No abstract

High temperature is known to cause some instability in polysaccharide-protein conjugated vaccines and studies under stress conditions may be useful in determining whether short-term accidental exposure to undesired conditions can compromise product quality. In this study, we examined the structural stability of three industrial batches of Brazilian Meningococcal C conjugate bulk (MPCT) incubated at 4, 37, and 55 °C for 5 weeks. The effect of exposure to the storage temperatures was monitored by HPLC-SEC, CZE, CD and NMR techniques. The immunological significance of any physicochemical changes observed in MPCT was determined by SBA and ELISA assays of serum from immunized mice. Fluorescence emission spectra at 4 and 37 °C were similar among all samples and compatible with the native fold of the carrier protein. Fluorescence spectra of MPCT stored at 55 °C decreased in intensity and had a significant red-shift, indicating conformational changes. Far-UV CD spectra revealed a trend toward loss of structural conformation as storage temperature was increased to 55 °C. The NMR data showed modified signal intensity of the aromatic and aliphatic residues, mainly for samples incubated at 55 °C, suggesting a partial loss of tertiary structure. About 50% free saccharide content was found in bulks stored at 55 °C, but no difference was observed in the IgG or SBA titers. The present study showed physicochemical methods alone are insufficient to predict the biological activity of a MPCT conjugate vaccine without extensive validation against immunological data. However, they provide a sensitive means of detecting changes induced in a vaccine exposed to adverse environmental condition.

Neisseria meningitidis group C is an encapsulated bacterium that causes several diseases and is associated with high mortality rates, thereby constituting a serious public health problem. Bio-Manguinhos/Fiocruz is developing a conjugate vaccine by covalent attachment of capsular polysaccharide to hydrazide-activated tetanus toxoid through reductive amination. It is necessary to quantify free components as a quality control process to prevent exacerbated adverse reactions and/or attenuation of vaccine immunogenicity. Thus, this study aimed to develop and validate a quality control method appropriate for the separation and quantification of free polysaccharide present in this conjugate N. meningitidis group C vaccine using CE. CZE was used to remove unbound polysaccharide, and the electrophoretic conditions were varied to optimize resolution. We were able to develop and validate the proposed method, which was linear and showed a matrix effect. Repeatability and partial reproducibility of the method were also evaluated. The robustness results showed that control of temperature is required for reliable results. The validated method will be used to evaluate the conjugate batches submitted for Phase III clinical studies and for routine quality control of the conjugate vaccine.

No abstract

Introduction: Inside the biopharmaceuticals class, monoclonal antibodies (mAbs) are prominent molecules with a large clinical application, including autoimmune diseases and different types of cancer. Is crucial to characterize their structural and physical-chemical properties, which can impact the product efficacy, safety, and compliance to regulatory requirements. LC-MS has been an essential tool in these evaluations, due its versatility, high sensibility, and precision. In the Brazilian scenario, the MS core facility of Fiocruz-Paraná, has offered shotgun proteomics analysis for a decade, and is now challenged by the increasing demand of therapeutical protein characterization. Here, we present sample preparation and LC-MS methods used to the characterization of mAbs including intact mass (IM), disulfide bond mapping (DBM) and peptide mapping (PM).Objectives: Develop LC-MS/MS methods for characterizing mAbs to support the development of biopharmaceuticals in Brazil by providing a portfolio of analysis applied to therapeutic proteins. Methodology:For the implementation of the analyses, the antibodies Opdivo, Keytruda, and the Reference Material 8671 NISTmAb were used. For PM and DBM analysis the antibodies were digested in urea with Lys-C, trypsin or Glu-C. For IM, the samples (untreated, reduced or deglycosylated) were diluted in 0.1% formic acid. Digested or undigested samples in the range of 200 to 1000 nanogram were injected into an Ultimate 3000 RSLC coupled to an Orbitrap Fusion Lumos (Thermo Scientific). The data were processed in Unidec, BioPharma Finder or Peaks DB softwares. Results:In IM analysis, the deconvoluted spectra showed the intact molecule comprising the glycoforms, and the light and heavy chains (reduced samples). DBM analysis addressed all nine predicted disulfide bonds and PM confirmed the amino acid sequences. Besides the sample preparation and LC-MS analysis steps, the data processing showed to be an important step for obtaining reliable results and requires optimization as well. Conclusion:To reach the presented results, different parameters were evaluated, enabling the core facility team to learn on analyzing mAbs by LC-MS. Other analysis should be implemented, such as glycan profiling and host cell proteins (HCP). Nonetheless, this work represents one step closer to strengthening competencies nationally, leading to the availability of a portfolio of analyses applied to monoclonal antibodies and support the development of innovators biopharmaceuticals and biosimilars.