Characterization of IgG1 Fc Deamidation at Asparagine 325 and Its Impact on Antibody-dependent Cell-mediated Cytotoxicity and FcγRIIIa Binding.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important mechanism of action for many therapeutic antibodies. A therapeutic immunoglobulin (Ig) G monoclonal antibody lost more than half of its ADCC activity after heat stress at 40 °C for 4 months. Size-exclusion and ion-exchange chromatography were used to fractionate various size and charge variants from the stressed IgG.

Characterization of IgG2 Disulfide Bonds with LC/MS/MS and Postcolumn Online Reduction.

The complication of IgG2 disulfide connections demands advances in techniques for disulfide bond determination. We have developed a new LC/MS/MS method for improved disulfide analysis. With postcolumn introduction of dithiothreitol (DTT) and ammonium hydroxide, each disulfide-containing peptide eluted out of LC in an acidic mobile phase can be rapidly reduced prior to MS analysis.

Qualification of a Quantitative Method for Monitoring Aspartate Isomerization of a Monoclonal Antibody by Focused Peptide Mapping.

Unlabelled: Aspartate (Asp) isomerization is a common post-translational modification of recombinant therapeutic proteins that can occur during manufacturing, storage, or administration. Asp isomerization in the complementarity-determining regions of a monoclonal antibody may affect the target binding and thus a sufficiently robust quality control method for routine monitoring is desirable. In this work, we utilized a liquid chromatography-mass spectrometry (LC/MS)-based approach to identify the Asp isomerization in the complementarity-determining regions of a therapeutic monoclonal antibody.

Characterization of virus-like particles in GARDASIL® by cryo transmission electron microscopy.

Cryo-transmission electron microscopy (cryoTEM) is a powerful characterization method for assessing the structural properties of biopharmaceutical nanoparticles, including Virus Like Particle-based vaccines. We demonstrate the method using the Human Papilloma Virus (HPV) VLPs in GARDASIL®. CryoTEM, coupled to automated data collection and analysis, was used to acquire images of the particles in their hydrated state, determine their morphological characteristics, and confirm the integrity of the particles when absorbed to aluminum adjuvant.

Toolbox for non-intrusive structural and functional analysis of recombinant VLP based vaccines: a case study with hepatitis B vaccine.

Background: Fundamental to vaccine development, manufacturing consistency, and product stability is an understanding of the vaccine structure-activity relationship. With the virus-like particle (VLP) approach for recombinant vaccines gaining popularity, there is growing demand for tools that define their key characteristics. We assessed a suite of non-intrusive VLP epitope structure and function characterization tools by application to the Hepatitis B surface antigen (rHBsAg) VLP-based vaccine.

In-depth process understanding of RECOMBIVAX HB® maturation and potential epitope improvements with redox treatment: multifaceted biochemical and immunochemical characterization.

Recombinant Hepatitis B surface antigen virus-like particles (VLPs) produced in yeast undergo spontaneous maturation during the vaccine production process, and the biophysical characteristics of the particles with respect to maturation were described in Zhao et al. (2006) [13]. Here we report additional biochemical and immunochemical characterization by various techniques, including the use of a panel of monoclonal antibodies (mAbs) that differ in their selectivity and conformation-sensitivity, for probing surface epitope structures.

Pharmacokinetic immunoassay methods in the presence of soluble target.

Soluble targets represent a special challenge when employing ligand binding assays to support pharmacokinetic analysis of monoclonal therapeutics. Target-engaged antibody is not available for binding in immunoassays employing anti-idiotype-specific antibodies or target for capture. We investigated several formats of total antibody assays that show reduced interference of soluble targets: direct target capture, indirect target capture and acid dissociation.

Measurement and decomposition kinetics of residual hydrogen peroxide in the presence of commonly used excipients and preservatives.

Quantitation of residual hydrogen peroxide (H(2)O(2)) and evaluation of the impact on product stability is necessary as unwanted H(2)O(2) can potentially be introduced during the manufacturing of pharmaceuticals, biologics, and vaccines. A sensitive and convenient microplate-based method with fluorescence detection for H(2)O(2) quantitation was recently reported (Towne et al., 2004, Anal Biochem 334: 290-296).

Applications of CE SDS gel in development of biopharmaceutical antibody-based products.

CE SDS gel technique offers many advantages over the traditional labor-intensive SDS PAGE slab gel technology. The CE-based method has increasingly been applied to many protein analysis applications. Specific examples are provided for monoclonal antibody (mAb), though the technique can be adapted to many other therapeutic protein products.

Maturation of recombinant hepatitis B virus surface antigen particles.

The major surface antigen of Hepatitis B virus (HBsAg) is a cysteine-rich, lipid-bound protein with 226 amino acids. Recombinant HBsAg (rHBsAg) with associated lipids can self-assemble into 22-nm immunogenic spherical particles, which are used in licensed Hepatitis B vaccines. Little is known about the structural evolvement or maturation upon assembly beyond an elevated level of disulfide formation.

Evaluation of the thermal stability of Gardasil.

The thermostability of GARDASIL (Merck & Co., Inc, Whitehouse Station, NJ, USA), a developmental vaccine against human papillomavirus (HPV), was evaluated using an enzyme immunoassay, referred to as the in vitro relative potency (IVRP) assay and differential scanning calorimetry (DSC). Gardasil samples were stored at temperatures ranging from 4 to 42 degrees C and tested for IVRP at various time points.

Enhanced sensitivity and precision in an enzyme-linked immunosorbent assay with fluorogenic substrates compared with commonly used chromogenic substrates.

Quantitative enzyme-linked immunosorbent assay (ELISA) is a widely used tool for analyzing biopharmaceutical and vaccine products. The superior sensitivity of the ELISA format is conferred by signal amplification through the enzymatic oxidation or hydrolysis of substrates to products with enhanced color or fluorescence. The extinction coefficient for a colored product or the quantum yield of a fluorescent product, coupled with the efficiency of the immobilized enzyme, is the determining factor for the sensitivity and precision of a given ELISA.

Visualization of discrete L1 oligomers in human papillomavirus 16 virus-like particles by gel electrophoresis with Coomassie staining.

The recombinant major capsid protein (L1) of human papillomavirus (HPV) can self-assemble into virus-like particles (VLPs) with 360 L1 molecules per VLP. These tightly associated L1 oligomers in the assembled VLPs were disrupted in a pH-, denaturant-, time-, and temperature-dependent fashion. With non-reducing Laemmli-type SDS-PAGE, primarily the monomeric L1 protein ( approximately 55 kDa) is observed when analyzing VLP preparations.

Quantitative nuclear magnetic resonance analysis and characterization of the derivatized Haemophilus influenzae type b polysaccharide intermediate for PedvaxHIB.

PedvaxHIB is a pediatric vaccine that protects children from severe disease caused by the gram-negative bacterium Haemophilus influenzae type b (Hib). The vaccine is made by chemically conjugating Hib capsular polysaccharide to the outer membrane protein complex of Neisseria meningitidis. The protein-conjugated vaccine has proven to be extremely effective in preventing invasive Hib disease in infants and young children.

Development of an adenovirus-shedding assay for the detection of adenoviral vector-based vaccine and gene therapy products in clinical specimens.

Adenoviral vectors are used widely as gene therapy and vaccine delivery systems. An adenovirus-shedding assay may be performed in clinical trials to monitor the safety of the vector and to investigate the potential relation between clinical symptoms and shed vector virus. This report describes the development and statistical performance of the shedding assay.

Memory T cells and vaccines.

T lymphocytes play a central role in the generation of a protective immune response in many microbial infections. After immunization, dendritic cells take up microbial antigens and traffic to draining lymph nodes where they present processed antigens to naïve T cells. These naïve T cells are stimulated to proliferate and differentiate into effector and memory T cells.

Overexpression, purification, and characterization of the periplasmic space thiamin-binding protein of the thiamin traffic ATPase in Escherichia coli.

Thiamin (Vitamin B(1)) transport in Escherichia coli occurs by the superfamily of traffic ATPases in which the initial receptor is the periplasmic binding protein. We have cloned the periplasmic thiamin-binding protein (TBP) of the E. coli periplasmic thiamin transport system and purified the overexpressed protein to apparent homogeneity.

Thiamine transport in Escherichia coli: the mechanism of inhibition by the sulfhydryl-specific modifier N-ethylmaleimide.

Active transport of thiamin (vitamin B(1)) into Escherichia coli occurs through a member of the superfamily of transporters known as ATP-binding cassette (ABC) transporters. Although it was demonstrated that the sulfhydryl-specific modifier N-ethylmaleimide (NEM) inhibited thiamin transport, the exact mechanism of this inhibition is unknown. Therefore, we have carried out a kinetic analysis of thiamin transport to determine the mechanism of inhibition by NEM.