Development of a biotechnology process for the production of a novel hyperglycosylated long-acting recombinant bovine follicle-stimulating hormone.

Follicle-stimulating hormone (FSH) is an important protein used for bovine ovarian hyperstimulation in multiple ovulation and embryo transfer technology (MOET). Several attempts to produce bovine FSH (bFSH) in recombinant systems have been reported, nonetheless, up to date, the most commonly used products are partially purified preparations derived from porcine or ovine (pFSH or oFSH) pituitaries. Here we describe the development of a biotechnology process to produce a novel, hyperglycosylated, long-acting recombinant bFSH (LA-rbFSH) by fusing copies of a highly O-glycosylated peptide. LA-rbFSH and a nonmodified version (rbFSH) were produced in suspension CHO cell cultures and purified by IMAC with high purity levels (>99%). LA-rbFSH presented a higher glycosylation degree and sialic acid content than rbFSH. It also demonstrated a notable improvement in pharmacokinetic properties after administration to rats, including a higher concentration in plasma and a significant (seven-fold) reduction in apparent clearance (CLapp). In addition, the in vivo specific bioactivity of LA-rbFSH in rats was 2.4-fold higher compared to rbFSH. These results postulate this new molecule as an attractive substitute for commercially available porcine pituitary-derived products.

Optimization and Validation of a Liquid Formulation for a New Recombinant Veterinary Product using QbD Approach.

Protein formulation and drug characterization are one of the most difficult and time-consuming tasks because of the complexity of biotherapeutic proteins. Hence, maintaining a protein drug in its active state typically requires preventing changes in its physical and chemical properties. Quality by Design (QbD) is a systematic approach emphasizing product and process understanding. Design of Experiments (DoE) is one of the most important QbD tools, allowing the possibility to modify the formulation attributes within a defined design space. Here, we report the validation of a RP-HPLC assay for recombinant equine chorionic gonadotropin (reCG) that demonstrated a high correlation with the in vivo potency biological assay. QbD concepts were then applied to obtain an optimized liquid formulation of reCG with a predefined quality product profile. The developed strategy demonstrates the importance of applying multivariable strategies as DoE to simplify formulation stages, improving the quality of the obtained results. Moreover, it is important to highlight that this is the first time that a liquid formulation is reported for an eCG molecule, since, up to now, the only eCG products available in the market for veterinary use consisted in partially purified preparations of pregnant mare serum gonadotropin (PMSG) presented as a lyophilized product.

Design and characterization of chimeric Rabies-SARS-CoV-2 virus-like particles for vaccine purposes.

Due to the high number of doses required to achieve adequate coverage in the context of COVID-19 pandemics, there is a great need for novel vaccine developments. In this field, there have been research approaches that focused on the production of SARS-CoV-2 virus-like particles. These are promising vaccine candidates as their structure is similar to that of native virions but they lack the genome, constituting a biosafe alternative. In order to produce these structures using mammal cells, it has been established that all four structural proteins must be expressed. Here we report the generation and characterization of a novel chimeric virus-like particle (VLP) that can be produced by the expression of a single novel fusion protein that contains SARS-CoV-2 spike (S) ectodomain fused to rabies glycoprotein membrane anchoring region in HEK293 cells. This protein is structurally similar to native S and can autonomously bud forming enveloped VLPs that resemble native virions both in size and in morphology, displaying S ectodomain and receptor binding domain (RBD) on their surface. As a proof of concept, we analyzed the immunogenicity of this vaccine candidate in mice and confirmed the generation of anti-S, anti-RBD, and neutralizing antibodies. KEY POINTS: • A novel fusion rabies glycoprotein containing S ectodomain was designed. • Fusion protein formed cVLPs that were morphologically similar to SARS-CoV-2 virions. • cVLPs induced anti-S, anti-RBD, and neutralizing antibodies in mice.

A COVID-19 vaccine candidate based on SARS-CoV-2 spike protein and immune-stimulating complexes.

Spike protein from SARS-CoV-2, the etiologic agent of the COVID-19 pandemic disease, constitutes a structural protein that proved to be the main responsible for neutralizing antibody production. Thus, its sequence is highly considered for the design of candidate vaccines. Animal cell culture represents the best option for the production of subunit vaccines based on recombinant proteins since they introduce post-translational modifications that are important to mimic the natural antigenic epitopes. Particularly, the human cell line HEK293T has been explored and used for the production of biotherapeutics since the products derived from them present human-like post-translational modifications that are important for the protein's activity and immunogenicity. The aim of this study was to produce and characterize a potential vaccine for COVID-19 based on the spike ectodomain (S-ED) of SARS-CoV-2 and two different adjuvants: aluminum hydroxide (AH) and immune-stimulating complexes (ISCOMs). The S-ED was produced in sHEK293T cells using a 1-L stirred tank bioreactor operated in perfusion mode and purified. S-ED characterization revealed the expected size and morphology. High N-glycan content was confirmed. S-ED-specific binding with the hACE2 (human angiotensin-converting enzyme 2) receptor was verified. The immunogenicity of S-ED was evaluated using AH and ISCOMs. Both formulations demonstrated the presence of anti-RBD antibodies in the plasma of immunized mice, being significantly higher for the latter adjuvant. Also, higher levels of IFN-γ and IL-4 were detected after the ex vivo immune stimulation of spleen-derived MNCs from ISCOMs immunized mice. Further analysis confirmed that S-ED/ISCOMs elicit neutralizing antibodies against SARS-CoV-2. KEY POINTS: Trimeric SARS-CoV-2 S-ED was produced in stable recombinant sHEK cells in serum-free medium. A novel S-ED vaccine formulation induced potent humoral and cellular immunity. S-ED formulated with ISCOMs adjuvant elicited a highly neutralizing antibody titer.

Physicochemical Characterization of a Recombinant eCG and Comparative Studies with PMSG Commercial Preparations.

Equine chorionic gonadotropin (eCG) is a glycoprotein hormone widely used in timed artificial ovulation (TAI) and superovulation protocols to improve the reproductive performance in livestock. Until recently, the only eCG products available in the market for veterinary use consisted in partially purified preparations of pregnant mare serum gonadotropin (PMSG). Here, a bioactive recombinant eCG (reCG) produced in suspension CHO-K1 cells was purified employing different chromatographic methods (hydrophobic interaction chromatography and reverse-phase (RP)-HPLC) and compared with a RP-HPLC-purified PMSG. To gain insight into the structural and functional characteristics of reCG, a bioinformatics analysis was performed. An exhaustive characterization comprising the determination of the purity degree, aggregates and nicked forms through SDS-PAGE, RP-HPLC and SEC-HPLC was performed. Higher order structures were studied by fluorescence spectroscopy and SEC-HPLC. Isoforms profile were analyzed by isoelectric focusing. Glycosylation analysis was performed through pulsed amperometric detection and PNGase F treatment following SDS-PAGE and weak anion exchange-HPLC. Slight differences between the purified recombinant hormones were found. However, recombinant molecules and PMSG exhibited variations in the glycosylation pattern. In fact, differences in sialic acid content between two commercial preparations of PMSG were also obtained, which could lead to differences in their biological potency. These results show the importance of having a standardized production process, as occurs in a recombinant protein bioprocess. Besides, our results reflect the importance of the glycan moieties on eCG conformation and hence in its biological activity, preventing denaturing processes such as aggregation.

Design and optimization of an IgG human ELISA assay reactive to recombinant RBD SARS-CoV-2 protein.

Serology assays are essential tools to mitigate the effect of COVID-19, help to identify previous SARS-CoV-2 infections or vaccination, and provide data for surveillance and epidemiologic studies. In this study, we report the production and purification process of the receptor-binding domain (RBD) of SARS-CoV-2 in HEK293 cells, which allowed the design, optimization, and validation of an indirect ELISA (iELISA) for the detection of human anti-RBD antibodies. To find the optimal conditions of this iELISA, a multivariate strategy was performed throughout design of experiments (DoE) and response surface methodology (RSM), one of the main tools of quality by design (QbD) approach. The adoption of this strategy helped to reduce the time and cost during the method development stage and to define an optimum condition within the analyzed design region. The assay was then validated, exhibiting a sensitivity of 94.24 (86.01-98.42%; 95% CI) and a specificity of 95.96% (89.98-98.89%; 95% CI). Besides, the degree of agreement between quality results assessed using kappa's value was 0.92. Hence, this iELISA represents a high-throughput technique, simple to perform, reliable, and feasible to be scaled up to satisfy the current demands. Since RBD is proposed as the coating antigen, the intended use of this iELISA is not only the detection of previous exposure to the virus, but also the possibility of detecting protective immunity. KEY POINTS: • RBD was produced in 1-L bioreactor and highly purified. • An iELISA assay was optimized applying QbD concepts. • The validation procedure demonstrated that this iELISA is accurate and precise.

Development of highly stable and de-immunized versions of recombinant alpha interferon: Promising candidates for the treatment of chronic and emerging viral diseases.

Human interferon alpha (hIFN-α) administration constitutes the current FDA approved therapy for chronic Hepatitis B and C virus infections. Additionally, hIFN-α treatment efficacy was recently demonstrated in patients with COVID-19. Thus, hIFN-α constitutes a therapeutic alternative for those countries where vaccination is inaccessible and for people who did not respond effectively to vaccination. However, hIFN-α2b exhibits a short plasma half-life resulting in the occurrence of severe side effects. To optimize the cytokine's pharmacokinetic profile, we developed a hyperglycosylated IFN, referred to as GMOP-IFN. Given the significant number of reports showing neutralizing antibodies (NAb) formation after hIFN-α administration, here we applied the DeFT (De-immunization of Functional Therapeutics) approach to develop functional, de-immunized versions of GMOP-IFN. Two GMOP-IFN variants exhibited significantly reduced ex vivo immunogenicity and null antiproliferative activity, while preserving antiviral function. The results obtained in this work indicate that the new de-immunized GMOP-IFN variants constitute promising candidates for antiviral therapy.

Development of a suitable manufacturing process for production of a bioactive recombinant equine chorionic gonadotropin (reCG) in CHO-K1 cells.

Equine chorionic gonadotropin (eCG) is a heterodimeric glycoprotein hormone produced by pregnant mares that has been used to improve reproductive performance in different domestic species. Several strategies to produce the hormone in a recombinant way have been reported; nevertheless, no approach has been able to produce a recombinant eCG (reCG) with significant in vivo bioactivity or in sufficient quantities for commercial purposes. For this reason, the only current product available on the market consists of partially purified preparations from serum of pregnant mares (PMSG). Herein, we describe a highly efficient process based on third-generation lentiviral vectors as delivery method for the production of reCG in suspension CHO-K1 cells, with productivities above 20 IU 106 cell-1.d-1 and 70% purification yields after one purification step. Importantly, reCG demonstrated biological activity in cattle, since around 30 µg of reCG were needed to exert the same biologic effect of 400 IU of PMSG in an ovulation synchronization protocol. The results obtained demonstrate that the developed strategy represents an attractive option for the production of reCG and constitutes an auspicious alternative for the replacement of animals as a source of PMSG.

Bifunctional GM-CSF-derived peptides as tools for O-glycoengineering and protein tagging.

Rapid development of effective biotherapeutics has been a concern during the last couple decades. In our work we designed two novel peptide tags, GMOP and mGMOP, derived from the N-terminal region of human granulocyte and macrophage colony stimulating factor (hGM-CSF), which contain four and six potential O-glycosylation sites, respectively. These peptide tags were fused to the N-terminus of human interferon-α2b (hIFN-α2b), a therapeutic antiviral and antiproliferative protein rapidly cleared from circulation. Two new molecules were obtained which, consistently with the presence of O-glycans, showed higher molecular masses, more negatively charged isoforms, and higher sialic acid content compared to wild-type IFN. In vitro bioactivity of purified chimeras revealed a similar antiviral specific biological activity (SBA) compared to unmodified IFN. A reduction of antiproliferative SBA was only observed for mGMOP-IFN. Pharmacokinetic studies in rats showed a notable improvement in terminal half-life (t1/2elim) (3.3 and 2.8 times-longer) and a marked reduction of the apparent clearance (CLapp, 3.7 and 4.1-fold lower for GMOP-IFN and mGMOP-IFN in comparison with native IFN, respectively). Furthermore, the in vitro thermal and plasma stability of both proteins was improved. Finally, a monoclonal antibody (mAb) that recognizes an N-terminal GM-CSF epitope was able to bind both chimeras in western blots and ELISAs. This demonstrates the potential of both peptides to behave as bifunctional tags to create novel long-acting biotherapeutics and to facilitate detection and purification.

Pharmacokinetics Versus In Vitro Antiproliferative Potency to Design a Novel Hyperglycosylated hIFN-α2 Biobetter.

PURPOSE: IFN4N is a glycoengineered version of recombinant human interferon alpha 2 (rhIFN-α2) that was modified to exhibit four N-glycosylation sites. It shows reduced in vitro specific biological activity (SBA) mainly due to R23 mutation by N23. However, it has improved pharmacokinetics and led to a high in vivo antitumor activity in mice. In order to prepare a new IFN-based biobetter, this work compares the influence of glycosylation (affecting pharmacokinetics) with the in vitro antiproliferative SBA on the in vivo efficacy. METHODS: Based on IFN4N, three groups of muteins were designed, produced, and characterized. Group A: variants with the same glycosylation degree (4N) but higher in vitro antiproliferative SBA (R23 restored); group B: muteins with higher glycosylation degree (5N) but similar in vitro antiproliferative activity; and group C: variants with improved glycosylation (5N and 6N) and in vitro antiproliferative bioactivity. RESULTS: Glycoengineering was successful for improving pharmacokinetics, and R23 restoration considerably increased in vitro antiproliferative activity of new muteins compared to IFN4N. Hyperglycosylation was able to improve the in vivo efficacy similarly to or even better than R23 restoration. Additionally, the highest glycosylated mutein exhibited the lowest immunogenicity. CONCLUSIONS: Hyperglycosylation constitutes a successful strategy to prepare a novel IFN biobetter.

Production of Therapeutic Enzymes by Lentivirus Transgenesis.

Since ERT for several LSDs treatment has emerged at the beginning of the 1980s with Orphan Drug approval, patients' expectancy and life quality have been improved. Most LSDs treatment are based on the replaced of mutated or deficient protein with the natural or recombinant protein.One of the main ERT drawback is the high drug prices. Therefore, different strategies trying to optimize the global ERT biotherapeutic production have been proposed. LVs, a gene delivery tool, can be proposed as an alternative method to generate stable cell lines in manufacturing of recombinant proteins. Since LVs have been used in human gene therapy, clinical trials, safety testing assays and procedures have been developed. Moreover, one of the main advantages of LVs strategy to obtain manufacturing cell line is the short period required as well as the high protein levels achieved.In this chapter, we will focus on LVs as a recombinant protein production platform and we will present a case study that employs LVs to express in a manufacturing cell line, alpha-Galactosidase A (rhαGAL), which is used as ERT for Fabry disease treatment.

Effect of ANITVNITV peptide fusion on the bioactivity and pharmacokinetics of human IFN-α2b and a hyper-N-glycosylated variant.

Different strategies have been developed and successfully applied to biotherapeutics in order to improve their in vivo efficacy. The genetic fusion to natural or synthetic glycosylated peptides constitutes a promising strategy since it conserves the protein sequence and results in the improvement of the pharmacokinetic properties. The ANITVNITV peptide described by Perlmann and coworkers presents 9 amino acids and 2 potential N-glycosylation sites. Its fusion to FSH resulted in the increase of the molecular mass and negative charge of the protein. Consequently, the pharmacokinetics was considerably improved. The aim of the present study was to compare the influence of ANITVNITV peptide fusion on the physicochemical, biological and pharmacokinetic properties of native hIFN-α2b (IFNwt), which contains a single O-glycosylation site, and a hyperglycosylated variant (IFN4N), that bears, in addition, 4 N-linked glycans. The resulting molecules, IFNwtNter and IFN4NNter, evidenced a higher molecular mass and negative charge compared to IFNwt and IFN4N, respectively. Therefore, the pharmacokinetic properties of the new molecules were significantly improved. The molecules obtained by the synthetic peptide fusion strategy evidenced a decrease in their in vitro antiviral specific biological activities (SBA). However, in vitro antiproliferative SBA was differentially modified for IFNwtNter and IFN4NNter in comparison with the parental molecules. For IFNwtNter, a reduction in the antiproliferative SBA was also observed. Remarkably, the addition of the ANITVNITV peptide to the N-terminus of IFN4N had a positive impact on its growth-inhibitory activity. This feature together with its improved pharmacokinetics encourages the development of IFN4NNter as an IFN-α based biobetter.

Strategies to Develop Therapeutic N- and O-Hyperglycosylated Proteins.

Glycoengineering by N- and/or O-hyperglycosylation represents a procedure to introduce potential sites for adding N- and/or O-glycosyl structures to proteins with the aim of producing biotherapeutics with improved pharmacodynamic and pharmacokinetic properties. In this chapter, a detailed description of the steps routinely performed to generate new proteins having high content of N- and/or O-glycosyl moieties is carried out. The rational strategy involves the initial stage of designing N- and/or O-hyperglycosylated muteins to be expressed by mammalian cells and includes the upstream and downstream processing stages necessary to develop hyperglycosylated versions of the proteins of interest with the purpose of beginning the long road toward producing biobetters.

High yield process for the production of active human α-galactosidase a in CHO-K1 cells through lentivirus transgenesis.

Fabry disease is an X-linked recessive disorder caused by a deficiency in lysosomal α-Galactosidase A. Currently, two enzyme replacement therapies (ERT) are available. However, access to orphan drugs continues to be limited by their high price. Selection of adequate high-expression systems still constitutes a challenge for alleviating the cost of treatments. Several strategies have been implemented, with varying success, trying to optimize the production process of recombinant human α-Galactosidase A (rhαGAL) in Chinese hamster ovary (CHO-K1) cells. Herein, we describe for the first time the application of a strategy based on third-generation lentiviral particles (LP) transduction of suspension CHO-K1 cells to obtain high-producing rhαGAL clones (3.5 to 59.4 pg cell-1 d-1 ). After two purification steps, the active enzyme was recovered (2.4 × 106 U mg-1 ) with 98% purity and 60% overall yield. Michaelis-Menten analysis demonstrated that rhαGAL was capable of hydrolyzing the synthetic substrate 4MU-α-Gal at a comparable rate to Fabrazyme®, the current CHO-derived ERT available for Fabry disease. In addition, rhαGAL presented the same mannose-6-phosphate (M6P) content, about 40% higher acid sialic amount and 33% reduced content of the immunogenic type of sialic acid (Neu5Gc) than the corresponding ones for Fabrazyme®. In comparison with other rhαGAL production processes reported to date, our approach achieves the highest rhαGAL productivity preserving adequate activity and glycosylation pattern. Even more, considering the improved glycosylation characteristics of rhαGAL, which might provide advantages regarding pharmacokinetics, our enzyme could be postulated as a promising alternative for therapeutic use in Fabry disease. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1334-1345, 2017.

Novel Poly(Diol Sebacate)s as Additives to Modify Paclitaxel Release From Poly(Lactic-co-Glycolic Acid) Thin Films.

Paclitaxel (PTX) incorporation in poly(lactic-co-glycolic acid) (PLGA) matrices produce films with high tensile rigidity and slow release that fail to deliver the required release rate for most biomedical applications such as in drug eluting stents and cancer treatments. To modify and improve this behavior, a set of poly(diol sebacate)s were synthesized and fully characterized as possible additives. The tensile properties of PLGA blends were evaluated as these materials could be used as coatings in drug eluting stent applications. A significant improvement in mechanical flexibility was observed with 20% additive content, as it reduced the Young's modulus value and increased the maximum deformation at break. PTX release was studied and correlated with the release of additive from PLGA films. An increase in the initial burst release phase was observed on all blends when compared to the control films of PLGA. Modulation of PTX release was achieved by altering the hydrophilicity degree of the additive or its percentage content on the blend. This supports the possibility that PTX was partitioned into the additive phase. Cytotoxicity analyses of novel additives were performed on mouse embryonic fibroblasts NIH/3T3.

Glycosylation and antiproliferative activity of hyperglycosylated IFN-α2 potentiate HEK293 cells as biofactories.

Both CHO and HEK cells are interesting hosts for the production of biotherapeutics due to their ability to introduce post-translational modifications such as glycosylation. Even though oligosaccharide structures attached to proteins are conserved among eukaryotes, many differences have been found between therapeutic glycoproteins expressed in hamster and human derived cells. In this work, a hyperglycosylated IFN-α2b mutein (IFN4N) was produced in CHO and HEK cell lines and an extensive characterization of their properties was performed. IFN4NCHO exhibited a higher average molecular mass and more acidic isoforms compared to IFN4NHEK. In agreement with these results, a 2-times higher sialic acid content was found for IFN4NCHO in comparison with the HEK-derived protein. This result was in agreement with monosaccharide quantification and glycan's analysis using WAX chromatography and HILIC coupled to mass spectrometry; all methods supported the existence of highly sialylated and also branched structures for IFN4NCHO glycans, in contrast with smaller and truncated structures among IFN4NHEK glycans. Unexpectedly, those remarkable differences in the glycosylation pattern had not a considerable impact on the clearance rate of both molecules in rats. In fact, although IFN4NHEK reached maximum plasma concentration 3-times faster than IFN4NCHO, their elimination profile did not differ significantly. Also, despite the in vitro antiviral specific biological activity of both proteins was the same, IFN4NHEK was more efficient as an antiproliferative agent in different tumor-derived cell lines. Accordingly, IFN4NHEK showed a higher in vivo antitumor activity in animal models. Our results show the importance of an appropriate host selection to set up a bioprocess and potentiate the use of HEK293 cells for the production of a new hyperglycosylated protein-based pharmaceutical.

Improvement of in vitro stability and pharmacokinetics of hIFN-α by fusing the carboxyl-terminal peptide of hCG ß-subunit.

Improving in vivo half-life and in vitro stability of protein-based therapeutics is a current challenge for the biopharmaceutical industry. In particular, recombinant human interferon alpha-2b (rhIFN-α2b), which belongs to a group of cytokines extensively used for the treatment of viral diseases and cancers, shows a poor stability in solution and an extremely short plasma half-life which determines a strict therapeutic regimen comprising high and repeated doses. In this work, we have used a strategy based on the fusion of the carboxyl-terminal peptide (CTP) of human chorionic gonadotropin (hCG) ß-subunit, bearing four O-linked oligosaccharide recognition sites, to each or both N- and C-terminal ends of rhIFN-α2b. Molecules containing from 5 (CTP-IFN and IFN-CTP) to 9 (CTP-IFN-CTP) O-glycosylation sites were efficiently expressed and secreted to CHO cells supernatants, and exhibited antiviral and antiproliferative bioactivities in vitro. Significant improvements in pharmacokinetics in rats were achieved through this approach, since the doubly CTP-modified IFN variant showed a 10-fold longer elimination half-life and a 19-fold decreased plasma apparent clearance compared to the wild-type cytokine. Moreover, CTP-IFN-CTP demonstrated a significant increase in in vitro thermal resistance and a higher stability against plasma protease inactivation, both features attributed to the stabilizing effects of the O-glycans provided by the CTP moiety. These results constitute the first report that postulates CTP as a tag for improving both the in vitro and in vivo stability of rhIFN-α2b which, in turn, would positively influence its in vivo bioactivity.

High performance collection of cerebrospinal fluid in rats: evaluation of erythropoietin penetration after osmotic opening of the blood-brain barrier.

An important issue to be considered when studying a new drug for treatment of central nervous system (CNS) diseases is its ability to cross the blood-brain barrier (BBB) and distribute throughout the brain. As cerebrospinal fluid (CSF) has demonstrated to be an invaluable reservoir to study CNS availability of therapeutic proteins, we have developed an improved method for CSF sampling from the cisterna magna of rats. The technique enables the simple and rapid collection of adequate quantities (50-75 µl) of blood-free CSF, rendering a high percentage of animal survival (99%) without clinic or neurological consequences. Its success in avoiding blood contamination of CSF lays in the use of a mixture of lidocaine/ephinephrine topically injected in the rat's suboccipital area and neck. Another relevant feature of the methodology is its low cost, since the puncture device can be easily assembled with cheap and available materials and, more importantly, neither expensive stereotaxic equipment nor frame is required. The present method is demonstrated by studying the CSF pharmacokinetics of recombinant human erythropoietin (rhEPO), a well-studied therapeutic candidate for neurological diseases. Moreover, we applied this technique to evaluate a strategy of osmotic disruption of the BBB to achieve a faster delivery of rhEPO into the CNS.

Isolation and characterization of a subset of erythropoietin glycoforms with cytoprotective but minimal erythropoietic activity.

Although historically used for the treatment of anemia, erythropoietin (EPO) has emerged as a neurotrophic and neuroprotective agent in different conditions of neuronal damage (traumatic brain injury, ischemia, spinal cord compression, peripheral neuropathy, retinal damage, epilepsy, Parkinson's Disease, among others). Nonetheless, EPO's therapeutic application is limited due to its hematological side-effects. With the aim of obtaining EPO derivatives resembling the hormone isolated from cells and tissues of neural origin, a novel combination of less acidic EPO glycoforms -designated as neuroepoetin (rhNEPO)- was purified to homogeneity from the supernatant of a CHO-producing cell line by a four-step chromatographic procedure. This simple and single process allowed us to prepare two EPO derivatives with distinct therapeutic expectations: the hematopoietic version and a minimally hematopoietic, but mainly in vitro cytoprotective, alternative. Further biological characterization showed that the in vivo erythropoietic activity of rhNEPO was 25-times lower than that of rhEPO. Interestingly, using different in vitro cytoprotective assays we found that this molecule exerts cytoprotection equivalent to, or better than, that of rhEPO in cells of neural phenotype. Furthermore, despite its shorter plasma half-life, rhNEPO was rapidly absorbed and promptly detected in the cerebrospinal fluid after intravenous administration in rats (5 min postinjection, in comparison with 30 min for rhEPO). Therefore, our results support the study of neuroepoetin as a potential drug for the treatment of neurological diseases, combining high cytoprotective activity with reduced hematological side-effects.