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.

Determination of Peptide Profile Consistency and Safety of Collagen Hydrolysates as Quality Attributes.

Collagen hydrolysates are dietary supplements used for nutritional and medical purposes. They are complex mixtures of low-molecular-weight peptides obtained from the enzymatic hydrolysis of collagen, which provide intrinsic batch-to-batch heterogeneity. In consequence, the quality of these products, which is related to the reproducibility of their mass distribution pattern, should be addressed. Here, we propose an analytical approach to determine the peptide pattern as a quality attribute of Colagenart®, a product containing collagen hydrolysate. In addition, we evaluated the safety by measuring the viability of two cell lines exposed to the product. The consistency of peptide distribution was determined using Size Exclusion Chromatography (SEC), Mass Spectrometry coupled to a reversed phase UPLC system (MS-RP-UPLC), and Shaped-pulse off-resonance water-presaturation proton nuclear magnetic resonance spectrometry [1 Hwater_presat NMR]. The mass distribution pattern determined by SEC was in a range from 1.35 to 17 kDa, and from 2 to 14 kDa by MS-RP-UPLC. [1 Hwater_presat NMR] showed the detailed spin-systems of the collagen hydrolysates components by global assignment of backbone Hα and NH, as well as side-chain proton resonances. Additionally, short-range intraresidue connectivity pathways of identified spin-regions were obtained by a 2D homonuclear shift correlation Shaped-pulse solvent suppression COSY scheme. Safety analysis of Colagenart® was evaluated in CaCo-2 and HepG2 cells at 2.5 and 25 µg/mL and no negative effects were observed. The results demonstrated batch-to-batch reproducibility, which evinces the utility of this approach to establish the consistency of the quality attributes of collagen hydrolysates. PRACTICAL APPLICATION: We propose state-of-the art analytical methodologies (SEC, MS, and NMR) to evaluate peptide profile and composition of collagen hydrolysates as quality attributes. These methodologies are suitable to be implemented for quality control purposes.

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.

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.

Transferon™, a peptide mixture with immunomodulatory properties is not immunogenic when administered with various adjuvants.

Transferon, a human dialyzable leukocyte extract (hDLE), is a biotherapeutic that comprises a complex mixture of low-molecular-weight peptides (< 10 kDa) and is used to treat diseases with an inflammatory component. Some biotherapeutics, including those composed of peptides, can induce anti-drug antibodies (ADA) that block or diminish their therapeutic effect. Nevertheless, few studies have evaluated peptide-derived drug immunogenicity. In this study, the immunogenicity of Transferon was examined in a murine model during an immunization scheme using the following adjuvants: Al(OH)3, incomplete Freund's adjuvant (IFA), or Titermax Gold. The inoculation scheme entailed three routes of administration (intraperitoneal, Day 1; subcutaneous, Day 7; and intramuscular, Day 14) using 200 µg Transferon/inoculation. Serum samples were collected on Day 21. Total IgG levels were quantitated by affinity chromatography, and specific antibodies against components of Transferon were analyzed by dot-blot and ELISA. Ovalbumin (OVA, 44 kDa) and peptides from hydrolyzed collagen (PFHC, < 17 kDa) were used as positive and negative controls, respectively, in the same inoculation scheme and analyses for Transferon. OVA, PFHC, and Transferon increased total IgG concentrations in mice. However, only IgG antibodies against OVA were detected. Based on the results, it is concluded that Transferon does not induce generation of specific antibodies against its components in this model, regardless of adjuvant and route of administration. These results support the safety of Transferon by confirming its inability to induce ADA in this animal model.