characterization – BioProcessing Journal

Gamma-Immunoglobulin Response Characterization, in COVID-19 Convalescent Patients, Against the Spike Protein S2 Subunit with Eight Linear Peptides for Monoclonal Antibody Generation

The SARS-CoV-2 spike protein S2 subunit plays an essential role in the virus-host cell membrane fusion process. Therefore, the subject of this study was to characterize the gamma-immunoglobulin (IgG) response, in a group of COVID-19 convalescent patients, against the S2 subunit with eight linear peptides to generate a monoclonal antibody (mAb) against the immunodominant linear peptide to be used for therapeutic and diagnostic purposes. Results of antibody percentages against assessed linear peptides were 100% for A21P73, A21P74, A21P75, A21P76, M20P51, M20P65, M20P83, and 66.7% for M20P85. Plasma samples were also used for purifying IgG to corroborate specificity against the same linear peptides, where results reproduced those applying plasmas directly to ELISA-plates. Within these peptides, A21P75 was chosen as immunodominant (100% of recognition with higher absorbance). The A21P75 linear peptide showed poor immunogenicity in mice (1:4000–8000 after four doses), allowing the generation of a CB.HS2A21P75 hybridoma for mAb production that recognized the A21P75 linear peptide with middle-to-high affinity constant (K_aff) (0.8×10⁸ M^-1).

This study concludes that the A21P75 linear peptide is the assessed immunodominant linear peptide for this COVID-19 convalescent patient group. This peptide is located in the HR1 region that plays an important role in SARS-CoV-2 host cell membrane fusion process and is highly conserved between SARS-CoV-2 and SARS-CoV. Thus, due to CB.S2A21P75 mAb specificity and K_aff, it might be the proper reagent to study inhibition of virus-host cell membrane fusion, and as a diagnostic reagent for coronavirus. Finally, the combination of A21P75 linear peptide with other peptides (e.g., receptor binding domain [RBD]) could be suitable reagents for the development of vaccines and therapeutic antibodies with virus infection-blocking capacity.

Biologics Production

cell membrane fusion characterization gamma-immunoglobulin response inhibitory therapeutics linear peptide monoclonal antibody subunit

Testing for Geographic Origin of Fetal Bovine Serum

Since its inception in 2006, the International Serum Industry Association (ISIA) has been focused on providing a more informative characterization standard for animal sera. A fundamental aspect of this effort has been the development of a program focused on product traceability from abattoir to end-user. This goal has been achieved in part by implementing the ISIA-sponsored audit program. Serum vendors determined to be compliant with all audit requirements are awarded ISIA Traceability Certifications. In conjunction with Oritain Global Ltd, ISIA has developed and implemented a method for establishing geographical origin of serum products. The method and its capability of determining geographical origin are described in this paper…

Regulatory

characterization chemical fingerprinting elemental analysis fetal bovine serum geographic origin product traceability standards

Characterization and Qualification of Cell Substrates for Manufacturing Viral Vaccines in the United States

Cell substrates are used in various stages of viral vaccine manufacturing, as in the isolation, selection, and propagation of the virus seed or virus vector stock, as well as for the amplification of the virus to produce the final vaccine product. The various stages of cell substrate use, including cell banking, are shown in a generic manufacturing scheme in Figure 1. Traditionally, viral vaccines have been produced in animal tissues, primary cell cultures, and cell lines that either have a finite life span, such as normal diploid cells, or a theoretically infinite life span, as achieved with continuous or immortalized neoplastic cells. The cell substrates used in viral vaccines currently licensed in the US are listed in Table 1…

Manufacturing Viral Vectors

cell substrates characterization FDA safety testing vaccines viral vaccines

A Summary of the Manufacture, Biochemical Characterization, and Virological Safety Demonstration of the Mouse mAb CB.Hep-1 Used to Produce the Hepatitis B Vaccine

Monoclonal antibodies (mAb) are highly selective molecules, and an unlimited amount of mAbs with equal quality can be produced using mammalian cell cultures and animals. These molecules have remarkable applications in biomedicine, diagnosis and therapy due to the ability to reproduce exactly the same binding properties. The mAbs have been generated against an ostensible set of compounds such as toxins, drugs, blood proteins, cancer cells, viruses, hormones, environmental pollutants, food products, metals and plant materials. In general, mAbs can also be used for creating sensitive tests to detect small amounts of substances, and in therapies, abzymes, and for isolating specific compounds from complex mixtures by immunoaffinity chromatography (IAC)…

Manufacturing

ascites method characterization immunoaffinity chromatography monoclonal antibody vaccines viral clearance viral safety

Profiling Intracellular Monoclonal Antibody Expression for Improved Cell Culture Characterization and Process Optimization

Many laboratories have utilized cell-free systems or prokaryotic systems designed to produce biological molecules with single polypeptide chains, limited folding requirements, and without glycosylation. The yeast systems are used to generate glycoproteins; however, their glycosylation profiles are vastly different from those of mammalian cells. Without significant glycoengineering, the yeast-produced recombinant glycoproteins may be unsuitable as therapeutic molecules. As such, the use of mammalian cells is still the preferred method to produce complex biological molecules…

Biologics Production

antibody expression characterization flow cytometry mammalian cell culture process development specific productivity

Authentication and Characterization of Animal Cell Lines: The Impact on Research

Propagation and culturing of animal cells is fundamental to biomedical research. Over the past decade, there has been an increased demand for cell lines for usage as both research tools and models by academic and industrial scientists. Cell culture is a critical tool in such areas as cell biology, gene therapy, genomics, transcriptomics and proteomics. The increased demand for cells and cell-based assays has triggered a remarkable boost in cell culture activities, which in turn has lead to a greater incidence of misidentified and contaminated cell cultures…

Research

animal cell line authentication characterization contaminant control microbial contamination morphology

Characterization of a Transgenic Murine Antibody Expressed in Nicotiana tabacum Plants

Despite the existence of effective vaccines against Hepatitis B virus, the infection with it remains an important problem worldwide due to its association with hepatocellular carcinoma. Several procedures have been used to purify the Hepatitis B surface antigen (HBsAg) for immunization purposes. Immuno-purification using HBsAg-specific murine monoclonal antibodies (MAbs) has been one of the most successful strategies for such a purpose due to the high antigen selectivity (high affinity) of MAbs…

Biologics Production

antibody expression characterization immunopurification plantibody purification transgenic plants vaccines

Product Characterization of Recombinant Proteins and Monoclonal Antibodies

The analytical characterization of recombinant protein therapeutic drug products has broadened to include the use of more sophisticated technologies. The expansion of technical abilities has translated into increasing the depth and breadth of our knowledge and understanding of the drug product intended for commercialization. With the availability of more precise methods, the regulatory expectations for understanding the characteristics of a protein therapeutic drug product are increasing. A thorough understanding of a therapeutic protein’s biochemical and biophysical characteristics is necessary to support investigational new drug (IND) applications and other drug regulatory filings…

Regulatory

analysis characterization monoclonal antibody recombinant proteins standards therapeutic proteins

An Investigative Comparison: Plant-Based MAbs for Hep B Surface Antigen and MAbs from Mice

Regulatory agencies such as the FDA require the structure and amino acid sequence characterization of recombinant monoclonal antibodies (MAbs) to grant marketing approval. Characterizing such complex, inherently heterogeneous molecules is a significant analytical challenge that requires a broad array of physicochemical tests. Mass spectrometry (MS) is an essential tool for characterizing protein identity, functions, substrate specificity and amino acid sequence (AAS) of recombinant MAb biotherapeutics as it complements, or in some cases supersedes the utility of traditional biological methods. For some of the most important proteomic applications, the high sensitivity and accuracy provided by modern MS has allowed the unequivocal protein characterization…

Biologics Production

characterization monoclonal antibody plant-based protein production plantibody recombinant antibodies

Fast On-Line Desalting of Proteins for Determination of Structural Variation Using Exact Mass Spectroscopy

Analytical tools for the characterization of protein identity and structure are fundamental to many fields of biochemical research. For the development of protein biopharmaceuticals, it is particularly important to measure modifications of the structure that may affect safety and efficacy. This application requires the analysis of large numbers of samples during process development. Small quantities of modifications must be detected in samples that are in the presence of more abundant native protein. Many kinds of analytical techniques are applied to this problem including peptide mapping, bioassays, liquid chromatography, spectroscopy, and so on. One of the most attractive tools is mass spectrometry, since essentially any change in the molecule is accompanied by a change in molecular weight. With the recent advent of readily accessible mass spectrometers capable of routine exact mass measurement, it is appropriate to consider the detailed requirements for this application…

Biologics Production

analysis characterization contaminant control desalting mass spectrometry

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