Salmonella Typhi OmpS1 and OmpS2 porins are potent protective immunogens with adjuvant properties.

Salmonella enterica serovar Typhi (S. Typhi) is the causal agent of typhoid fever, a disease that primarily affects developing countries. Various antigens from this bacterium have been reported to be targets of the immune response. Recently, the S. Typhi genome has been shown to encode two porins--OmpS1 and OmpS2--which are expressed at low levels under in vitro culture conditions. In this study, we demonstrate that immunizing mice with either OmpS1 or OmpS2 induced production of specific, long-term antibody titres and conferred protection against S. Typhi challenge; in particular, OmpS1 was more immunogenic and conferred greater protective effects than OmpS2. We also found that OmpS1 is a Toll-like receptor 4 (TLR4) agonist, whereas OmpS2 is a TLR2 and TLR4 agonist. Both porins induced the production of tumour necrosis factor and interleukin-6, and OmpS2 was also able to induce interleukin-10 production. Furthermore, OmpS1 induced the over-expression of MHC II molecules in dendritic cells and OmpS2 induced the over-expression of CD40 molecules in macrophages and dendritic cells. Co-immunization of OmpS1 or OmpS2 with ovalbumin (OVA) increased anti-OVA antibody titres, the duration and isotype diversity of the OVA-specific antibody response, and the proliferation of T lymphocytes. These porins also had adjuvant effects on the antibody response when co-immunized with either the Vi capsular antigen from S. Typhi or inactivated 2009 pandemic influenza A(H1N1) virus [A(H1N1)pdm09]. Taken together, the data indicate that OmpS1 and OmpS2, despite being expressed at low levels under in vitro culture conditions, are potent protective immunogens with intrinsic adjuvant properties.

Taking advantage of a high-throughput flow cytometer for the implementation of an ADCC assay for regulatory compliance.

Technological advances allowed the development of high-throughput instruments such as IntelliCyt iQue Screener PLUS®. Here, we took advantage of this technology to transfer a previously validated cytotoxicity assay. The evaluated parameters were cell permeability, caspase activation and phosphatidyl serine exposure. The assay was accurate (r2 = 0.90), precise (%CV ≤ 18.90) and specific. These results showed that this technology is suitable to be used in control quality environments. In addition, the automation provided a faster acquisition and analysis of data with precise and accurate results. This application could be implemented to evaluate another in vitro mechanism of action of different biotherapeutics.

Validation of a cell-based colorimetric reporter gene assay for the evaluation of Type I Interferons.

The biotherapeutic type I interferons (IFN-I) are indicated to treat several diseases. These products are regulated to guarantee safety and efficacy through critical quality attributes. For this purpose, the development of robust assays is required, followed by its validation to demonstrate their suitability for its intended purpose. Despite there are some commercial kits to evaluate IFN-I signaling, these are focused on measuring in vitro biological response instead of their validation, which is a pharmaceutical industry requirement. The aim of this work was to validate the HEK-Blue IFN-α/ß system evaluating the biological activity of IFN-α/ß under good laboratory practices, according to international standards. Our results demonstrated that HEK-Blue IFN-α/ß system comply with accuracy (r2>0.95) precision (CV < 20%) and specificity for both IFN-α/ß; confirming that this assay is robust for this biotherapeutics' evaluation. Thereby, this bioassay could be implemented as a complementary method to the classical anti-proliferative and anti-viral assays under quality control environments.

Validation of an ADCC assay using human primary natural killer cells to evaluate biotherapeutic products bearing an Fc region.

The development of biotherapeutics requires continuous improvement in analytical methodologies for the assessment of their quality attributes. A subset of biotherapeutics is designed to interact with specific antigens that are exposed on the membranes of target cells or circulating in a soluble form, and effector functions are achieved via recognition of their Fc region by effector cells that induce mechanisms such as antibody-dependent cell-mediated cytotoxicity (ADCC). Thus, ADCC induction is a critical quality attribute (CQA) that must be evaluated to ensure biotherapeutic efficacy. Induction of ADCC can be evaluated by employing effector cells from different sources, such as peripheral blood mononuclear cells (PBMC) and genetically modified cell lines (e.g., transfected NKs or Jurkat cells), and different approaches can be used for detection and results interpretation depending on the type of effector cells used. In this regard, validation of the assays is relevant to ensure the reliability of the results according to the intended purpose. Herein, we show the standardization and validation of ADCC assays to test the potency of three biotherapeutic proteins using primary NK cells obtained from fresh blood as effector cells and detecting cell death by flow cytometry. The advantage of using primary NKs instead of modified cells is that the response is closer to that occurring in vivo since cytotoxicity is evaluated in a direct manner. Our results indicate that in all cases, the assays exhibited a characteristic sigmoidal dose/response curve complying with accurate, precise and specific parameters. Thereby, the validated ADCC assay is an appropriate alternative to evaluate the biological activities of these type of biotherapeutics.

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

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

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 adalimumab's affinity towards membrane-bound TNFα (mTNFα) 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 mTNFα. 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.

Safety and immunogenicity of a virus-like particle pandemic influenza A (H1N1) 2009 vaccine in a blinded, randomized, placebo-controlled trial of adults in Mexico.

Virus-like particles (VLPs) can be rapidly developed from influenza virus genetic sequences in order to supply vaccine after the onset of a pandemic. The safety and immunogenicity of one or two doses of a recombinant A (H1N1) 2009 influenza VLP vaccine was evaluated in a two-stage, Phase 2, randomized, double-blind, placebo-controlled study conducted in 4563 healthy adults, 18-64 years of age, during the H1N1 2009 pandemic in Mexico. In Part A, 1013 subjects were randomized into four treatment groups (5 µg, 15 µg, or 45 µg hemagglutinin [HA] VLP vaccine or placebo) and vaccinated 21 days apart, with sera collected on Days 1, 14 and 36 for hemagglutination inhibition (HAI) testing. After review of safety and immunogenicity data from Part A, additional subjects were immunized with a single dose of 15 µg VLP vaccine (N=2537) or placebo (N=1011) and assessed for safety in Part B. Results showed the H1N1 2009 VLP vaccine was safe and well-tolerated. Systemic solicited events were similar between placebo and VLP vaccinated groups with no vaccine-related serious adverse events. Dose response trends for solicited local adverse events were observed, with higher incidences of local pain, swelling, tenderness, and redness reported in the higher VLP dose groups (15 µg and 45 µg) compared to the placebo and 5 µg VLP groups following both vaccinations. Although the majority of local AEs were mild in severity, a dose trend in events of moderate or greater severity was also noted for these solicited events. The VLP vaccine groups demonstrated robust HAI immune responses after a single vaccination, with high rates of seroprotection (≥ 40 HAI titer) in 82-92% of all subjects and in 64-85% of subjects who were seronegative at the time of immunization. HAI geometric mean titers (GMTs), geometric mean ratios (GMRs) and seroconversion rates were also all statistically higher in the VLP groups compared to placebo for both post-baseline time points. Based on these data, additional clinical trials are in development to evaluate influenza vaccine candidate antigens manufactured using Spodoptera frugiperda (Sf9)/baculovirus-based VLP technology.