A comprehensive study of glucose and oxygen gradients in a scaled-down model of recombinant HuGM-CSF production in thermoinduced Escherichia coli fed-batch cultures.

The effect of gradients of elevated glucose and low dissolved oxygen in the addition zone of fed-batch E. coli thermoinduced recombinant high cell density cultures can be evaluated through two-compartment scale-down models. Here, glucose was fed in the inlet of a plug flow bioreactor (PFB) connected to a stirred tank bioreactor (STB). E. coli cells diminished growth from 48.2 ± 2.2 g/L in the stage of RP production if compared to control (STB) with STB-PFB experiments, when residence time inside the PFB was 25 s (34.1 ± 3.5 g/L) and 40 s (25.6 ± 5.1 g/L), respectively. The recombinant granulocyte-macrophage colony-stimulating factor (rHuGM-CSF) production decreased from 34 ± 7% of RP in inclusion bodies (IB) in control cultures to 21 ± 8%, and 7 ± 4% during the thermoinduction production phase when increasing residence time inside the PFB to 25 s and 40 s, respectively. This, along with the accumulation of acetic and formic acid (up to 4 g/L), indicates metabolic redirection of central carbon routes through metabolic flow and mixed acid fermentation. Special care must be taken when producing a recombinant protein in heat-induced E. coli, because the yield and productivity of the protein decreases as the size of the bioreactors increases, especially if they are carried at high cell density.

Thermoinduced recombinant E. coli grew less in a two-compartment scale-down model.Heat-inducible E. coli cultures at a large scale significantly decrease recombinant protein production.The accumulation of acetic and formic acid increases when E. coli is exposed to glucose and oxygen gradients.The axial flow pattern inside the PFB mimics glucose and dissolved oxygen gradients at the industrial scale.

Lipid-Like Biofilm from a Clinical Brain Isolate of Aspergillus terreus: Quantification, Structural Characterization and Stages of the Formation Cycle.

Invasive infections caused by filamentous fungi have increased considerably due to the alteration of the host's immune response. Aspergillus terreus is considered an emerging pathogen and has shown resistance to amphotericin B treatment, resulting in high mortality. The development of fungal biofilm is a virulence factor, and it has been described in some cases of invasive aspergillosis. In addition, although the general composition of fungal biofilms is known, findings related to biofilms of a lipid nature are rarely reported. In this study, we present the identification of a clinical strain of A. terreus by microbiological and molecular tools, also its in vitro biofilm development capacity: (i) Biofilm formation was quantified by Crystal Violet and reduction of tetrazolium salts assays, and simultaneously the stages of biofilm development were described by Scanning Electron Microscopy in High Resolution (SEM-HR). (ii) Characterization of the organizational structure of the biofilm was performed by SEM-HR. The hyphal networks developed on the surface, the abundant air channels created between the ECM (extracellular matrix) and the hyphae fused in anastomosis were described. Also, the presence of microhyphae is reported. (iii) The chemical composition of the ECM was analyzed by SEM-HR and CLSM (Confocal Laser Scanning Microscopy). Proteins, carbohydrates, nucleic acids and a relevant presence of lipid components were identified. Some structures of apparent waxy appearance were highlighted by SEM-HR and backscatter-electron diffraction, for which CLSM was previously performed. To our knowledge, this work is the first description of a lipid-type biofilm in filamentous fungi, specifically of the species A. terreus from a clinical isolate.

Photodegradation of Rituximab and Critical Evaluation of Its Sensibility to Electromagnetic Radiation.

Rituximab is a monoclonal antibody used in the treatment of lymphoma non-Hodgkin. This mAb is photosensitive as it is administrated to the patient by infusion/perfusion; therefore, photostability is a decisive factor in the efficacy of this biologic. To better understand the photodegradation mechanisms of Rituximab, this biologic was exposed to different irradiance conditions. We show in this study that this mAb photodegrade proportionally to irradiance intensities. The main modifications of Rituximab by irradiance correlate to the increase in aggregates, decrease in its Tm, acidic charge variants, oxidation of the Trp (36) in the heavy chain, and decrease in complement-dependent cytotoxicity (CDC) potency. To understand the relationship between real-life photo-exposition conditions and ICH standardized light tests, a full characterization was set up. Worst photo-stress cases were evaluated, 1 and 2 h under direct sunlight through a window, mimicking the ID65 electromagnetic radiation profile. Our results show that only exposition to direct sun irradiance during 2 h, (≈ 150 kluxes•h), increases critically soluble and insoluble aggregates, diminishing Tm, increasing acidic charge variants, and the photooxidation of the Trp (36) in the heavy chain measured by peptide mapping-RP-UPLC-MS. A decrease in CDC below 80% resulted under this condition, which correlates with physicochemical analyses. While inside light-room exposition (similar to ICH test) and ICH conditions do not have any contribution to the degradation of Rituximab measured by these physicochemical and biological analytical methods. These results indicate that exposition of Rituximab to below ≈ 75 kluxes•h of sun light cannot photodegrade critically this biologic inside of its primary container.

The pre-induction temperature affects recombinant HuGM-CSF aggregation in thermoinducible Escherichia coli.

The overproduction of recombinant proteins in Escherichia coli leads to insoluble aggregates of proteins called inclusion bodies (IBs). IBs are considered dynamic entities that harbor high percentages of the recombinant protein, which can be found in different conformational states. The production conditions influence the properties of IBs and recombinant protein recovery and solubilization. The E. coli growth in thermoinduced systems is generally carried out at 30 °C and then recombinant protein production at 42 °C. Since the heat shock response in E. coli is triggered above 34 °C, the synthesis of heat shock proteins can modify the yields of the recombinant protein and the structural quality of IBs. The objective of this work was to evaluate the effect of different pre-induction temperatures (30 and 34 °C) on the growth of E. coli W3110 producing the human granulocyte-macrophage colony-stimulating factor (rHuGM-CSF) and on the IBs structure in a λpL/pR-cI857 thermoinducible system. The recombinant E. coli cultures growing at 34 °C showed a ~ 69% increase in the specific growth rate compared to cultures grown at 30 °C. The amount of rHuGM-CSF in IBs was significantly higher in cultures grown at 34 °C. Main folding chaperones (DnaK and GroEL) were associated with IBs and their co-chaperones (DnaJ and GroES) with the soluble protein fraction. Finally, IBs from cultures that grew at 34 °C had a lower content of amyloid-like structure and were more sensitive to proteolytic degradation than IBs obtained from cultures at 30 °C. Our study presents evidence that increasing the pre-induction temperature in a thermoinduced system allows obtaining higher recombinant protein and reducing amyloid contents of the IBs. KEY POINTS: • Pre-induction temperature determines inclusion bodies architecture • In pre-induction (above 34 °C), the heat shock response increases recombinant protein production • Inclusion bodies at higher pre-induction temperature show a lower amyloid content.

Microbial Warfare on Three Fronts: Mixed Biofilm of Aspergillus fumigatus and Staphylococcus aureus on Primary Cultures of Human Limbo-Corneal Fibroblasts.

Background: Coinfections with fungi and bacteria in ocular pathologies are increasing at an alarming rate. Two of the main etiologic agents of infections on the corneal surface, such as Aspergillus fumigatus and Staphylococcus aureus, can form a biofilm. However, mixed fungal-bacterial biofilms are rarely reported in ocular infections. The implementation of cell cultures as a study model related to biofilm microbial keratitis will allow understanding the pathogenesis in the cornea. The cornea maintains a pathogen-free ocular surface in which human limbo-corneal fibroblast cells are part of its cell regeneration process. There are no reports of biofilm formation assays on limbo-corneal fibroblasts, as well as their behavior with a polymicrobial infection. Objective: To determine the capacity of biofilm formation during this fungal-bacterial interaction on primary limbo-corneal fibroblast monolayers. Results: The biofilm on the limbo-corneal fibroblast culture was analyzed by assessing biomass production and determining metabolic activity. Furthermore, the mixed biofilm effect on this cell culture was observed with several microscopy techniques. The single and mixed biofilm was higher on the limbo-corneal fibroblast monolayer than on abiotic surfaces. The A. fumigatus biofilm on the human limbo-corneal fibroblast culture showed a considerable decrease compared to the S. aureus biofilm on the limbo-corneal fibroblast monolayer. Moreover, the mixed biofilm had a lower density than that of the single biofilm. Antibiosis between A. fumigatus and S. aureus persisted during the challenge to limbo-corneal fibroblasts, but it seems that the fungus was more effectively inhibited. Conclusion: This is the first report of mixed fungal-bacterial biofilm production and morphological characterization on the limbo-corneal fibroblast monolayer. Three antibiosis behaviors were observed between fungi, bacteria, and limbo-corneal fibroblasts. The mycophagy effect over A. fumigatus by S. aureus was exacerbated on the limbo-corneal fibroblast monolayer. During fungal-bacterial interactions, it appears that limbo-corneal fibroblasts showed some phagocytic activity, demonstrating tripartite relationships during coinfection.

Functional analysis of glycosylation in Etanercept: Effects over potency and stability.

Etanercept is a biotechnological product that has a complex glycosylation profile. To elucidate Etanercept glycosylation effect over biological activity and stability, we deglycosylated sequentially this molecule. Sequential deglycosylation was performed to understand which glycans are critical for Etanercept folding and activity. Extended study showed that gross glycosylation differences, affect thermal stability, hydrodynamic radius, pI, CDC, ADCC, protection against oxidation and charge surface exposition with any effect (within biological assay dispersion) over TNFα neutralization, indicating which glycoforms have a critical effect over Etanercept ADCC, CDC and stability. In this regard, complete remotion of sialic acids have a predominant importance over pI, ADCC, CDC and surface charge while N and O glycosylation over thermal stability, hydrophobicity, aggregation and protection against oxidation. Our research suggest that gross differences in the glycosylation profile are relevant for the stability and biological main activities of Etanercept, and that significant differences that affect the activities related to this fusion protein could be detected with proper analytical methods and stability studies.

Detection and quantification of leached ethylene glycol in biopharmaceuticals by RP-UHPLC.

Biopharmaceuticals are in direct contact with different plastic materials, which can contribute to process-related impurities. Polyethylene terephthalate glycol (PETG) is used for storage and transportation of biopharmaceuticals and it is synthetized from the poly-condensation reaction between ethylene glycol, 1,4-cyclodimethanol and dimethyl terephthalate. PETG bottles are analyzed for such impurities prior to release; however, the nature of the pharmaceutical matrix can extract impurities, so it is important to measure these contaminants in biopharmaceuticals. This study shows a liquid chromatography method for the quantification of ethylene glycol in PETG materials as an alternative to the standard USP colorimetric method. The method is based on the derivatization of ethylene glycol with benzoyl chloride in a Schotten-Baumann reaction. We present a comprehensive method development and validation. The method allows the detection and quantification of leached and extracted ethylene glycol directly in biopharmaceuticals after years of storage in contact with PETG bottles. Results showed residual ethylene glycol in drug substances to a level of ≈  0.1-0.5 µg/mL exposed during 2-6 years of storage in PETG bottles and ≈ 0.2-0.9 µg/mL in biopharmaceuticals. Graphical abstract Biopharmaceuticals must be free or low concentration for leachables, FR-UHPLC-UV analysis is a precise and accurate analytical method for ethylene glycol measurement. This leachable is commonly present in products in direct contact with PETG plastic.

Nanoparticles for Protein Sensing in Primary Containers: Interaction Analysis and Application.

Silver nanoparticles (AgNPs) are known to interact with proteins, leading to modifications of the plasmonic absorption that can be used to monitor this interaction, entailing a promising application for sensing adsorption of therapeutic proteins in primary containers. First, transmission electron microscopy in combination with plasmonic absorption and light scattering responses were used to characterize AgNPs and protein-AgNP complexes, including its concentration dependence, using two therapeutic molecules as models: a monoclonal antibody (mAb) and a synthetic copolymer (SC). Upon interaction, a protein corona was formed around AgNPs with the consequent shifting and broadening of their characteristic surface plasmon resonance (SPR) band (400 nm) to 410 nm and longer wavelenghts. Additional studies revealed secondary and three-dimensional structure modifications of model proteins upon interaction with AgNPs by circular dichroism and fluorescence techniques, respectively. Based on the modification of the SPR condition of AgNPs upon interaction with proteins, we developed a novel protein-sensing application of AgNPs in primary containers. This strategy was used to conduct a compatibility assessment of model proteins towards five commercially available prefillable glass syringe (PFS) models. mAb- and SC-exposed PFSs showed that 74 and 94% of cases were positive for protein adsorption, respectively. Interestingly, protein adsorption on 15% of total tested PFSs was negligible (below the nanogram level). Our results highlight the need of a case-by-case compatibility assessment of therapeutic proteins and their primary containers. This strategy has the potential to be easily applied on other containers and implemented during early-stage product development by pharmaceutical companies and for routine use during batch release by packaging manufacturers.

Process signatures in glatiramer acetate synthesis: structural and functional relationships.

Glatiramer Acetate (GA) is an immunomodulatory medicine approved for the treatment of multiple sclerosis, whose mechanisms of action are yet to be fully elucidated. GA is comprised of a complex mixture of polypeptides with different amino acid sequences and structures. The lack of sensible information about physicochemical characteristics of GA has contributed to its comprehensiveness complexity. Consequently, an unambiguous determination of distinctive attributes that define GA is of highest relevance towards dissecting its identity. Herein we conducted a study of characteristic GA heterogeneities throughout its manufacturing process (process signatures), revealing a strong impact of critical process parameters (CPPs) on the reactivity of amino acid precursors; reaction initiation and polymerization velocities; and peptide solubility, susceptibility to hydrolysis, and size-exclusion properties. Further, distinctive GA heterogeneities were correlated to defined immunological and toxicological profiles, revealing that GA possesses a unique repertoire of active constituents (epitopes) responsible of its immunological responses, whose modification lead to altered profiles. This novel approach established CPPs influence on intact GA peptide mixture, whose physicochemical identity cannot longer rely on reduced properties (based on complete or partial GA degradation), providing advanced knowledge on GA structural and functional relationships to ensure a consistent manufacturing of safe and effective products.

Design of a strong cation exchange methodology for the evaluation of charge heterogeneity in glatiramer acetate.

Complex pharmaceuticals are in demand of competent analytical methods able to analyze charge heterogeneity as a critical quality attribute (CQA), in compliance with current regulatory expectations. A notorious example is glatiramer acetate (GA), a complex polypeptide mixture useful for the treatment of relapsing-remitting multiple sclerosis. This pharmaceutical challenges the current state of analytical technology in terms of the capacity to study their constituent species. Thus, a strong cation exchange methodology was designed under the lifecycle approach to support the establishment of GA identity, trough the evaluation of its chromatographic profile, which acts as a charge heterogeneity fingerprint. In this regard, a maximum relative margin of error of 5% for relative retention time and symmetry factor were proposed for the analytical target profile. The methodology met the proposed requirements after precision and specificity tests results, the former comprised of sensitivity and selectivity. Subsequently, method validation was conducted and showed that the method is able to differentiate between intact GA and heterogeneity profiles coming from stressed, fractioned or process-modified samples. In summary, these results provide evidence that the method is adequate to assess charge heterogeneity as a CQA of this complex pharmaceutical.

Characterization and comparability of biosimilars: a filgrastim case of study and regulatory perspectives for Latin America

Background: Developing countries have an estimate of ten times more approved biosimilars than developed countries. This disparity demands the need of an objective regulation that incorporates health policies according to the technological and economical capabilities of each country. One of the challenges lies on the establishment of comparability principles based on a physicochemical and biological characterization that should determine the extent of additional non-clinical and clinical studies. This is particularly relevant for licensed biosimilars in developing countries, which have an extensive clinical experience since their approval as generics' in some cases more than a decade. To exemplify the current status of biosimilars in Mexico' a characterization exercise was conducted on licensed filgrastim biosimilars using pharmacopeial and extended characterization methodologies. Results: Most of the evaluated products complied with the pharmacopeial criteria and showed comparability in their Critical Quality Attributes (CQAs) towards the reference product. These results were expected in accordance with their equivalent performance during their licensing as generics. Accordingly' a rational approval and registration renewal scheme for biosimilars is proposed, that considers the proper identification of CQAs and its thoroughly evaluation using selected techniques. Conclusions: This approach provides support to diminish uncertainty of exhibiting different pharmacological profiles and narrows or even avoids the necessity of comparative clinical studies. Ultimately, this proposal is intended to improve the accessibility to high quality biosimilars in Latin America and other developing countries.

New Alternatives for Autoimmune Disease Treatments: Physicochemical and Clinical Comparability of Biosimilar Etanercept.

Etanercept is a recombinant fusion protein approved for the treatment of TNF-α mediated diseases such as rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, and ankylosing spondylitis. Herein, we present an evaluation of the physicochemical and biological properties of a biosimilar etanercept and its reference product followed by a clinical study in patients diagnosed with RA intended to demonstrate comparability of their immunomodulatory activity. Identity analyses showed a total correspondence of the primary and higher-order structure between the two products. In regard to intrinsic heterogeneity, both products showed to be highly heterogenous; however the biosimilar etanercept exhibited similar charge and glycan heterogeneity intervals compared to the reference product. Apoptosis inhibition assay also showed that, despite the high degree of heterogeneity exhibited by both products, no significant differences exist in their in vitro activity. Finally, the clinical assessment conducted in RA-diagnosed patients did not show significant differences in the evaluated pharmacodynamic markers of both products. Collectively, the results from the comparability exercise provide convincing evidence that the evaluated biosimilar etanercept can be considered an effective alternative for the treatment of RA.

Optimization of a recombinant human growth hormone purification process using quality by design.

This work describes a strategy to optimize a downstream processing of a recombinant human growth hormone (rhGH) by incorporating a quality by design approach toward meeting higher quality specifications. The optimized process minimized the presence of impurities and degradation by-products during manufacturing by the establishment of in-process controls. Capillary zone electrophoresis, reverse phase, and size-exclusion chromatographies were used as analytical techniques to establish new critical process parameters for the solubilization, capture, and intermediate purification steps aiming to maintain rhGH quality by complying with pharmacopeial specifications. The results indicated that the implemented improvements in the process allowed the optimization of the specific recovery and purification of rhGH without compromising its quality. In addition, this optimization facilitated the stringent removal of the remaining impurities in further polishing stages, as demonstrated by the analysis of the obtained active pharmaceutical ingredient.

Theoretical approximations and experimental extinction coefficients of biopharmaceuticals.

UV spectrophotometric measurement is a widely accepted and standardized routine analysis for quantitation of highly purified proteins; however, the reliability of the results strictly depends on the accuracy of the employed extinction coefficients. In this work, an experimental estimation of the differential refractive index (dn/dc), based on dry weight measurements, was performed in order to determine accurate extinction coefficients for four biotherapeutic proteins and one synthetic copolymer after separation in a size-exclusion ultra-performance liquid chromatograph coupled to an ultraviolet, multiangle light scattering and refractive index (SE-UPLC-UV-MALS-RI) multidetection system. The results showed small deviations with respect to theoretical values, calculated from the specific amino acid sequences, for all the studied immunoglobulins. Nevertheless, for proteins like etanercept and glatiramer acetate, several considerations, such as glycan content, partial specific volume, polarizability, and higher order structure, should be considered to properly calculate theoretical extinction coefficient values. Herein, these values were assessed with simple approximations. The precision of the experimentally obtained extinction coefficients, and its convergence towards the theoretical values, makes them useful for characterization and comparability exercises. Also, these values provide insight into the absorbance and scattering properties of the evaluated proteins. Overall, this methodology is capable of providing accurate extinction coefficients useful for development studies.

Pharmacokinetic Comparability of a Biosimilar Trastuzumab Anticipated from Its Physicochemical and Biological Characterization.

Comparability between a biosimilar and its reference product requires the evaluation of critical quality attributes that may impact on its pharmacological response. Herein we present a physicochemical characterization of a biosimilar trastuzumab focused on the attributes related to the pharmacokinetic response. Capillary isoelectrofocusing (cIEF) and cation exchange chromatography (CEX) were used to evaluate charge heterogeneity; glycosylation profiles were assessed through hydrophilic interaction liquid chromatography (HILIC); aggregates content was evaluated through size exclusion chromatography (SEC) while binding affinity to FcRn was evaluated using isothermal titration calorimetry (ITC). The biosimilar trastuzumab and its reference product exhibited a high degree of similarity for the evaluated attributes. In regard to the pharmacokinetic parameters, randomized, double blind, and two-arm parallel and prospective study was employed after the administration of a single intravenous dose in healthy volunteers. No significant differences were found between the pharmacokinetic profiles of both products. Our results confirm that similarity of the critical quality attributes between a biosimilar product, obtained from a different manufacturing process, and the reference product resulted in comparable pharmacokinetic profiles, diminishing the uncertainty related to the biosimilar's safety and efficacy.

Physicochemical and biological characterization of a biosimilar trastuzumab.

According to the World Health Organization, the incidence of malignant neoplasms and endocrine, blood, and immune disorders will increase in the upcoming decades along with the demand of affordable treatments. In response to this need, the development of biosimilar drugs is increasing worldwide. The approval of biosimilars relies on the compliance with international guidelines, starting with the demonstration of similarity in their physicochemical and functional properties against the reference product. Subsequent clinical studies are performed to demonstrate similar pharmacological behavior and to diminish the uncertainty related to their safety and efficacy. Herein we present a comparability exercise between a biosimilar trastuzumab and its reference product, by using a hierarchical strategy with an orthogonal approach, to assess the physicochemical and biological attributes with potential impact on its pharmacokinetics, pharmacodynamics, and immunogenicity. Our results showed that the high degree of similarity in the physicochemical attributes of the biosimilar trastuzumab with respect to the reference product resulted in comparable biological activity, demonstrating that a controlled process is able to provide consistently the expected product. These results also constitute the basis for the design of subsequent delimited pharmacological studies, as they diminish the uncertainty of exhibiting different profiles.

Assessment of physicochemical properties of rituximab related to its immunomodulatory activity.

Rituximab is a chimeric monoclonal antibody employed for the treatment of CD20-positive B-cell non-Hodgkin's lymphoma, chronic lymphocytic leukemia, rheumatoid arthritis, granulomatosis with polyangiitis and microscopic polyangiitis. It binds specifically to the CD20 antigen expressed on pre-B and consequently on mature B-lymphocytes of both normal and malignant cells, inhibiting their proliferation through apoptosis, CDC, and ADCC mechanisms. The immunomodulatory activity of rituximab is closely related to critical quality attributes that characterize its chemical composition and spatial configuration, which determine the recognition of CD20 and the binding to receptors or factors involved in its effector functions, while regulating the potential immunogenic response. Herein, we present a physicochemical and biological characterization followed by a pharmacodynamics and immunogenicity study to demonstrate comparability between two products containing rituximab. The physicochemical and biological characterization revealed that both products fit within the same response intervals exhibiting the same degree of variability. With regard to clinical response, both products depleted CD20+ B-cells until posttreatment recovery and no meaningful differences were found in their pharmacodynamic profiles. The evaluation of anti-chimeric antibodies did not show differential immunogenicity among products. Overall, these data confirm that similarity of critical quality attributes results in a comparable immunomodulatory activity.

Analysis of therapeutic proteins and peptides using multiangle light scattering coupled to ultra high performance liquid chromatography.

Analysis of the physical properties of biotherapeutic proteins is crucial throughout all the stages of their lifecycle. Herein, we used size-exclusion ultra high performance liquid chromatography coupled to multiangle light scattering and refractive index detection systems to determine the molar mass, mass-average molar mass, molar-mass dispersity and hydrodynamic radius of two monoclonal antibodies (rituximab and trastuzumab), a fusion protein (etanercept), and a synthetic copolymer (glatiramer acetate) employed as models. A customized instrument configuration was set to diminish band-broadening effects and enhance sensitivity throughout detectors. The customized configuration showed a performance improvement with respect to the high-performance liquid chromatography standard configuration, as observed by a 3 h column conditioning and a higher resolution analysis in 20 min. Analysis of the two monoclonal antibodies showed averaged values of 148.0 kDa for mass-average molar mass and 5.4 nm for hydrodynamic radius, whereas for etanercept these values were 124.2 kDa and 6.9 nm, respectively. Molar-mass dispersity was 1.000 on average for these proteins. Regarding glatiramer acetate, a molar mass range from 3 to 45 kDa and a molar-mass dispersity of 1.304 were consistent with its intrinsic peptide diversity, and its mass-average molar mass was 10.4 kDa. Overall, this method demonstrated an accurate determination of molar mass, overcoming the difficulties of size-exclusion chromatography.

Validation of three viable-cell counting methods: Manual, semi-automated, and automated.

A viable cell count is essential to evaluate the kinetics of cell growth. Since the hemocytometer was first used for counting blood cells, several variants of the methodology have been developed towards reducing the time of analysis and improving accuracy through automation of both sample preparation and counting. The successful implementation of automated techniques relies in the adjustment of cell staining, image display parameters and cell morphology to obtain equivalent precision, accuracy and linearity with respect to the hemocytometer. In this study we conducted the validation of three trypan blue exclusion-based methods: manual, semi-automated, and fully automated; which were used for the estimation of density and viability of cells employed for the biosynthesis and bioassays of recombinant proteins. Our results showed that the evaluated attributes remained within the same range for the automated methods with respect to the manual, providing an efficient alternative for analyzing a huge number of samples.