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Representative studies of the application of chromatographic techniques for the purification of distinct classes of recombinant therapeutic IFNs.
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The advent of biopharmaceuticals in modern medicine brought enormous benefits to the treatment of numerous human diseases and improved the well-being of many people worldwide. First introduced in the market in the early 1980s, the number of approved biopharmaceutical products has been steadily increasing, with therapeutic proteins, antibodies, and...
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... chromatographic methods generally applied to the isolation and purification of IFNs include (i) affinity chromatography, (ii) IEX, (iii) SEC, (iv) reverse-phase, and (v) HIC. Representative studies of the chromatographic purification of IFNs are overviewed in Table 5, and the separation principles of each method are schematized in Figure 7. Abbreviations: AC-Affinity chromatography; AEX-Anion-exchange chromatography; CEX-Cation-exchange chromatography; HIC-Hydrophobic interaction chromatography; IAC-Immunoaffinity chromatography; IMAC-Immobilized metal-affinity chromatography; NR-Not reported; RP-Reverse-phase chromatography; SEC-Size exclusion chromatography. Affinity chromatography allows a specific type of protein to be isolated from a mixture of proteins contaminants and is based on the affinity of proteins to specific ligands, for instance, metal cations or antibodies, respectively, in IMAC or immunoaffinity chromatography [40]. ...Context 2
... and coauthors [77] used the Ni-NTA (nitrilotriacetic acid) IMAC column for the purification of 8xHis-tagged extracellular recombinant IFNγ from P. pastoris GS115 crude broth. The authors found that the purity of IFNγ was positively correlated with the production levels: the codonoptimized version of IFNγ-GS-IFNγ opt -, the IFNγ co-expressed with PDI-GS-IFNγ-PDIand the native version, GS-IFNγ-were obtained, respectively, with purities of 80%, 63.83%, and 56.5% (Table 5) [77]. This study reinforces the premise defended by several researchers in which the purification yield seems to be proportional to the initial concentration of IFN and its purity [4]. ...Context 3
... and coauthors [77] used the Ni-NTA (nitrilotriacetic acid) IMAC column for the purification of 8xHis-tagged extracellular recombinant IFNγ from P. pastoris GS115 crude broth. The authors found that the purity of IFNγ was positively correlated with the production levels: the codon-optimized version of IFNγ-GS-IFNγ opt -, the IFNγ co-expressed with PDI-GS-IFNγ-PDI-and the native version, GS-IFNγ-were obtained, respectively, with purities of 80%, 63.83%, and 56.5% (Table 5) [77]. This study reinforces the premise defended by several researchers in which the purification yield seems to be proportional to the initial concentration of IFN and its purity [4]. ...Citations
... It is a small protein composed of 165 amino acids that comprise a helical secondary structure consisting of 5 alpha helices [3]. It has a considerable market value as an anti-viral medication that prevents infections and boosts natural immunity [4,5]. It also has a high demand among academic professionals for scientific research. ...
... It also has a high demand among academic professionals for scientific research. However, in vitro production of native IFN in mammalian cells for therapeutic purposes is challenging and expensive [4,6]. As a result, biotech companies and academic professionals have lately exploited recombinant-DNA technology to heterologously generate IFNs in a variety of host species, including Escherichia coli, yeast, and plants [4,7,8]. ...
... However, in vitro production of native IFN in mammalian cells for therapeutic purposes is challenging and expensive [4,6]. As a result, biotech companies and academic professionals have lately exploited recombinant-DNA technology to heterologously generate IFNs in a variety of host species, including Escherichia coli, yeast, and plants [4,7,8]. The production of active recombinant-IFNs in these heterologous host species presents several challenges due to IFN's susceptibility to heat-stress, protein misfolding, and structural instability (degradation) via the host cell proteasome-mediated recombinant protein removal processes [9,10]. ...
... Nanoparticles are nanoscale structures that can be either capsules or spheres, depending on their internal constitution [27]. These systems simplify the administration of IFNs, improve therapeutic efficacy, and reduce dose-related side effects without decreasing biological activity or altering the protein structure [28]. The development of new delivery strategies allowing local and controlled release of IFN-α through biological activity potentiated with another cytokine that regulates its action, as is the case with IFN-γ, would allow achieving both an optimal preventive and therapeutic response to immune system activation [29]. ...
... The use of biopharmaceuticals has contributed to shortening patients' recovery time and improving their quality of life [28]. Recombinant proteins and antibodies are the most abundant therapeutic bioproducts on the market [164]. ...
Background: Interferons (IFNs) are cytokines involved in the immune response with a synergistic regulatory effect on the immune response. They are therapeutics for various viral and proliferative conditions, with proven safety and efficacy. Their clinical application is challenging due to the molecules’ size, degradation, and pharmacokinetics. We are working on new drug delivery systems that provide adequate therapeutic concentrations for these cytokines and prolong their half-life in the circulation, such as nanoformulations. Methods: Through nanoencapsulation using electrospray technology and biocompatible and biodegradable polymers, we are developing a controlled release system based on nanoparticles for viral infections of the respiratory tract. Results: We developed a controlled release system for viral respiratory tract infections. A prototype nanoparticle with a core was created, which hydrolyzed the polyvinylpyrrolidone (PVP) shell , releasing the active ingredients interferon-alpha (IFN-α) and interferon-gamma (IFN-γ). The chitosan (QS) core degraded slowly, with a controlled release of IFN-α. The primary and rapid effect of the interferon combination ensured an antiviral and immunoregulatory response from day one, induced by IFN-α and enhanced by IFN-γ. The multilayer design demonstrated an optimal toxicity profile. Conclusions: This formulation is an inhaled dry powder intended for the non-invasive intranasal route. The product does not require a cold chain and has the potential for self-administration in the face of emerging viral infections. This novel drug has applications in multiple infectious, oncological, and autoimmune conditions, and further development is proposed for its therapeutic potential. This prototype would ensure greater bioavailability, controlled release, fewer adverse effects, and robust biological action through the simultaneous action of both molecules.
... Nanoparticles are nanoscale structures that can be either capsules or spheres, depending on their internal constitution [15]. These systems simplify the administration of IFNs, improve therapeutic efficacy, and reduce dose-related side effects without decreasing biological activity or altering the protein structure [16]. The development of new delivery strategies allowing local and controlled release of interferon-alpha (IFN-α) through biological activity potentiated with another cytokine that regulates its action, as is the case with interferon-gamma (IFN-γ), would allow achieving both an optimal preventive and therapeutic response to immune system activation [17]. ...
... PVP increased cell proliferation at the latter concentrations, which had greater relevance to the formulation without active ingredients because PVP domains provide a suitable environment for cell growth [102,103]. 16 Cell viability was lower in the protein-encapsulated formulations than in the empty formulations, especially in the HeLa cell line, showing the encapsulated interferons' anticancer action [104]. This finding is related to the release kinetics assay that showed an initial abrupt release of the encapsulated proteins in the first hours and decreased cell viability on HeLa cells at early concentrations. ...
... The use of biopharmaceuticals has contributed to shortening patients' recovery time and improving their quality of life [16]. Recombinant proteins and antibodies are the most abundant therapeutic bioproducts on the market [146]. ...
Interferons (IFNs) are cytokines involved in the immune response with synergistic regulatory action. They are therapeutics for various viral and proliferative conditions, with proven safety and efficacy. Their clinical application presents difficulties due to the molecules' size, degradation, and pharmacokinetics. We developed a controlled release system for viral respiratory tract infections. A core-shell nanoparticle was prototyped, which hydrolyzed the shell (polyvinylpyrrolidone), releasing the active ingredients IFN-α and IFN-γ. The core (chitosan) degraded slowly, with a controlled release of IFN-α. The primary and rapid effect of the combination of interferons ensured an antiviral and immunoregulatory response from day one, induced by IFN-α and enhanced by IFN-γ. The multilayer design demonstrated an optimal toxicity profile. This formulation is an inhaled dry powder targeted for the non-invasive intranasal route. This prototype would ensure greater bioavailability, controlled release, fewer adverse effects, and robust biological action thanks to the simultaneous impact of both molecules.
... In the preparation of inclusion-body IFN-α2b from E. coli, other proteins derived from unbroken cells (most likely) or E. coli cytoplasmatic proteins co-precipitated or trapped during aggregate formation [15] are also present, such as serum albumin [14]. Several hosts have been applied to produce all classes of interferon molecules, such as Escherichia coli, Bacillus subtilis, Lactococcus lactistext, Pichia pastoristext, Saccharomyces cerevisiae, Trichoderma reesei, Yarrowia lipolytica, insect cells, plants, transgenic mice, and mammalian cells [6,87]. For example, the in vivo efficacy of IFN beta increases by its natural glycosylation [88], and since mammalian cell lines are the best hosts for obtaining recombinant proteins with native glycosylation patterns, they represent the best compromise between yield and quality. ...
Interferon alpha-2b (IFN-α2b) is an essential cytokine widely used in the treatment of chronic hepatitis C and hairy cell leukemia, and serum albumin is the most abundant plasma protein with numerous physiological functions. Effective single-step aqueous biphasic system (ABS) extraction for the simultaneous purification of IFN-α2b and BSA (serum albumin protein) was developed in this work. Effects of the ionic liquid (IL)-based ABS functionalization, fluorinated ILs (FILs; [C 2C 1Im][C 4F 9SO 3] and [N 1112(OH)][C 4F 9SO 3]) vs. mere fluoro-containing IL ([C 4C 1Im][CF 3SO 3]), in combination with sucrose or [N 1112(OH)][H 2PO 4] (well-known globular protein stabilizers), or high-charge-density salt K 3PO 4 were investigated. The effects of phase pH, phase water content (%wt), phase composition (%wt), and phase volume ratio were investigated. The phase pH was found to have a significant effect on IFN-α2b and BSA partition. Experimental results show that simultaneous single-step purification was achieved with a high yield (extraction efficiency up to 100%) for both proteins and a purification factor of IFN-α2b high in the enriched IFN-α2b phase (up to 23.22) and low in the BSA-enriched phase (down to 0.00). SDS-PAGE analysis confirmed the purity of both recovered proteins. The stability and structure of IFN-α2b and BSA were preserved or even improved (FIL-rich phase) during the purification step, as evaluated by CD spectroscopy and DSC. Binding studies of IFN-α2b and BSA with the ABS phase-forming components were assessed by MST, showing the strong interaction between FILs aggregates and both proteins. In view of their biocompatibility, customizable properties, and selectivity, FIL-based ABSs are suggested as an improved purification step that could facilitate the development of biologics.
... The main applications of AAs are as adjuvants, to improve the immunological response to a given antigen (Azuar et al., 2021;Skwarczynski et al., 2020;Lim et al., 2019); stabilizers, to avoid losses and degradation of BPs during the lyophilization process (Castro et al., 2021;Idrees et al., 2020;Mohammed, Coombes, Perrie, 2007); nanoparticles (Vrieling et al., 2019); and buffers (Wlodarczyk et al., 2018). ...
A simple, rapid, precise, accurate and sustainable spectrofluorimetric method (SFM) was developed, validated and applied for the determination of 4-aminobenzoic acid and aromatic aminoacids (phenylalanine, tryptophan and tyrosine). These compounds are used in biopharmaceutical formulations and therefore must be analyzed by quality control laboratories to meet the criteriaestablished in pharmacopoeias. In general, potentiometric titration (PT) is described in the compendia as the official analytical technique. However, this method showed low sensitivity and selectivity, and moreover was performed with a non-aqueous solvent (acetic acid), which led to higher consumption of reagents and consequently to the formation of residues. Therefore, the SFM was developed in aqueous medium at pH 7.2 using phosphate buffer. It was successfully validated according to the ICH guidelines and showed good linearity range (r > 0.999), specificity, accuracy and precision (within and between days) and robustness. The test results were compared between the SFM and PT using raw material samples, while according to the F- and t- tests at 95% confidence level, no statistical difference was found between the methods.
... While expression systems based on bacterial and mammalian cell cultures are dominant production of most recombinant proteins, including interferons (Castro et al. 2021), each of them has several limitations, e.g., existence of bacterial endotoxins, possible contamination with human pathogens, oncogenes or prions, and recombinant protein aggregation into insoluble inclusion bodies. These disadvantages can be overcome by alternative expression system, such as plant expression system. ...
Human interferon (hINF) alpha 2b is clinically important pharmaceutical product included in combinatory therapy against chronic hepatitis C and B and complex therapy against several cancer diseases. Here, we created the genetic constructions, based on genome elements of potato virus X (PVX), carrying the infα2b gene for transient expression in plant cells. The created plasmid vector constructions were tested through Agrobacterium-mediated transient gene expression method in two plant species—Nicotiana benthamiana and Ocimum basilicum (sweet basil). Production of recombinant hINF alpha 2b was more efficient in N. benthamiana than that in O. basilicum plants. The average yield of hINF alpha 2b produced in N. benthamiana plants was 0.56 mg/g of fresh leaf weight (FW) or 6% of the total soluble cell proteins (TSP). The maximal level reached up to 1.2 mg/g FW or 9% TSP. We estimated that about 0.67 mg of hINF can be obtained from one N. benthamiana plant. The yield of hINF alpha 2b obtained with the PVX-based expression cassette was about 80 times higher than the yield of hINF alpha 2b obtained with a simple expression cassette in which the infα2b gene was controlled by the 35S promoter of cauliflower mosaic virus.
Key points
• PVX-based expression vectors provide efficient transient expression of infα2b gene
• N. benthamiana plants can produce human interferon alpha 2b at high levels
• The yield of the hINF α2b reached up to 1.2 mg/g of fresh leaf weight
... Interferons are class II α-helical cytokine proteins secreted by lymphocytes, leukocytes, and fibroblasts that can be used in anticancer and antiviral treatments [1]. Interferons are categorized into Type I (including IFN-α, IFN-β, IFN-κ, IFN-ω, and IFN-), Type II (IFN-γ ), or Type III (encompassing IFN-λ1, IFN-λ2, IFN-λ3, and IFN-λ4) based on their antigenic properties and their biological and chemical characteristics [2,3]. ...
... Post-viral infection type I IF is expressed with innate antiviral activity inhibiting viral replication and aids in inducing long-term immunity via adaptive immune responses [91]. Therapeutic IFs were initially derived from leukocytes and lymphoblastoid cell lines; RDNA technology, however, has quickly encouraged the use of recombinant IF production in large-scale bioreactors [92]. Many expression systems (Table 2), including E. coli and yeast, can be used to produce recombinant cytokines such as interferons [40]. ...
... P. pastoris and Y. lipolytica are yeast expression systems for the production of interferon-α for the treatment of hepatitis B and C [95]. Currently, E. coli and P. pastoris are the most widely used expression systems for producing clinical IFs; IF β is produced in CHO cells as its activity increases with glycosylation [92]. Proteins produced by P. pastoris are secreted intracellularly or extracellularly where protein degradation can be an issue; protein protease-deficient strains, e.g., SMD 1168, can be used to overcome this [96]. ...
... mAb against SARS-CoV-2 targeting the spike protein have demonstrated efficacy in vitro [101]. Indeed, there are approximately 70 mAbs in development for treatment of SARS-CoV-2, with four agents granted emergency use authorization by the FDA as antibody cocktails [92]. Yet, despite promising results in animal models, current research shows anti-SARS-CoV-2 mAbs are ineffective against newer Omicron variants and its subvariants [102]. ...
Emerging, re-emerging and zoonotic viral pathogens represent a serious threat to human health, resulting in morbidity, mortality and potentially economic instability at a global scale. Certainly, the recent emergence of the novel SARS-CoV-2 virus (and its variants) highlighted the impact of such pathogens, with the pandemic creating unprecedented and continued demands for the accelerated production of antiviral therapeutics. With limited effective small molecule therapies available for metaphylaxis, vaccination programs have been the mainstay against virulent viral species. Traditional vaccines remain highly effective at providing high antibody titres, but are, however, slow to manufacture in times of emergency. The limitations of traditional vaccine modalities may be overcome by novel strategies, as outlined herein. To prevent future disease outbreaks, paradigm shift changes in manufacturing and distribution are necessary to advance the production of vaccines, monoclonal antibodies, cytokines and other antiviral therapies. Accelerated paths for antivirals have been made possible due to advances in bioprocessing, leading to the production of novel antiviral agents. This review outlines the role of bioprocessing in the production of biologics and advances in mitigating viral infectious disease. In an era of emerging viral diseases and the proliferation of antimicrobial resistance, this review provides insight into a significant method of antiviral agent production which is key to protecting public health.
... The codon sequences of the IFN genes, which are available in online database sequences and have been previously discovered and designed by the farmer's research can be used for insertion for the application through vector (pET28a expression vector) insertion in the bacteria. E. coli is a natural choice for the commercial production of interferon (Castro et al., 2021). In case when the codon sequence of the IFN genes is online not available, it can design in any advanced molecular laboratory [33]. ...
... DS-PAGE which stands for Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis is often used to check the purity and concentration of the IFN proteins. Digital gel doc, reading machine of SDS-PAGE, analyses the test sample with help of the band's density and with [6,16].Lowry and Bradford's assays are also used to determine the concentration of IFN protein by calculating the relative value of the test sample with the standard samples HPSEC which stands for High-Performance Size-Exclusion Chromatography is used to concentrate the isolated recombinant IFN proteins [35]. ...
This review examines the use of interferon as a potential drug against SARS-CoV-2 and other animal coronaviruses. The production of interferon is also discussed. Despite the ongoing global pandemic of COVID-19, there are currently no effective drugs available to treat SARS-CoV-2 and its new variants. The review aims to determine if interferon could be an effective treatment option. The role and importance of interferon in viral drug development and production are summarized using recent research and literature.
... Being a bridge between innate and adaptive immunity, being essential in host defense and involved in cancer and autoimmunity, placed interferon in the limited list of master cytokines. As such it is being continuously revisited and is a source for drug discovery (33). ...
Human urinary proteins are a goldmine of natural proteins a feature that simplifies their translation to biologics. Combining this goldmine together with the ligand-affinity-chromatography (LAC) purification method, proved a winning formula in their isolation. LAC specificity, efficiency, simplicity and inherent indispensability in the search for predictable and unpredictable proteins, is superior to other separation techniques. Unlimited amounts of recombinant cytokines and monoclonal antibodies (mAb) accelerated the “triumph”. My approach concluded 35 years of worldwide pursuit for Type I IFN receptor (IFNAR2) and advanced the understanding of the signal transduction of this Type of IFN. TNF, IFNγ and IL-6 as baits enabled the isolation of their corresponding soluble receptors and N-terminal amino acid sequence of the isolated proteins facilitated the cloning of their cell surface counterparts. IL-18, IL-32, and heparanase as the baits yielded the corresponding unpredictable proteins: the antidote IL-18 Binding Protein (IL-18BP), the enzyme Proteinase 3 (PR3) and the hormone Resistin. IFNβ proved beneficial in Multiple Sclerosis and is a blockbuster drug, Rebif®. TNF mAbs translated into Remicade® to treat Crohn’s disease. Enbrel® based on TBPII is for Rheumatoid Arthritis. Both are blockbusters. Tadekinig alfa™, a recombinant IL-18BP, is in phase III clinical study for inflammatory and autoimmune diseases. Seven years of continuous compassionate use of Tadekinig alfa™ in children born with mutations (NLRC4, XIAP) proved life-saving and is an example of tailored made medicine. IL-18 is a checkpoint biomarker in cancer and IL-18BP is planned recently to target cytokine storms resulting from CAR-T treatment and in COVID 19.
