Carlos A. López-Morales

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

Graphical abstract

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-Hodgkins 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.

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

HighlightsStrong cation exchange method to assess glatiramer acetate charge heterogeneity.Relative retention time and symmetry factor are proposed for quality assessment.The method was challenged through Analytical Target Profile and Validation tests.The method is able to discern between glatiramer acetate and non‐desired heterogeneity profiles.The method was validated and is adequate for its intended purpose. ABSTRACT 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.

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.

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 biosimilars safety and efficacy.

Development and validation of a bioassay to evaluate binding of adalimumab to cell membrane-anchored TNFα using flow cytometry detection

HIGHLIGHTSA bioassay to assess binding of adalimumab in vitro was developed and validated.The bioassay assesses binding of adalimumab towards TNF‐&agr; receptors in rCHO cells.The developed bioassay invokes flow‐cytometry as the detection method of binding.The validation demonstrates that the bioassay is suitable for its intended purpose.The validated assay can be used for characterization or batch release in industry. ABSTRACT Physicochemical and structural properties of proteins used as active pharmaceutical ingredients of biopharmaceuticals are determinant to carry out their biological activity. In this regard, the assays intended to evaluate functionality of biopharmaceuticals provide confirmatory evidence that they contain the appropriate physicochemical properties and structural conformation. The validation of the methodologies used for the assessment of critical quality attributes of biopharmaceuticals is a key requirement for manufacturing under GMP environments. Herein we present the development and validation of a flow cytometry‐based methodology for the evaluation of adalimumabs affinity towards membrane‐bound TNF&agr; (mTNF&agr;) on recombinant CHO cells. This in vitro methodology measures the interaction between an in‐solution antibody and its target molecule onto the cell surface through a fluorescent signal. The characteristics evaluated during the validation exercise showed that this methodology is suitable for its intended purpose. The assay demonstrated to be accurate (r2=0.92, slope=1.20), precise (%CV≤18.31) and specific (curve fitting, r2=0.986–0.997) to evaluate binding of adalimumab to mTNF&agr;. The results obtained here provide evidence that detection by flow cytometry is a viable alternative for bioassays used in the pharmaceutical industry. In addition, this methodology could be standardized for the evaluation of other biomolecules acting through the same mechanism of action.

Regulatory Pathway for Licensing Biotherapeutics in Mexico

Biotherapeutic products which are derived from living organisms using recombinant DNA technology significantly contribute to the progress in the treatment of life-threatening and chronic diseases. The worldwide sale of biological drugs in 2016 was near US

Performance of an immobilized recombinant leucine aminopeptidase after storage in ethanol–water solution

263,700 million. In Latin America, where monoclonal antibodies market was worth US

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

7000 million, being Mexico the second largest market. Approval is one of the key aspects which influences the market of medicinal products, thus it is responsibility of the regulatory authority to establish a regulatory framework that ensure safety and efficacy of the products, and it is responsibility of the applicants to provide a high quality dossier in accordance with the registration requirements of the country. The applicants submitting registration requests in Mexico need to be aware of the requirements. Similar to many other countries, Mexico has adopted the Common Technical Document (CTD) structure for organizing dossier of the medicinal product for submission into main modules (i.e., quality, non-clinical, and clinical). This facilitates the submission process of medicinal products following a logical sequence aligned to the International Council on Harmonisation (ICH) guidelines. Moreover, this structure improves the transparency and clarity of the dossier in process of evaluation of medicinal products. In Mexico, the Ministry of Health has published a regulation, NOM-257-SSA1-2014, which established the general requirements to be followed by applicants to complete the registration of biotherapeutics. This regulation stipulates that the evaluation process is supported by a regulatory framework involving Good Manufacturing Practices, labeling, stability, clinical trials, biocomparability studies, pharmacovigilance, and a technical evaluation performed by a multidisciplinary team of experts in biotherapeutics development. Additionally, the Mexican regulatory agency, COFEPRIS, has published specific guidelines to facilitate the application process. Despite the availability of this information, the scope is limited to regulatory and administrative purposes, rather than technical-scientific supporting knowledge. The aim of this article is to provide concise information to improve and promote communication between industry and regulatory agencies. Herein, we describe the current process of COFEPRIS in regulating biotherapeutics in Mexico. This process explains the basis for the organization and structure of the technical-scientific information of biotherapeutics required for registration application.