monoclonal antibody – 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

Monoclonal and Polyclonal Antibodies as Biological Reagents for SARS-CoV-2 Diagnosis Through Nucleocapsid Protein Detection

SARS-CoV-2 is an enveloped, positive-strand RNA virus that contains four structural proteins: spike, envelope, membrane, and nucleocapsid (N-protein). The N-protein participates in virus RNA packaging and particle release, is conserved within SARS-CoV-2 isolates, is highly immunogenic, and is abundantly expressed during SARS-CoV-2 infection. For these reasons, the N-protein could be used as a marker for detecting SARS-CoV-2 in early infection when antibodies against SARS-CoV-2 have not been produced yet. This paper describes the production and characterization of mouse monoclonal antibodies (mAb) and rabbit polyclonal antibodies (pAb) specific for the M20P19 peptide (N-protein linear epitope) for detection purposes. For this study, B-cell hybridomas were generated from mice independently immunized with two different M20P19 peptide-carrier protein conjugates: (1) meningococcal protein P64K; and (2) the keyhole limpet hemocyanin (KLH). Rabbits were also independently immunized with these two immunogens. Study results demonstrated that the M20P19 peptide was very immunogenic in mice and rabbits, and both mAb and pAb specifically recognized the non-conjugated M20P19 peptide, conjugated M20P19 peptide, and N-protein with high affinity and specificity, which could allow SARS-CoV-2 detection by different analytical techniques. This study corroborated that specific and high affinity constant mAb and pAb against the M20P19 peptide can be used as biological reagents for specific and rapid SARS-CoV-2 detection, mainly in tissue samples.

Biologics Production

diagnostics monoclonal antibody nucleocapsid protein detection polyclonal antibody serologic testing tissue sample pathology analysis

Novel Lipid Delivery Mechanism of Cell-Ess Increases Higher Order Glycoforms and the Consistency of Glycan Patterns on Monoclonal Antibodies Produced by CHO Cells

Glycosylation drives protein quality and therapeutic benefits in protein-based therapies. Recently, there has been a push in the pharmaceutical industry to improve the consistency and quality of the glycan patterns on therapeutic proteins like monoclonal antibodies. Post-translational modification begins in the endoplasmic reticulum but is finished in the Golgi where more complex glycans are added. In this study, the addition of lipids via a novel mechanism provided by the medium supplement, Cell-Ess®, improves the consistency in glycan patterns so that they are more reproducible between product batches. The effect of media supplementation with Cell-Ess on the variation of glycan patterns was measured in two different media formulations across two separate experiments. Supplementation with Cell-Ess resulted in a statistically significant reduction in the variation of glycoforms when measured by the standard error of the mean. In addition, to improved consistency, there were increased higher glycoforms or galactosylation. There was also significantly more total galactosylation and significantly fewer lower glycoforms for antibodies produced by CHO cells supplemented with Cell-Ess. These data taken together suggest that the addition of lipids via Cell-Ess results in a more functional Golgi and an associated improvement of protein quality and consistency…

Biologics Production Uncategorized

galactosylation glycosylation lipids media supplement monoclonal antibody post-translational modification

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

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

Effects of Tobacco Extract and Temperature On the Stability of the Monoclonal Antibody CB.Hep-1 Expressed in Transgenic Tobacco Plants

Proteins are widely used in research, medicine and industry, but its extraction from their natural sources can be difficult, tedious and expensive. Therefore, a simple and inexpensive system that allows large-scale production of safe recombinant proteins will always be highly desirable. Traditional production systems that use microbial, insect and mammalian cell cultures have drawbacks, in terms of cost, scalability and product safety. Several studies have shown that molecular farming in plants has many practical, economic, and safety advantages as compared to these conventional methods. Thus, the use of plants for recombinant protein synthesis is gaining wide acceptance…

Biologics Production

molecular farming monoclonal antibody plantibody proteolytic degradation recombinant proteins stability transgenic plants

Solvent Modulation of Protein Interactions In Affinity Chromatography

A variety of affinity chromatography mechanisms exist that exploit either immobilized proteins or small molecule ligands. Among them, protein-A chromatography is widely used as a platform technology for a commercial-scale manufacturing of therapeutic monoclonal antibodies. Antigen, dye and specific ligand-columns are used to purify target proteins for research uses and diagnostic applications. It uses specific interactions between affinity columns and target proteins. In general, the samples containing a protein of interest, such as cell culture media, serum, and tissue or cell lysates, are loaded on to the affinity columns; the proteins will normally bind to the column due to high affinity…

Biologics Production

affinity chromatography antigen chromatography aqueous arginine solution ligand-affinity chromatography monoclonal antibody protein elution

Characterization and Release of Raw Materials Used in Upstream Processes for Production of Monoclonal Antibodies by Mammalian Cell Culture

Since the mid-1970’s, when Kohler and Milstein first discovered the process by which myeloma cells and splenocytes could be fused to produce monoclonal antibodies (MAbs), a whole new world of important therapeutic, prophylactic and diagnostic products has opened up, bringing in huge benefits for patients and manufacturers. The total sales of therapeutic MAbs reached more than $13 billion in 2005. Sixteen of the 18 FDA-approved MAbs came to the market after 1997, and over 150 are currently in clinical development, suggesting their increasing medical importance and the remarkable, recent advancements in development technology…

Biologics Production

characterization growth promotion test mammalian cell culture monoclonal antibody raw materials raw materials qualification

The Quest for a Generic IgG Purification Process

The outstanding success and safety record of first generation monoclonal products has created an immense increase in the number of product candidates that need to be evaluated clinically. The concept of platform purification has emerged in response to this need. A platform is a semigeneric, multistep purification procedure that can be applied to a wide range of monoclonal antibodies without extensive method-scouting and optimization. This approach can substantially accelerate process development and hasten inception of clinical trials…

Biologics Production

cation exchange chromatography hydrophobic interaction chromatography hydroxyapatite chromatography immunoglobulin G monoclonal antibody platform purification

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