CD and infrared spectroscopies (Kypr et al., 68 2009; Sosnick et al., 2000; Tsuboi, 1970). 69 16 The tertiary and quaternary structures of nucleic acids have overall lower stability 70 than proteins, and the determination of their higher-level structure was challenging. The 71 traditional methods to directly assess tertiary and quaternary structures of a nucleic acid 72 include X-ray crystallography (Lin et al., 2011), neutron and X-ray scatterings (Oliver 73 et al., 2019), and NMR (Liu et al., 2021). However, cryo-EM is a fairly recent method 74 that has allowed the determination of novel structural forms of nucleic acids (Ma et al., 75 2022). Hence, even more motifs for nucleic acids may be found soon. There are also 76 indirect methods to estimate changes to the tertiary and quaternary structures of nucleic 77 acids, such as electrophoresis and chromatography (size determination) (Largy and 78 Mergny, 2014; Wei et al., 2022), fluorescence (probes and labeling) (Juskowiak, 2011; 79 Michel et al., 2020), UV absorbance (usually, at a λ of 260 nm) (Barbas et al., 2007), 80 and computational modeling (Feng et al., 2022; Ponce-Salvatierra et al., 2019). 81 The thermal stability of nucleic acids can be evaluated by similar thermodynamic 82 parameters to proteins, such as Tm, ∆Hm, ΔS, and ΔG (Rozners et al., 2015). The Tm can 83 also be determined but some of the same methods, such as ITC, DSF, and DSC, or an 84 association of controlled heating and CD, UV, or infrared spectroscopies (Rozners et 85 al., 2015; Silvers et al., 2015). Their activity will also depend on the intended 86 application of the biopharmaceutical. For example, you can test nucleic acid vaccine 87 stability by verifying its potency, immunogenicity, and immunomodulation with in vivo 88 or in vitro studies (Chavda et al., 2021; Chen et al., 2022), and viral vectors by their 89 transduction efficiency (Chen et al., 2018). 90 As presented in this section, there are multiple parameters and methods used to 91 evaluate the stability of biopharmaceuticals. When designing an experiment to assess or 92 improve biological drugs, it is necessary to consider its intended application, as certain 93 formulations can increase one type of stability to the detriment of another. For example, 94 2.4 M of cholinium dihydrogen phosphate ([Ch]H₂PO₄) improves the thermal stability 95 of lysozyme (Lys) to the detriment of its activity (Weaver et al., 2012), while 0.5 M of 96 N-butylpyridinium chloride has the opposite effect (Yamamoto et al., 2011). Moreover, 97 the same IL can enhance the stability of one specific biopharmaceutical and impair 98 another. For instance, around 0.3 - 0.5 M of 1-butyl-3-methylimidazolium acetate 99 ([C₄MIm][CH₃COO]) improves the structural stability of insulin (Todinova et al., 2016) 100 but will decrease it for α-chymotrypsin (CT) (Kumar et al., 2015). Concentration range 101 is also relevant when developing the stabilizing formulations, as the same IL can have 102 different effects on the same biopharmaceutical depending on the concentration range, 103 17 such as [Ch]H₂PO₄ maintaining the structural integrity of lysozyme (Lys) at 1.2 M but 104 decreasing it at 2.4 M (Weaver et al., 2012). With this in mind, the next section will 105 discuss the most prevalent problems related to the instability of biopharmaceuticals and 106 briefly explore the uses and limitations of conventional additives and solvents for the 107 stabilization of biologics. 108 3.1. Stabilization of biopharmaceuticals with additives and solvents 109 The low stability of biopharmaceuticals in adverse conditions (e.g., low and high 110 pH, extreme temperatures, and the presence of proteases, nucleases, and other 111 denaturing compounds) limits their systematic application, oral and transdermal 112 delivery, production, storage, and transportation (Manning et al., 2010). Therefore, the 113 pharmaceutical industry is constantly searching for additives, formulations, or structural 114 modifications that enhance the resistance of macromolecules to denaturation. However, 115 although engineering recombinant protein and nucleic acids is a highly effective method 116 to improve their stability (Jiang, 2019), this strategy is long and costly, as modified 117 biopharmaceuticals (e.g., biobetters) are treated as new medicines by most regulations 118 and require all safety and clinical trials of novel drugs (Kesik‐Brodacka, 2018). 119 Although all additives in pharmaceuticals must be Generally Recognized As Safe 120 (GRAS) in the USA or have an equivalent classification in other countries (Manchanda 121 et al., 2018), after being declared safe for medical use in humans, they can be applied to 122 several pharmaceutical formulations with fewer tests than novel medicines (Use, 2007). 123 Hence, the discovery of additives and solvents to stabilize and improve the delivery of 124 biopharmaceuticals can streamline the development of more effective medicinal 125 formulations. 126 The stabilization of macromolecules relies on obtaining a balance between 127 stabilizing and destabilizing forces that maintain their native folding. In solution, the 128 stabilizing forces are mainly due to the intramolecular interactions of the 129 macromolecule and the interactions between them and the solvents or other solutes in 130 the environment (Veríssimo et al., 2022). When the solute-macromolecules interactions 131 are positive, the additives can either form a hydration layer around the macromolecules 132 or directly bind to them, helping to prevent unfolding and aggregation. However, if the 133 macromolecule is lyophilized, the stabilization process will be due to the direct binding 134 of the additives to the protein (Ohtake et al., 2011). As for the destabilizing forces, they 135 18 will be caused by the increase of the entropy of unfolding, leading to denaturation, loss 136 of activity, and aggregation (Veríssimo et al., 2022). 137 There are already different classes of molecules used as excipients of 138 biopharmaceuticals, particularly vaccines. For example, sugars, amino acids, proteins, 139 polyols, polymers, surfactants, salts, and organic molecules (Butreddy et al., 2021). 140 These excipients can be applied as preservatives to prevent contamination (e.g., 141 thimerosal), as adjuvants to improve activity (e.g., aluminum salts to stimulate the 142 immune response in vaccines), or as stabilizers to preserve biopharmaceuticals during 143 processing or storage (Centers for Disease Control and Prevention, 2019). However, 144 although lyophilized protein pharmaceuticals and vaccines can be stored for months at 145 room temperature or in the fridge (Remmele et al., 2012), nucleic acids usually require 146 storage at –70 °C (Uddin and Roni, 2021) and many biopharmaceuticals can lose their 147 potency a few hours after reconstitution (Center for Drug Evaluation and Research, 148 2018). 149 Thus, the development of formulations to improve the stability of 150 biopharmaceuticals is still a field of interest in medicine and public health. In this 151 context, ILs and DESs appear as new classes of compounds with unique properties that 152 have been applied as additives to stabilize biomolecules (Egorova et al., 2021). Thus, 153 the next section will explore the suitability of ILs and DESs for pharmaceutical 154 formulations. 155 4. ILs and DESs 156 Green solvents are one of the hot topics of green chemistry (Mussagy et al., 2022b) 157 because they are commonly considered more environmentally friendly and less toxic 158 alternatives than traditional solvents (Mussagy et al., 2020; Veríssimo et al., 2022). 159 Green solvents are considered green because they have a lower environmental impact 160 and a reduced risk of exposure to human health and the environment. ILs and DESs are 161 newer classes of green solvents designed to be even more environmentally friendly and 162 sustainable than traditional solvents, characterized by low toxicity, low flammability, 163 and low volatility (Mussagy et al., 2022a; Quintana et al., 2022). 164 ILs are a type of salt that is liquid at room temperature, consisting of a mixture of 165 cations and anions held together by intramolecular interactions such as ionic bonds, 166 hydrogen bonds, and van der Waals forces (Mamusa et al., 2023; Myrdek et al., 2021). 167 ILs are considered an ideal mixture due to the combination of two significant properties: 168 19 the liquid state of molecular liquids and the ionic character of ionic compounds (Kaur et 169 al., 2022). Unlike traditional salts (i.e., most of them solid at room temperature), ILs are 170 composed of ions that have unique properties such as low volatility, high thermal 171 stability, and high solvating power (Rashid, 2021). ILs are often used as green solvents 172 in a wide range of applications including pharmaceuticals for the formulation of 173 biopharmaceuticals (Kaur et al., 2022; Quintana et al., 2022). DESs are eutectic 174 compounds usually formed by mixing a low molecular weight organic compound 175 (Hydrogen Bond Acceptor - HBA) with a salt (Hydrogen Bond Donor - HBD) via non-176 covalent interactions such as hydrogen bonding (Sun et al., 2022). Since DESs are 177 mostly composed of ionic species, they are now widely acknowledged as a new class of 178 IL analogs because they share the same properties and characteristics as ILs (Afonso et 179 al., 2023). In addition, if the HBD and HBA components of the DES are suitably 180 selected, they can be designed to be highly biodegradable and sustainable for further 181 application in biopharmaceuticals (Hong et al., 2020). 182 ILs and DES are highly designable and tunable solvents that generally share many 183 properties, such as a wide range of low volatility, thermal stability, high solvating 184 power, and ability to dissolve organic or inorganic compounds with significant interest 185 in the biopharmaceutical industry (Sun et al., 2022). However, there are relevant 186 distinctions between ILs and DESs, particularly for industrial applications. ILs are 187 typically formed from a mix of organic heterocyclic cations and anions, whereas DESs 188 are usually based on environmentally benign hydrogen bond donors and acceptors 189 (Płotka-Wasylka et al., 2020). Moreover, the synthesis of ILs often involves multiple 190 chemical steps, making the process more complex and resource-intensive. In contrast, 191 DESs are synthesized through more sustainable and direct methods, such as mixing 192 hydrogen bond donors with acceptors under mild conditions. In terms of application 193 scope, ILs are versatile, with consolidated applications in specialized fields such as 194 electrochemistry and catalysis (Quintana et al., 2022). On the other hand, DES 195 applications are still limited to research state due to their novelty, but they are gaining 196 traction in fields that prioritize environmental impact and safety, like biotechnology and 197 pharmaceuticals (Shamshina and Rogers, 2023). Thus, while ILs and DESs share 198 specific properties, their distinct synthesis processes, sustainability profiles, and 199 application scopes set them apart, particularly in industrial and environmental 200 applications. 201 20 In the last years, ILs and DES have been studied for their potential in drug 202 production and delivery, extraction, and purification of biopharmaceuticals, and as 203 reaction media for chemical synthesis (Quintana et al., 2022). For example, ILs and 204 DESs can be employed as alternative mediums to water and volatile organic solvents to 205 improve chemical and biological reactions, such as amino acid and peptide ligations 206 (Nolan et al., 2022). Furthermore, due to their high selectivity and multiple interactions 207 with target compounds and analytes (Makoś et al., 2018; Momotko et al., 2022, 2021), 208 ILs and DESs are frequently applied for the selective separation of biomolecules 209 (Castro-Muñoz et al., 2022; Khajavian et al., 2022). Some ILs and DESs even present 210 pharmaceutical properties, such as antimicrobial and anticancer activity (Ibsen et al., 211 2018; Veríssismo et al., 2023). 212 Despite the interesting properties of ILs and DES, it is also important to consider 213 some potential drawbacks and limitations of these green solvents such as high cost, 214 some extent of volatility, high viscosity, low moderate instability in the presence of 215 some acids and bases, and limited availability in the market (Chen and Mu, 2021) 216 (Figure 2). Furthermore, the solubility of some biopharmaceuticals in ILs and DESs 217 can be limited, leading to difficulties in the development of pharmaceutical formulations 218 (Veríssimo et al., 2022). The aforementioned drawbacks cannot be ignored, and to 219 enhance the sustainability of the processes and overcome some of these limitations, the 220 design of these solvents must be thoroughly evaluated. 221 222 Figure 2. Differences and similarities of ILs and DESs. 223 21 Although ILs and DESs are generally regarded as green solvents, they are also 224 classes of very diverse chemicals due to the countless combinations of cations and 225 anions to form ILs and mixtures of substances capable of resulting in DESs. For 226 example, the most prevalent ions in the first generation of ILs were the anions 227 hexafluorophosphate, tetrafluoroborate, and bis[(trifluoromethyl)sulfonyl]imide and the 228 cations imidazolium, pyridinium, and pyrrolidinium (Veríssimo et al., 2022). These 229 aromatic cations and hydrophobic anions overall presented high terrestrial and aquatic 230 toxicity and low compatibility with biological molecules (Cho et al., 2021; Greer et al., 231 2020). Depending on the composition and solubility of the ILs and DESs, they can also 232 exhibit microbial toxicity (Marchel et al., 2022a), contribute to atmospheric pollution 233 and cross-contamination (Janjhi et al., 2023), and contaminate water sources (Marchel 234 et al., 2023). Even for ILs and DESs not regarded as toxic, researchers must consider 235 the whole life cycle of the compound when projecting their industrial application. For 236 example, some DESs could promote eutrophication (i.e., excessive nutrient enrichment 237 of water bodies) even though they present no general toxicity, leading to harmful algal 238 blooms and oxygen depletion, adversely affecting aquatic ecosystems (Vieira Sanches 239 et al., 2023). This phenomenon was demonstrated by Vieira Sanches et al. in a study 240 evaluating the effect of choline-, betaine- and proline-based DESs in marine 241 microorganisms and invertebrates (Vieira Sanches et al., 2023). Therefore, researchers 242 should not overlook the environmental footprint and potential toxicity of ILs and DESs. 243 Nonetheless, the extensive array of ion and compound combinations forming ILs 244 and DESs is estimated to surpass one million substances (Bakis et al., 2021), allowing 245 the potential to design chemicals with a wide range of properties. Hence, the tunability 246 of the ILs and DESs properties and a driven approach by researchers to develop solvents 247 with low toxicities, negligible vapor pressure, and non-flammable to favor industrial 248 applications can explain why they are usually labeled as green solvents despite the 249 presence of classes with high toxicity. 250 Recently, some efforts have been made to develop new classes of biocompatible 251 ILs and DES to reduce their potential toxic effects and improve sustainability (Chen and 252 Mu, 2021). These new classes of solvents, such as cholinium-based ILs ([Ch]ILs) and 253 DESs, are designed to be non-toxic and environmentally friendly, making them suitable 254 for use in the stabilization and formulation of several active pharmaceutical ingredients 255 (APIs) (Li et al., 2022). Cholinium-based ILs and DESs are among the most promising 256 22 alternatives, as they are derived from vitamin B8, a quaternary ammonium cation, and 257 usually present low toxicity and cost, high biodegradability, and improved delivery and 258 solubilization of APIs (Boethling et al., 2007; Kunz and Häckl, 2016; Pereira et al., 259 2016; Petkovic et al., 2010; Santos et al., 2015; Ventura et al., 2014). Furthermore, 260 researchers can select anions and compounds with low toxicity to pair with choline, 261 such as specific carboxylic acids and amino acids, to develop biocompatible ILs and 262 DESs. Other types of new non-toxic and biodegradable ammonium-based ILs [e.g., 263 triethylammonium phosphate ([N0,2,2,2]PO₄), trimethylammonium dihydrogen phosphate 264 ([N0,1,1,1]H₂PO₄), trimethylammonium acetate ([N0,1,1,1][CH₃COO])], synthesized using 265 biodegradable and renewable resources have been used as a promising alternative to 266 conventional solvents (e.g., ethanol, acetone, ethers) (Moshikur et al., 2020; Silva et al., 267 2021). 268 By applying bio-based ILs and DESs, the concerns with biocompatibility and 269 biodegradability can be addressed. However, even bio-based compounds can present 270 cytotoxicity, particularly substances that can interact with the lipidic membranes of 271 cells. For example, ILs and DESs with long alkyl side changes can present a 272 hydrophobic or surface-active nature, which will impact their biocompatibility and 273 interaction with biological systems. Nonetheless, the surfactant nature and cytotoxicity 274 of certain ILs and DESs can be exploited for therapeutical and delivery purposes, such 275 as antibacterial and antifungal treatments and transdermal delivery of APIs (Gonçalves 276 et al., 2021; Moshikur et al., 2020; Sivapragasam et al., 2019; Tanner et al., 2018; Wu et 277 al., 2021). The use of ILs and DESs to act in synergy with APIs, either by enhancing 278 their activity or improving their administration and distribution, has been the focal point 279 of research for their applications in the medical field recently. 280 In the market, around 50 % of available drugs are administrated in their salt form 281 mainly because it is the most preferred method to enhance the physicochemical 282 properties of an active compound. So, the development of biocompatible salts (such as 283 ILs or DESs) of the targeted APIs can be a suitable and effective approach to overcome 284 some current drawbacks (solubility and melting temperature) on drug processing and 285 bioavailability faced by the industry of biopharmaceuticals (Ferraz et al., 2011). ILs and 286 DESs have many potential applications in drug delivery, such as improving thermal 287 stability of biomolecules, controlling drug release, eliminating polymorphism, tailoring 288 their solubility and increasing dissolution, modulating surfactant properties of systems, 289 23 enhancing permeability of APIs, and modulating cytotoxicity on tumor cells 290 (Shamshina and Rogers, 2023; Veríssimo et al., 2022; Wu et al., 2021). DESs have also 291 been extensively investigated as alternative solvents for the solubilization of APIs, 292 particularly in the context of topical formulations (Smith et al., 2014). For example, the 293 solubility of ibuprofen can be significantly improved, over 5,400 times in DESs 294 compared to its solubility in water (Lu et al., 2016). Additionally, another type of DES 295 composed of cholinium chloride ([Ch]Cl) and glycolic acid has shown substantial 296 solubility enhancements for itraconazole, piroxicam, lidocaine, and posaconazole, with 297 improvements of 6700x, 430x, 28x, and 6400x respectively, compared to their solubility 298 in water (Li and Lee, 2016). Some fatty acids (lauric and palmitic acids) combined with 299 ibuprofen and lidocaine have been successfully used for formulations of API-DESs for 300 transdermal delivery purposes (Benessam et al., 2013; Nazzal et al., 2002). 301 Despite the growing interest in new green solvents for biopharmaceutical 302 applications, the use of ILs or DESs as APIs in formulations is still in the early stages of 303 development, and further research is needed to fully elucidate their properties and 304 applications in these fields. For example, although there are many ILs and DESs with 305 the capacity to improve the stability of biomolecules, there are also reports of these 306 compounds impairing the structural integrity of macromolecules such as proteins 307 (Veríssimo et al., 2022), nucleic acids (Mandal et al., 2020), and polymers (Tan et al., 308 2018) depending on their structure, interactions, and environmental conditions under 309 study. Furthermore, it is also necessary to assess their toxicity, bioavailability, and 310 dissolution, as well as investigate the irritancy and skin permeation of these 311 formulations. 312 Finally, understanding the stability of the ILs and DESs is another fundamental 313 parameter to allow their application, as degraded compounds in pharmaceutical 314 formulations can lose their function or be detrimental to the safety and efficacy of the 315 medicine. For example, the thermal or long-term degradation of ILs and DESs can 316 cause the formation of toxic byproducts even of initially biocompatible compounds, as 317 demonstrated by Marchel et al. (Marchel et al., 2022b). Moreover, there are other safety 318 concerns related to the overheating of ILs and DESs, such as sample and equipment 319 degradation, increased chemical and biosafety risks, and higher processing costs to add 320 cooling systems to avoid overheating. Hence, in addition to guaranteeing the structural 321 integrity of ILs and DESs at the expected conditions for use of the biopharmaceuticals, 322 24 long-term stability studies and stress assays [e.g., the thermal stability of ILs and DESs 323 (Cao and Mu, 2014; Chen et al., 2018), chemical stability (Wang et al., 2017)] should 324 also be performed to confirm the potential range of application of these substances. 325 To determine the safety and effectiveness of the clinical use of ILs and DESs, it 326 will be crucial to conduct both in vitro and in vivo studies, starting with non-clinical 327 trials and progressing to clinical trials. As our understanding of the role of ILs and 328 DESs expands, it is expected that a growing number of cation/anion and HBD/HBA 329 combinations will be utilized to create APIs, resulting in a multitude of options for 330 design and product efficacy. With this in mind, the next section will disclose the state-331 of-the-art on the use of ILs and DESs to enhance the stability and delivery of 332 biopharmaceuticals. 333 5. Stability of biopharmaceuticals in ILs and DESs 334 The effect of ILs and DESs on the stability of protein and nucleic acid 335 biopharmaceuticals will be presented in Table 2 (biopharmaceuticals in ILs) and Table 336 3 (biopharmaceuticals in DESs). For each biopharmaceutical, we will include its 337 therapeutical use and the effects of the ILs and DESs solutions considering 338 concentration ranges and different stability types (i.e., structural, thermal, aggregation, 339 and activity) and effects (e.g., solubilization, improved delivery, long-term 340 preservation). The symbol (↑) indicates an increase in stability, (=) represents that the 341 solution had a similar effect to the control, and (↓) means a decrease in stability. 342 For proteins and peptides, we will also include their molecular weight (MW), 343 grand average hydrophobicity index (GRAVY), and instability index (II) calculated 344 using their UniProt sequence using the Expasy ProtParam tool (Artimo et al., 2012; 345 ExPASy, 2018; UniProt, 2020), and their structure in Figure 3 and 4. For GRAVY, 346 values below zero represent hydrophilic amino acid chains, while GRAVY above zero 347 means lipophilic chains. For the instability index, values below 40 indicate a stable 348 protein (Veríssimo et al., 2022). The properties of each macromolecule will be 349 discussed in their respective subsection. The type or source of the protein 350 biopharmaceuticals will also be provided in Tables 2 and 3 when available, considering 351 this can also impact their stability and properties. Finally, we also provide the chemical 352 structures of the cations and anions for the ILs and the components of the DESs from 353 Tables 2 and 3 in Figure 4, so reader can refer to them during the discussions in the 354 next subsections. 355 25 Tables 2 and 3 present the properties, use, and stability (according to stability 356 types) of biopharmaceuticals in the presence of different IL and DES solutions (i.e., 357 classes and concentrations), respectively. 358 359 Table 2. Stability (structural, thermal, activity, aggregation, or simulation) of biopharmaceutical in 360 different concentrations of ILs and effect of ILs on their delivery. Specific information for each protein 361 and peptide, namely, UniProt of the most usual variant, molecular weight (MW), instability index (II), 362 GRAVY‡, and therapeutical use of the biopharmaceuticals are also presented in the table. 363 364 Biopharmaceutical Variant or class ILs Conc.* (Stability) [Effect] Ref. Peptides Glucagon-like peptide 1 (GLP-1) Cholinium-based ILs UniProt: P55095, MW: 3.3 kDa Native human GLP-1 [Ch][C₉H₁₅COO] # 0.7 - 3.7 M (20 - 100 w%) (= Structural, activity), [Improved pharmacokinetics and sustained release] (Agatemor et al., 2021) II: 17.7 (stable), GRAVY: -0.230 Use: Hormone for treatment of type 2 diabetes. Interleukin-2 (IL-2) Cholinium-based ILs UniProt: P60568; Mw: 15.2 kDa; II: 53.4 (unstable); GRAVY: -0.198 DES- ALANYL-1, SER-125 human IL-2 [Ch]H₂PO₄ 0.030 - 0.185 M (↑ Thermal) (Weaver et al., 2012) Use: Cytokine for immunotherapy. [Ch]H₂PO₄ 0.030 - 0.185 M (= Structural) Insulin Ammonium-based ILs UniProt: P01308 (A and B chains) Zn-free insulin [N0,2,2,2]PO₄, [N0,1,1,1]HSO4, [N0,2,2,2]SO₄, [N0,1,1,1]H2PO4, [N0,1,1,1][CH₃COO] 0.5 - 2.0 M (↑ Thermal, ↓ aggregation) (Kumar and Venkatesu, 2013) (Human insulin) Cholinium-based ILs MW: 5.8 kDa Insulin from porcine pancreas [Ch][Gln], [Ch]₂[Asn] 0.0008 M (↑ Thermal) (Guncheva et al., 2019) II: 13.61 (stable) [Ch][Asn] 0.0008 M (= Thermal) GRAVY: 0.218 [Ch][Arg], [Ch]₂[Gln], [Ch][Lys] 0.0008 M (↓ Thermal, structural) Use: Hormone for treatment of diabetes. [Ch][Gln], [Ch]₂[Asn], [Ch][Asn] 0.0008 M (↓ Structural) FITC-insulin [Ch][C₉H₁₅COO] 1.85 - 3.70 M (= Structural, activity), [↑ Transdermal permeation] (Banerjee et al., 2017) Human insulin [Ch][C₉H₁₅COO] 3.7 M (100%) (↑ Structural long-term, = activity), [↑ Oral intake, paracellular transportation, sustained (Banerjee et al., 2018) 26 activity; ↓ enzymatic degradation] [Ch][C₉H₁₅COO] 0.04 - 0.19 M [↑ Oral intake, paracellular transportation] (Peng et al., 2020) Imidazolium-based ILs Porcine insulin [C₄MIm][CH₃COO], [C₄MIm][CF₃COO], [C₄MIm][N(CN)₂] (ILs in KCl/HCl pH 2) 0.3 M (↑ Structural, thermal) (Todinova et al., 2016) [C₄MIm]Cl, [C₄MIm][SCN], (ILs in KCl/HCl pH 2) 0.3 M (= Structural, thermal) [C₄MIm][C(CN)₃] in KCl/HCl pH 2 0.3 M (↓ Structural, thermal) Zn-free insulin [C₄MIm]Cl, [C₄MIm]Br 0.01 - 0.04 M (↑ Structural) (Kumar and Venkatesu, 2014) [C₄MIm]Cl, [C₄MIm]Br 0.01 - 0.04 M (↓ Thermal) [C₄MIm][SCN], [C₄MIm]HSO₄, [C₄MIm]I, [C₄MIm][CH₃COO] 0.01 - 0.04 M (↓ Structural, thermal) Porcine insulin (experiment) and human insulin (simulation) [C₂MIm][CH₃COO] 3 - 6 M (50 - 90 wt%) (↑ Simulation, thermal) (D. Li et al., 2019) Human insulin [C₂MIm][CH₃COO], [C₄MIm]Cl, [C₄MIm]NO₃, [C₄MIm][CH₃SO₃], [C₄MIm][N(CN)2], [C₄MIm][CH₃COO], [C₆MIm][CH₃COO], [C₈MIm][CH₃COO], [C₁₀MIm][CH₃COO], [C₁₂MIm][CH₃COO] ~ 1.5 – 7.0 M (75 - 100 wt %) (↑ Simulation) Ovalbumin epitope (OVA) Cholinium-based ILs Sequence: SIINFEKL; Mw: 1.0 kDa OVA257-264 SIINFEKL (H-2 Kb) [Ch][C₁₂:0], [Ch][C₁₈:₁] ~ 0.5 - 0.7 M (20 wt%) [Skin penetration enhancer] (Tahara et al., 2020) Use: Cancer antigen for immunostimulation of therapeutic cancer vaccination. [Ch][C₁₈:₁] 0.2 M (8 wt%) (↑ Activity) [Solubilizer] Proteins Immunoglobulin G1 (IgG1) Cholinium-based ILs IgG from human serum [Ch][CH₃COO] 0.0005 - 0.0025 M (↑ Structural, thermal) (Dhiman et al., 2022) Unitprot: P01837 [Ch][CH₃COO], [Ch][C₅H₇O₅COO] 0.0005 - 0.0015 M (↓ Aggregation) 27 MW: 142.6 kDa [Ch][C₅H₇O₅COO], [Ch]H₂PO₄ 0.0005 - 0.0025 M (= Structural, thermal) II: 37.16 (stable) [Ch][CH₃COO], [Ch][C₅H₇O₅COO] 0.0020 - 0.0025 M (↑ Aggregation) GRAVY: -0.442 [Ch]H₂PO₄ 0.0005 - 0.0025 M (↑ Aggregation) Use: Immunization, immunotherapy, infection treatments, hematologic and autoimmune diseases treatments, and others. IgG from rabbit serum [Ch][HOCH₂COO], [Ch][C₅H₇O₄COO] ~ 0.9 - 1.4 M (25 wt%) (= Structural, aggregation) (Mondal et al., 2016) [Ch][CH₃COCOO] 1.3 M (25 wt%) (= Aggregation) [Ch][C₉H₈NCOO], [Ch][CH₃COCOO] ~ 1.1 - 1.3 M (25 wt%) (↓ Structural) [Ch][C₉H₈NCOO] 1.1 M (25 wt%) (↑ Aggregation) Antihuman TNF-α mouse IgG1 [Ch][HOCH₂COO] ~ 1 - 4 M (20 - 70 v%) (↑ Activity) (Angsantikul et al., 2021) [Ch][HOCH₂COO] ~ 3 M (50 v%) (= Structural) [Ch][HOCH₂COO] ~ 4 - 5 M (80 - 90 v%) (↓ Activity) Imidazolium-based ILs Human IgG1 [C₂MIm]Cl, [C₄MIm]Cl, [C₆MIm], [C₈MIm]Cl 0.04 M (↑ Structural, ↓ aggregation) (Rawat and Bohidar, 2015) IgG from rabbit serum [C₄MIm][CH₃BzSO₃], [C₄MIm][N(CN)₂], [C₄MIm][CH₃COO], [C₂MIm]Br, [C₄MIm]Br, [C₄MIm]Cl ~ 0.1 - 0.3 M (5 wt%) (= Structural) (Ferreira et al., 2016) Ammonium-based ILs [N₁,₁,₁,₁]Br, [N₂,₂,₂,₂]Br, [N₃,₃,₃,₃]Br ~ 0.1 - 0.2 M (5 wt%) (= Structural) (Ferreira et al., 2016) [N₄,₄,₄,₄]Br ~ 0.2 M (5 wt%) (↓ Structural) Phosphonium-based ILs [P₄,₄,₄,₄]Br ~ 0.1 M (5 wt%) (↓ Structural, ↑ aggregation) (Ferreira et al., 2016) L-Asparaginase (L- ASNase) Cholinium-based ILs Uniprot: P00805 (Using tetrameric form), MW: 137.7 L-ASNase EC 3.5.1.1 [Ch][CH₃COO], [Ch][C₂H₅COO], [Ch][C₃H₇COO] 0.001–0.050 mol IL/mol total (↑ Activity) (Magri et al., 2019) 28 (tetramer) II: 19.84 (stable) [Ch][C₅H₁₁COO] 0.001–0.010 mol IL/mol total (↑ Activity) GRAVY: -0.194 Treatments of acute lymphoblastic leukemia and lymphoblastic lymphoma. [Ch][C₅H₁₁COO] 0.025–0.050 mol IL/mol total (↓ Activity) Nucleic acids siRNA against CD45 Use: Treatment of autoimmune diseases. siRNA Cholinium-based ILs [Ch]H₂PO₄ 1.0 - 2.5 M (20 - 50 wt%) (↑ Structural, activity, thermal) [Long-term storage] (Mazid et al., 2014) siRNA against NFKBIZ Use: Treatment of psoriasis. siRNA Cholinium-based ILs [Ch][PhC₂H₅COO] ~ 2 M (50 v%) (↑ Structural) (Mandal et al., 2020) [Ch][C₉H₁₅COO] ~ 2 M (50 v%) (↓ Structural) [Ch][C₉H₁₅COO], [Ch][PhC₂H₅COO] ~ 2 M (50 v%) [Skin penetration enhancer] [Ch][C₉H₁₅COO]:[Ch][PhC₂H₅COO] ~ 1 M each (25 v% each) (↑ Structural, simulation, activity) / [Skin penetration enhancer] siRNA against GAPDH and MMP12 Use: Treatment of skin diseases. siRNA Ammonium IL-Robed siRNA [N1,1,(Bz),8]–siRNA1, [N1,1,(Bz),14]– siRNA1, [N1,1,(Bz),18]–siRNA1 0.00005 M [Skin penetration and cell internalization enhancer] (Zakrewsky and Mitragotri, 2016) STAT6 decoy oligonucleotide Use: Treatment of skin inflammation. Oligonucleotide Lidocainum-based IL ILTS® Not disclosed [Skin penetration enhancer] (Handa et al., 2019; Kubota et al., 2016) ‡ GRAVY - grand average hydrophobicity index, below 0 indicates that the protein sequence is 365 hydrophilic. * Approximate conversions (when possible) to molar (M) using MW and density (when 366 available on the manufacturer’s site or literature). # There is still an ongoing discussion about whether 367 choline and geranic acid 1:1 ([Ch][C₉H₁₅COO]) leads to the formation of an IL or a DES (Rogers and 368 Gurau, 2018). Considering the suggestion of Rogers and Gurau (Rogers and Gurau, 2018), 369 [Ch][C₉H₁₅COO] was addressed in this review as an IL. 370 371 Table 3. Stability (structural, thermal, activity, aggregation, or simulation) of biopharmaceuticals in 372 different concentrations of DESs and the effect of DESs on their delivery. Specific information for each 373 protein, namely, UniProt of the most usual variant, molecular weight (MW), instability index (II), 374 GRAVY‡, and therapeutical use of the biopharmaceuticals are also presented in the table. 375 376 Biopharmaceutical Variant or class DESs Conc.* (Stability) [Effect] Ref. Peptides Insulin Cholinium-based DESs 29 UniProt: P01308 (A and B chains) (Human insulin) MW: 5.8 kDa II: 13.61 (stable) GRAVY: 0.218 FITC-insulin [Ch][C₉H₁₅COO]– [C₉H₁₅COOH], [Ch][C₉H₁₅COO]– [C₉H₁₅COOH]₃ ~ 1.7 - 2.7 M (100 %) [↑ Transdermal permeation] (Tanner et al., 2018) Use: Hormone for treatment of diabetes. Human insulin [Ch][C₉H₁₅COO]– [C₉H₁₅COOH] ~ 2.7 M (100%) [↑ Transdermal permeation] (Jorge et al., 2020) Proteins Immunoglobulin G1 (IgG1) Cholinium-based DESs Unitprot: P01837 IgG from human serum [Ch]Cl–[CO(NH₂)₂], [Ch]Cl– [C₃H₅(OH)₃], [Ch]Cl– [(CH₂OH)₂] ~ 0.2 - 1.5 M (5 - 30 wt%) (↑ Thermal) (Dhiman et al., 2023) MW: 142.6 kDa [Ch]Cl–[CO(NH₂)₂] 1.5 M (30 wt%) [↑ Long-term stability] II: 37.16 (stable) [Ch]Cl–[CO(NH₂)₂], [Ch]Cl– [C₃H₅(OH)₃] ~ 0.2 - 0.8 M (5 - 15 wt%) (↓ Aggregation) GRAVY: -0.442 [Ch]Cl–[(CH₂OH)₂] ~ 0.3 - 0.5 M (5 - 10 wt%) (↓ Aggregation) Use: Immunization, immunotherapy, infection treatments, hematologic and autoimmune diseases treatments, and others. [Ch]Cl–[CO(NH₂)₂], [Ch]Cl– [C₃H₅(OH)₃], [Ch]Cl– [(CH₂OH)₂] ~ 0.2 - 0.8 M (5 - 15 wt%) (= Structural) [Ch]Cl–[C₃H₅(OH)₃], [Ch]Cl– [(CH₂OH)₂] ~ 1.3 - 1.5 M (30 wt%) [= Long-term stability] [Ch]Cl–[(CH₂OH)₂] ~ 0.8 - 2.5 M (15 - 50 wt%) (↑ Aggregation) [Ch]Cl–[CO(NH₂)₂], [Ch]Cl– [C₃H₅(OH)₃] (~ 1.3 - 2.5 M) 30 - 50 wt% (↑ Aggregation) [Ch]Cl–[CO(NH₂)₂], [Ch]Cl– [C₃H₅(OH)₃], [Ch]Cl– [(CH₂OH)₂] (~ 1.3 - 2.5 M) 30 - 50 wt% (↓ Structural) FITC-IgG [Ch][HOCH₂COO]–[Ch] 0.03 - 0.08 M [↑ Paracellular transportation] (Angsantikul et al., 2021) Antihuman TNF-α mouse IgG1 [Ch][HOCH₂COO]–[Ch] ~ 1.4 M (50 v%) [↑ Oral intake] [Ch][HOCH₂COO]–[Ch] ~ 0.7 – 2.5 M (20 - 70 v%) (↑ Activity) [Ch][HOCH₂COO]–[Ch], [Ch][HOCH₂COO]– [HOCH₂COOH] ~ 2 M (50 v%) (= Structural) [Ch][HOCH₂COO]–[Ch] ~ 3.0 – 3.5 M (80 - 90 v%) (↓ Activity) [Ch][HOCH₂COO]– [HOCH₂COOH] ~ 0.8 - 3.5 M (20 - 90 v%) (↓ Activity) 30 L-Asparaginase (L- ASNase) Poly(vinyl pyrrolidone)- based DESs Uniprot: P00805 (Using tetrameric form), MW: 137.7 (tetramer), II: 19.84 (stable), GRAVY: - 0.194 Use: Treatments of acute lymphoblastic leukemia and lymphoblastic lymphoma. Pure L- ASNase and L-ASNase from E. coli extract [(C6H9NO)n]–[CH₂(COOH)₂] 0.1 M (0.02 g/mL) (↓ Structural, = activity) [Adsorption of L- ASNase from E. coli extracts] (Li et al., 2019) ‡ GRAVY - grand average hydrophobicity index, below 0 indicates that the protein sequence is 377 hydrophilic. * Approximate conversions (when possible) to molar (M) using MW and density (when 378 available on the manufacturer’s site or literature). 379 380 381 As observed in Tables 2 and 3, the effect of ILs and DESs on biopharmaceuticals 382 has been demonstrated in several studies. The interactions between ILs and proteins will 383 not only rely on the IL class, but the properties of the biomolecule, concentration range, 384 and type of stability evaluated. Furthermore, specific ILs and DESs can also enhance 385 the delivery or solubility of biopharmaceuticals. Considering the impacts of each IL and 386 DES on the macromolecules are associated with their structure and specific activity, we 387 will discuss each biopharmaceutical individually. Figure 3 shows the structure of each 388 of the proteins and Figure 4 from the ILs and DESs constituents from Tables 2 and 3. 389 The next subsections present a detailed examination of the structure, use, drawbacks, 390 and interaction of ILs and DESs with biopharmaceuticals. 391 392 31 Figure 3. Structure of protein biopharmaceuticals. A) Glucagon-like peptide 1 (GLP-1), B) Insulin, C) 393 Interleukin-2 (IL-2), D) Ovalbumin epitope (OVA), E) Immunoglobulin G1 (IgG1), and F) L-394 asparaginase (L-ASNase). Images of the proteins were produced with the PDB structures using UCSF 395 Chimera 1.14 (Berman et al., 2002; Pettersen et al., 2004). 396 397 Figure 4. Chemical structures of cations and anions of ionic liquids (ILs) and constituents of deep 398 eutectic solvents (DESs). A) Ammonium, B) imidazolium, C) cholinium, D) phosphonium, and E) 399 lidocainum cations of ILS. F) Organic acid, G) inorganic, and H) amino acid anions of ILs. I) DESs 400 constituents. 401 402 5.1 Biopharmaceuticals in ILs and DESs 403 5.1.1. Glucagon-like peptide 1 (GLP-1) 404 GLP-1 is an intestinal hormone produced as a response to meal ingestion (Müller 405 et al., 2019). Its release enhances insulin secretion and helps to normalize the 406 postprandial glycemic response and achieve blood glucose homeostasis (Müller et al., 407 2019). As presented in Figure 3.A and Table 2, GLP-1 is a small (3.3 kDa) and 408 hydrophilic (GRAVY: -0.230) peptide formed by a continuous α-helix. Furthermore, 409 32 GLP-1 pI is 4.6 and is found in a negatively charged monomeric state at physiological 410 pH (Kaasalainen et al., 2015). As a biopharmaceutical, GLP-1 and GLP-1 receptor 411 agonists are applied to lower glucose levels in patients with type 2 diabetes (Walsh, 412 2010). However, GLP-1 is quickly degraded by the circulatory enzyme dipeptidyl 413 peptidase-4 (DPP-4) within minutes of its intravenous administration and around 1 h 414 after subcutaneous administration, which hinders its therapeutical use (Agatemor et al., 415 2021). 416 To improve the delivery and pharmacokinetics of native human GLP-1, Agatemor 417 et al. (Agatemor et al., 2021) used cholinium geranate (i.e., geranate also called 3,7-418 dimethyl-2,6-octadienoate) 1:1 ([Ch][C₉H₁₅COO]) as a vehicle for the subcutaneous 419 administration of this biopharmaceutical in rats. [Ch][C₉H₁₅COO] preserved the 420 structural integrity and activity of the peptide from low to high concentrations (0.7 to 421 3.7 M). Furthermore, it improved GLP-1 pharmacokinetics by reducing its degradation 422 by DPP-4 and possibly by a sustained release due to entrapment of the 423 biopharmaceutical. We must note that there is still an ongoing discussion about whether 424 choline and geranic acid systems lead to the formation of DESs or IL with a complex 425 anion (e.g., CAGE as [choline][geranate2(H)]), as pointed out by Rogers and Gurau 426 (Rogers and Gurau, 2018). Moreover, this discussion is not limited to choline and 427 geranic acid systems, as the distinction between certain DESs and ILs can be complex 428 to discern for specific classes, with some authors even considering DESs an IL subclass 429 (Płotka-Wasylka et al., 2020). As ILs or DESs, choline and geranic acid systems 430 showed remarkable potential as vehicles to boost the delivery of pharmaceuticals such 431 as GLP-1. 432 5.1.2. Interleukin-2 (IL-2) 433 IL-2 is a cytokine that immunomodulates regulatory T cells and effector 434 lymphocytes (Arenas-Ramirez et al., 2015). As presented in Figure 3.B and Table 2, 435 the IL-2 is a small (15.2 kDa) and hydrophilic (GRAVY: -0.198) peptide comprised of 436 four α-helices in a bundle. In chemotherapy, IL-2 is used as an immunostimulant to 437 activate T cells against cancer cells, already approved to treat unresectable metastatic 438 renal cell carcinoma and stage IV melanoma (Weaver et al., 2012). As with other 439 protein-based biopharmaceuticals, its stability can be an issue for application, storage, 440 and transportation. For example, according to the supplier R&D systems, IL-2 protein 441 should be used within 24 hours after reconstitution in water (R&D Systems, 2023). 442 33 Hence, the discovery of additives to stabilize IL-2 can enhance and expand its use in 443 medicine. 444 To improve the thermal stability of human IL-2, Eckstein et al. (Weaver et al., 445 2012) applied the IL [Ch]H₂PO₄ at low concentrations. From 0.030 to 0.185 M, 446 [Ch]H₂PO₄ preserved the structural integrity of IL-2 and increased its thermal stability. 447 According to the authors, the complementary electrostatic interactions between the IL 448 and the surface of the peptide likely offer protection against IL-2 thermal denaturation 449 without altering its secondary or tertiary protein structure. Therefore, cholinium-based 450 ILs can be applied as additives in very low concentrations to improve the thermal 451 stability of therapeutic peptides without altering their conformation. 452 5.1.3. Insulin 453 Insulin is an endogenous hormone peptide produced in the pancreas and is 454 responsible for blood glucose homeostasis (Mayer et al., 2007). Insulin was the first 455 recombinant biopharmaceutical, and it is still used for the treatment of diabetes (Walsh, 456 2013). As presented in Figure 3.C, insulin has two polypeptide chains (A and B) linked 457 by disulfide bonds (Brange and Langkjœr, 1993), with A chain formed by two 458 antiparallel α-helices and B chain comprised of an α-helix with a turn and β-strand 459 (Brange and Langkjœr, 1993). Insulin presents low solubility in water at neutral pH, but 460 it is possible to solubilize it up to 0.17 mM at pH 2 (Sigma-Aldrich, 2014). 461 Furthermore, this peptide is a weak dimer and can dimerize above 10-6 M and form 462 hexamers at 2 mM (Brange and Langkjœr, 1993). Regarding its stability, insulin is 463 considered stable and can be stored for one month at room temperature before 464 reconstitution (Center for Drug Evaluation and Research, 2018). However, insulin can 465 lose its potency when diluted or maintained at extreme temperatures and should be 466 discarded if exposed to these conditions (Center for Drug Evaluation and Research, 467 2018). Additionally, there are multiple efforts to develop oral and transdermal delivery 468 systems for insulin, due to the discomfort and distress caused by its main administration 469 via subcutaneous injection (Xiao et al., 2020; Zhang et al., 2019). In this sense, ILs and 470 DESs can help to improve insulin medical use by improving its solubility and stability 471 and in the development of novel oral and transdermal systems for drug delivery. 472 For the thermal stability of insulin, researchers found that different IL classes (i.e., 473 ammonium, cholinium, and imidazolium) and concentration ranges (i.e., 0.0008 to 6 M) 474 can improve its thermodynamic parameters. For example, [N0,2,2,2]PO₄, 475 34 trimethylammonium hydrogen sulfate ([N0,1,1,1]HSO₄), triethylammonium sulfate 476 ([N0,2,2,2]SO₄), [N0,1,1,1]H₂PO₄, and [N0,1,1,1][CH₃COO] from 0.5 to 2.0 M increased the 477 Tm of insulin and decreased its aggregation (Kumar and Venkatesu, 2013). Moreover, 478 dilute solutions of cholinium L-glutaminate ([Ch][Gln]) and dicholinium L-asparaginate 479 ([Ch]₂[Asn]) (at 0.0008 M) (Guncheva et al., 2019), and [C₄MIm][CH₃COO], 1-butyl-3-480 methylimidazolium trifluoroacetate ([C₄MIm][CF₃COO]), and 1-butyl-3-481 methylimidazolium dicyanamide ([C₄MIm][N(CN)₂]) (0.3 M at pH 2) (Todinova et al., 482 2016) also enhanced the thermal stability of insulin. Additionally, high concentrations 483 of 1-ethyl-3-methylimidazolium acetate ([C₂MIm][CH₃COO]) (3 to 6 M) had a similar 484 positive effect on the peptide (Li et al., 2019). There were also ILs at low concentrations 485 that did not affect the thermal stability of insulin, such as 0.0008 M of cholinium L-486 asparaginate ([Ch][Asn]) (Guncheva et al., 2019), and 0.3 M of 1-butyl-3-487 methylimidazolium chloride ([C₄MIm]Cl) and 1-butyl-3-methylimidazolium 488 thiocyanate ([C₄MIm][SCN]) at acidic pH (Todinova et al., 2016). However, even 489 concentrations below 0.04 M of specific cholinium and imidazolium ILs decreased the 490 Tm of insulin, including cholinium L-argininate ([Ch][Arg]), dicholinium L-glutaminate 491 ([Ch]₂[Gln]), cholinium L-lysinate ([Ch][Lys]),(Guncheva et al., 2019) [C₄MIm][SCN], 492 1-butyl-3-methylimidazolium hydrogen sulfate ([C₄MIm]HSO₄), 1-butyl-3-493 methylimidazolium iodide ([C₄MIm]I), [C₄MIm][CH₃COO] (Kumar and Venkatesu, 494 2014), and 1-butyl-3-methylimidazolium tricyanomethanide ([C₄MIm][C(CN)₃]) in 495 acidic pH (Todinova et al., 2016). Noteworthy, [C₄MIm][SCN] at low concentrations 496 (below 0.3 M) had different impacts on the thermal stability of insulin at different pH. 497 For example, this IL had no impact on insulin Tm at acidic pH (Todinova et al., 2016) 498 but decreased it at neutral conditions (Kumar and Venkatesu, 2014). Therefore, the 499 composition of the IL, its concentration range, and the environment will impact the 500 effect of ILs on protein thermal stability. 501 As for the effect of ILs on the structural stability of insulin in ILs, it varied 502 according to the IL class and concentration range of the solutions. Low concentrations 503 of different imidazolium ILs were detrimental to insulin native structure, such as 0.3 M 504 of [C₄MIm][C(CN)₃], [C₄MIm]Cl, [C₄MIm][SCN] at pH 2 (Todinova et al., 2016), and 505 [C₄MIm][SCN], [C₄MIm]HSO₄, [C₄MIm]I, and [C₄MIm][CH₃COO] from 0.01 to 0.04 506 M (Kumar and Venkatesu, 2014). The exceptions to this trend were 507 [C₄MIm][CH₃COO], [C₄MIm][CF₃COO], [C₄MIm][N(CN)₂] at 0.3 M and pH 2 508 (Todinova et al., 2016) and [C₄MIm]Cl and[C₄MIm]Br from 0.01 to 0.04 M (Kumar 509 35 and Venkatesu, 2014) that preserved the structure of insulin. There were also dilute 510 imidazolium ILs that did not alter insulin structure, such as [C₄MIm]Cl and 511 [C₄MIm][SCN] at 0.3 M and pH 2 (Todinova et al., 2016). As for cholinium ILs, high 512 concentrations (1.8 to 3.7 M) of [Ch][C₉H₁₅COO] maintained the short-term and 513 improved the long-term structural integrity and activity of insulin (Banerjee et al., 2018, 514 2017). However, dilute cholinium IL solutions (0.0008 M of [Ch][Arg], [Ch]₂[Gln], 515 [Ch][Lys], [Ch][Gln], [Ch]₂[Asn], [Ch][Asn] also impaired the structural stability of the 516 peptide (Guncheva et al., 2019). Interestingly, although [Ch][Gln], [Ch]₂[Asn], and 517 [Ch][Asn] caused a partial unfolding of insulin, they still increased its thermal stability, 518 confirming a certain environment can be positive to a protein stability parameter in 519 detriment of another (Guncheva et al., 2019). 520 Simulation tools such as MD were also used to predict the structural stability of 521 insulin in ILs. For example, Li et al. (Li et al., 2019) estimated with MD that high 522 concentrations of imidazolium ILs (75 to 100 wt%) stabilize insulin native state, 523 particularly the ILs with shorter alkyl chains and weak hydrogen bonding. The ILs 524 included [C₂MIm][CH₃COO], [C₄MIm]Cl, 1-butyl-3-methylimidazolium nitrate 525 ([C₄MIm]NO₃), 1-butyl-3-methylimidazolium methanesulfonate ([C₄MIm][CH₃SO₃]), 526 [C₄MIm][N(CN)2], [C₄MIm][CH₃COO], 1-hexyl-3-methylimidazolium acetate 527 ([C₆MIm][CH₃COO]), 1-methyl-3-octylimidazolium acetate ([C₈MIm][CH₃COO]), 1-528 decyl-3-methylimidazolium acetate [C₁₀MIm][CH₃COO], and 1-dodecyl-3-529 methylimidazolium acetate ([C₁₂MIm][CH₃COO]). Moreover, the MD demonstrated 530 that electrostatic interactions are the main forces responsible for the ability of the ILs to 531 stabilize the peptide. 532 Regarding the application of ILs and DESs in pharmaceutical formulations for 533 improved delivery, cholinium-based ILs and DESs have been successfully applied by 534 multiple groups for transdermal and oral administration of insulin. For instance, high 535 concentrations (above 1 M to neat) of cholinium geranate ILs and DESs [i.e., IL 536 [Ch][C₉H₁₅COO]; DESs cholinium geranate 1:2 ([Ch][C₉H₁₅COO]–[C₉H₁₅COOH]) and 537 cholinium geranate 1:4 ([Ch][C₉H₁₅COO]–[C₉H₁₅COOH]₃)] improved the transdermal 538 permeation of insulin in three different studies (Banerjee et al., 2018; Jorge et al., 2020; 539 Tanner et al., 2018). Moreover, [Ch][C₉H₁₅COO] also increased the oral intake and 540 paracellular transportation from 0.04 to 3.7 M (neat IL) in two other works (Banerjee et 541 al., 2018; Peng et al., 2020). Another advantage of cholinium geranate ILs and DESs is 542 36 their overall biocompatibility, which makes them remarkable candidates for the 543 development of biopharmaceutical delivery systems (Riaz et al., 2022). 544 For insulin, both ILs and DESs presented remarkable applications to improve its 545 delivery by enhancing its transdermal permeation. However, the data is still limited 546 regarding the impact of DESs on the stability of this biopharmaceutical. Hence, we 547 suggest following studies on the topic also account for the effect of DESs on the 548 stability of insulin, considering specific ILs improved while others impaired the stability 549 of this peptide. 550 Considering the extensive number of studies on the effect of ILs on insulin, we 551 evaluated the entries in Tables 2 and 3 to determine the amount of IL solutions that 552 increased or maintained the stability of insulin according to the stability type, IL class, 553 and concentration range. The results as percentages of the total for each variable are 554 presented in Figure 5. This method was chosen to try to establish trends regarding the 555 effect of different conditions on insulin stability. It should be noted that this analysis 556 does not try to be a definitive answer regarding the effects of ILs and DESs on insulin, 557 but it aims to find tendencies and knowledge gaps in this topic and be a guide for future 558 research. 559 560 561 Figure 5. A) Percentage of IL and DES solutions that maintained or increased the different types of 562 stability of insulin (structural, thermal, activity, and aggregation). For aggregation, improvement of 563 stability represents ILs that decrease protein aggregation. B) Percentage of IL and DES solutions that 564 maintained or increased the stability of insulin according to their class. C) Percentage of IL and DES 565 solutions that maintained or increased the stability of insulin according to their concentration (< 1 or ≥ 1 566 M). n = total number of samples for each condition. 567 568 As can be seen in Figure 5, most IL and DES solutions had a positive effect on 569 insulin stability (69 %, n = 64, with n representing the number of samples for each 570 condition evaluated). Specifically, half the IL and DES solutions improved the 571 structural stability of insulin (n = 22), 68 % increased or maintained its thermal stability 572 (n = 28), and 100 % decreased its aggregation (n = 10). Although 100 % also 573 maintained or improved insulin activity, the number of samples is still too low for a 574 37 conclusion (n = 4). Hence, we suggest that future studies also include the effect of ILs 575 on the biological activity of insulin. Regarding the effect of different classes, 576 ammonium IL had the most positive effect on insulin stability (100 %, n = 28), followed 577 by imidazolium (61 %, n = 20), and cholinium-based ILs and DESs (44 %, n = 16). 578 However, it should be noted that cholinium ILs and DESs also had a functional effect 579 on insulin oral and transdermal delivery, sustained release, activity, and long-term 580 stabilization, demonstrating its potential for application. As for the concentration range, 581 higher concentrations of ILs and DESs were more compatible with insulin than lower 582 (100 % and n = 19, 56 % and n = 45, respectively). 583 Therefore, many biocompatible IL and DES solutions can be applied to enhance 584 the stability, activity, and delivery of insulin. The development of more stable insulin 585 formulations with the possibility of oral and transdermal delivery can improve 586 prognosis and patient adherence to diabetes treatments. 587 5.1.4. Ovalbumin epitope (OVA) 588 OVA is a glycopeptide and the major protein constituent from chicken egg whites. 589 Furthermore, OVA is a small (1 kDa) peptide formed by β-sheets and β-turns (Figure 590 3.D) (Tahara et al., 2020). Because it is mildly immunogenic, OVA is used as an 591 antigen in vaccines to improve immunogenic response. However, OVA and most other 592 antigens used as adjuvants in vaccines have low solubility in oil-based formulations for 593 transdermal delivery (Tahara et al., 2020). Considering it is possible to design 594 amphipathic ILs, they can be a suitable media to solubilize and improve the skin 595 permeation of OVA. 596 To increase the solubility and skin penetration of OVA, Tahara et al. (Tahara et 597 al., 2020) applied cholinium fatty acid-based ILs as additives from 0.2 to 0.7 M. The IL 598 cholinium cis-9-octadecenoate ([Ch][C₁₈:₁]) and cholinium dodecanoate ([Ch][C₁₂:0]) at 599 20 wt% (0.5 and 0.7 M, respectively) enhanced the skin penetration of OVA, while 600 [Ch][C₁₈:₁] also improved the solubility and activity (suppression of tumor growth in 601 vivo) of OVA at 0.2 M. [Ch][C₁₈:₁] increased 28-fold the delivery of OVA when 602 compared with the control using an aqueous vehicle. Furthermore, [Ch][C₁₈:₁] was 603 found to be biocompatible with dendritic cells and to not cause skin irritation. Thus, 604 cholinium fatty-acid ILs can be used for the development of novel transdermal drug 605 delivery systems due to their biocompatibility with cells and skin and their ability to 606 help in the dissolution and skin penetration of hydrophilic biopharmaceuticals. 607 38 5.1.5. Immunoglobulin G1 (IgG1) 608 IgG1 is the most abundant immunoglobulin in humans and is one of the main 609 antibodies for mediating the humoral response against infections. As presented in 610 Figure 3.E, IgG1 is a large globular tetrameric protein (around 140 kDa) with two 611 heavy chains and two light chains (Harris et al., 1998). The identical heavy chains 612 connect at the base of the protein structure forming a Y shape (Fc region, conserved 613 portion). The top portion of the Y is the Fab region, which binds to antigens and is 614 highly variable. In the medical field, IgG1 is the main representative of monoclonal 615 antibodies, the largest class of biopharmaceuticals (Angsantikul et al., 2021). They are 616 used to treat several illnesses, such as cancers, infections, inflammations, and 617 autoimmune diseases. However, because IgG1 is a large and complex protein, it is 618 prone to unfolding and aggregation, losing its affinity towards its specific antigens and 619 in consequence, its function (Manning et al., 2010). Moreover, antibodies are not 620 resistant or have poor absorption in oral administration (Angsantikul et al., 2021). Thus, 621 the development of formulations to enhance the stability and delivery of 622 immunoglobulins can improve and expand their application in medicine. 623 Researchers have applied cholinium ILs and DESs to enhance the thermal stability 624 of IgG1. For example, Dhiman et al. used low concentrations of cholinium acetate 625 ([Ch][CH₃COO]) to increase the Tm of IgG1 from human serum (Dhiman et al., 2022). 626 In a follow-up study, the group saw that low to high concentrations (around 0.2 to 1.5 627 M) of cholinium DESs [i.e., choline chloride and carbamide ([Ch]Cl–[CO(NH₂)₂]), 628 choline chloride and glycerol ([Ch]Cl–[C₃H₅(OH)₃]), choline chloride and ethylene 629 glycol ([Ch]Cl–[(CH₂OH)₂])] also have a positive effect on the thermal stability of this 630 protein (Dhiman et al., 2023). Therefore, cholinium ILs and DESs are suitable 631 compounds to decrease the thermal unfolding of antibodies. 632 There are also several studies on the effects of ILs and DESs on the structural 633 stability of IgG1. In addition to evaluating the effect of cholinium ILs on IgG1 Tm, 634 Dhiman et al. showed that 0.0005 to 0.0025 M of [Ch][CH₃COO], cholinium citrate 635 (i.e., citrate also known as 2-hydroxypropane-1,2,3-tricarboxylate) 636 ([Ch][C₅H₇O₅COO]), and [Ch]H₂PO₄ and around 0.2 to 0.8 M [Ch]Cl–[CO(NH₂)₂], 637 [Ch]Cl–[C₃H₅(OH)₃], and [Ch]Cl–[(CH₂OH)₂] preserved the native structure of this 638 antibody (Dhiman et al., 2023, 2022). In a similar approach, Mondal et al. (Mondal et 639 al., 2016) and Angsantikul et al. demonstrated that high concentrations of cholinium 640 39 ILs and DESs can also maintain the structural integrity of IgG1 (Angsantikul et al., 641 2021). Specifically, Mondal et al. used around 0.9 to 1.4 M of cholinium 642 hydroxyacetate ([Ch][HOCH₂COO]) (i.e., also called cholinium glycolate) and 643 [Ch][C₅H₇O₄COO] with IgG from rabbit serum, (Mondal et al., 2016) while 644 Angsantikul et al. applied around 1.8 to 2.8 M of [Ch][HOCH₂COO], cholinium 645 hydroxyacetic acid 2:1 ([Ch][HOCH₂COO]–[Ch]), and cholinium hydroxyacetic acid 646 1:2 ([Ch][HOCH₂COO]–[HOCH₂COOH]) to maintain the structure of antihuman TNF-647 α mouse IgG1 (Angsantikul et al., 2021). With similar results, Rawat and Bohidar 648 (Rawat and Bohidar, 2015) showed that dilution solutions (0.04 M) of the imidazolium 649 ILs 1-ethyl-3-methylimidazolium chloride ([C₂MIm]Cl), [C₄MIm]Cl, 1-hexyl-3-650 methylimidazolium chloride ([C₆MIm]Cl), and 1-octyl-3-methylimidazolium chloride 651 ([C₈MIm]Cl) can increase the structural stability of human IgG1. For Ferreira et al. 652 (Ferreira et al., 2016), low concentrations (around 0.1 to 0.3 M) of imidazolium and 653 ammonium ILs also maintain the structure of IgG1 from rabbit serum. The tested ILs 654 included 1-butyl-3-methylimidazolium tosylate ([C₄MIm][CH₃BzSO₃]), 655 [C₄MIm][N(CN)₂], [C₄MIm][CH₃COO], 1-ethyl-3-methylimidazolium bromide 656 ([C₂MIm]Br), 1-butyl-3-methylimidazolium bromide ([C₄MIm]Br), [C₄MIm]Cl, 657 [N₁,₁,₁,₁]Br, [N₂,₂,₂,₂]Br, and [N₃,₃,₃,₃]Br. However, high concentrations (around 1.1 to 658 2.5 M) of certain cholinium ILs and even dilute solutions (0.1 to 0.2 M) of bulkier 659 phosphonium and ammonium ILs caused partial unfolding of the antibodies. The dilute 660 IL solutions that decrease the structural stability of IgG1 were [Ch]Cl–[CO(NH₂)₂], 661 [Ch]Cl–[C₃H₅(OH)₃], [Ch]Cl–[(CH₂OH)₂] (Dhiman et al., 2023), cholinium indole-3-662 acetate ([Ch][C₉H₈NCOO]), cholinium 2-oxopropanoate ([Ch][CH₃COCOO]) (Mondal 663 et al., 2016), tetrabutylphosphonium bromide ([P₄,₄,₄,₄]Br), and tetrabutylammonium 664 bromide ([N₄,₄,₄,₄]Br) (Ferreira et al., 2016). Overall, dilute solutions of ILs and DESs 665 are better at improving or maintaining the native structure of IgG1 than concentrated 666 solutions or low concentrations of bulkier ILs and DESs. 667 Regarding the effect of ILs and DESs on the activity of antibodies, there is only 668 one study on this topic. Angsantikul et al. (Angsantikul et al., 2021) observed that 669 around 0.7 to 3.9 M of [Ch][HOCH₂COO] and [Ch][HOCH₂COO]–[Ch] improved the 670 antigen binding capacity of antihuman TNF-α mouse IgG1, while around 0.8 to 3.5 M 671 of [Ch][HOCH₂COO]–[HOCH₂COOH] impaired it. As there is still only one study on 672 the impact of ILs and DESs on IgG1 activity, we recommend other studies to address 673 this variable. 674 40 The ILs and DESs can also affect the long-term storage and delivery of 675 antibodies. For example, Dhiman et al. (Dhiman et al., 2023) observed that 1.5 M of 676 [Ch]Cl–[CO(NH₂)₂] can preserve IgG1 for 20 days in solution, while there is 677 aggregation and partial unfolding in phosphate buffer for the same time. In another 678 approach, Angsantikul et al. (Angsantikul et al., 2021) were able to improve the 679 delivery of antihuman TNF-α mouse IgG1, a monoclonal antibody for the treatment of 680 gastrointestinal infections and inflammatory bowel disease, using cholinium DESs. The 681 researchers enhanced the paracellular transportation of the antibody with solutions from 682 0.03 to 0.08 M of [Ch][HOCH₂COO]–[Ch] and enhanced IgG1 delivery into the 683 intestinal mucosa and systemic circulation with 1.4 M [Ch][HOCH₂COO]–[Ch] 684 solution. Furthermore, [Ch][HOCH₂COO]–[Ch] is also biocompatible with Caco-2 685 Cells and rats, causing no damage to their gastrointestinal tissue and normal liver and 686 kidney functions. Thus, cholinium DES solutions are suitable and biocompatible 687 vehicles for the long-term storage and improved delivery of IgG1. 688 As previously presented for insulin, Figure 6 shows the percentage of IL and 689 DES solutions that increased or maintained the stability of IgG1 insulin according to the 690 stability type, IL class, and concentration range. 691 692 Figure 6. A) Percentage of IL and DES solutions that maintained or increased the different types of 693 stability (structural, thermal, activity, and aggregation) of IgG1. For aggregation, improvement of stability 694 represents ILs that decrease protein aggregation. B) Percentage of IL and DES solutions that maintained 695 or increased the stability of IgG1 according to their class. C) Percentage of IL and DEs solutions that 696 maintained or increased the stability of IgG1 according to their concentration (<1 or ≥ 1 M). n = total 697 number of samples for each condition. 698 699 In Figure 6.A, most IL and DES solutions improved the stability of IgG1 (73 %, n 700 = 74), with 81 % improving or preserving IgG1 structural (n = 32) and 100 % its 701 thermal stability (n = 12), 55 % reducing its aggregation (n = 22), and 50 % enhancing 702 or maintaining its activity (n = 8). As for insulin, only one study with 8 solutions 703 included the effect of ILs and DESs on the biological activity of the protein. Thus, we 704 suggest more studies include this parameter in their research design, considering the 705 41 maintenance of the activity of the biopharmaceutical is vital to allow its medical 706 application. 707 Regarding the different classes in Figure 6.B, ammonium-, cholinium- and 708 imidazolium-based IL and DESs solutions had a positive effect on IgG1, with 75 % (n = 709 4), 69 % (n = 54), and 100 % (n = 14) preserving its stability, respectively. Furthermore, 710 it should be noted that again, cholinium ILs and DESs also could improve the long-term 711 preservation and oral delivery of the biopharmaceutical, showing potential outside of 712 only enhancing protein stability. 713 As for the concentration range in Figure 6.C, dilute solutions (82 %, n = 51) were 714 better at preserving IgG1 than concentrated ILs (52 %, n = 23). Interestingly, this trend 715 is the opposite of what was observed for insulin, showing the nature of the protein will 716 change the tendencies and types of interactions between proteins and ILs and DESs. 717 As discussed, cholinium-based ILs and DESs have been investigated for their 718 potential to enhance the stability and delivery of IgG1. Both classes were able to 719 increase the thermal stability of IgG1. However, their effect on the structural integrity of 720 the protein differed based on concentration and the specific solvent. Generally, dilute 721 solutions of both ILs and DESs are more successful at maintaining the native structure 722 of IgG1 than concentrated solutions. Nonetheless, certain concentrated cholinium ILs 723 and even some dilute solutions of bulkier ILs have caused partial unfolding of the 724 antibodies. On the delivery front, cholinium DESs have showcased a distinct advantage, 725 demonstrating their ability to not only improve the long-term storage of IgG1 but also 726 enhance its delivery into the intestinal mucosa and systemic circulation. This suggests 727 that while both ILs and DESs can aid in the stabilization of IgG1, cholinium-based 728 DESs might offer superior advantages for antibody delivery applications. 729 In conclusion, ILs and DESs can be applied to improve the stability, activity, long-730 term preservation, and oral delivery of antibodies. These novel formulations can 731 potentially help in expanding and increasing access to antibodies for the treatment of 732 life-threatening diseases such as cancers, infections, and auto-immune disorders. 733 5.1.6. L-Asparaginase (L-ASNase) 734 L-ASNase is an enzyme used for the treatment of acute lymphoblastic leukemia 735 and other types of cancer (Stecher et al., 1999) and in the food industry as an acrylamide 736 mitigation agent (Bento et al., 2022). There are multiple L-ASNase types, with types I 737 and II being more applied in biotechnology. Type II is a bacterial periplasmic or 738 42 membrane-associated enzyme with a high affinity for L-asparagine and low activity 739 towards L-glutamine, which is ideal for applications as a pharmaceutical (Castro et al., 740 2021). Type I still has an affinity towards L-asparagine, but its hydrolysis of L-741 glutamine limits its application to the food industry. The L-ASNase structure is 742 tetrameric with two connected α/β domains (i.e., N-terminal and C-terminal) for each of 743 its four subunits, as shown in Figure 3.C. The N-terminal domain presents eight-744 stranded mixed β-sheets with four α-helices, and the C-terminal of four-stranded 745 parallel β-sheets with four α-helices (Swain et al., 1993). The anti-leukemia property of 746 L-ASNase is based on the hydrolysis of L-asparagine and the depletion of this amino 747 acid necessary for the survival of cancer cells (Van Trimpont et al., 2022). However, 748 different sources and environments can alter the catalytic activity of L-ANSase, 749 improve or impair its pharmaceutical use, and reduce or increase its adverse effects. 750 Regarding its stability, Elspar® (L-ASNase biopharmaceutical from Escherichia 751 coli) can be maintained for up to 7 days in solution in the fridge (Stecher et al., 1999), 752 but its activity decreases substantially after a few hours at 50 °C (Magri et al., 2019). L-753 ASNase from other sources can vary in properties and stability, hence, it is necessary to 754 take this information into account when comparing the enzyme variants (Bento et al., 755 2022). Furthermore, L-ASNase is usually obtained from microorganisms, which 756 generate many contaminants that can cause unwanted clinical effects, such as 757 hyperglycemia and hepatotoxicity (Li et al., 2019). The purification of L-ASNase to 758 medical grade levels is complex and costly, raising the price of this biopharmaceutical 759 and limiting its access (Dos Santos et al., 2018). Thus, the development of technologies 760 to enhance L-ASNase stability, activity, and extraction can improve its clinical use in 761 cancer treatment. 762 ILs can be used as additives to improve the hydrolysis of L-asparagine by L-763 ASNase, as observed in a study by Magri et al. that worked with L-ASNase EC 3.5.1.1 764 produced by E. coli (Magri et al., 2019). The ILs [Ch][CH₃COO], cholinium propanoate 765 ([Ch][C₂H₅COO]), cholinium butanoate ([Ch][C₃H₇COO]) improved asparagine 766 hydrolysis from 0.001 to 0.050 mol IL/mol total and cholinium hexanoate 767 ([Ch][C₅H₁₁COO]) from 0.001 to 0.010 mol IL/mol total at room temperature. However, 768 higher concentrations of [Ch][C₅H₁₁COO] (0.025 to 0.050 mol IL/mol total) or the 769 increase of temperature to 50 °C impaired L-ASNase catalysis for all ILs when 770 compared to the buffer. 771 43 The recovery and preservation of biopharmaceuticals during their upstream 772 processing is also a topic of relevance for the pharmaceutical industry. With this in 773 mind, Li et al. (Li et al., 2019) applied the DES poly(vinyl pyrrolidone) and 774 propanedioic acid 1:1 ([(C6H9NO)n]–[CH₂(COOH)₂]) to adsorb L-ASNase from E. coli 775 extract. The addition of 0.1 M of [(C6H9NO)n]–[CH₂(COOH)₂] to the bacterial extract 776 facilitates the separation of the enzyme from their complex medium. Even though there 777 was a slight alteration in the protein structure, the catalytic activity of the protein was 778 preserved. 779 Considering the goals of application of the ILs and DESs with L-ASNase were 780 very