Structure-Based Correlation of Light-Induced Histidine Reactivity in A Model Protein.

Light is known to induce covalently linked aggregates in proteins. These aggregates can be immunogenic and are of concern for drug product development in the biotechnology industry. Histidine (His) is proposed to be a key residue in cross-link generation ( Pattison , D.

Determination of critical quality attributes for monoclonal antibodies using quality by design principles.

Quality by design (QbD) is a global regulatory initiative with the goal of enhancing pharmaceutical development through the proactive design of pharmaceutical manufacturing process and controls to consistently deliver the intended performance of the product. The principles of pharmaceutical development relevant to QbD are described in the ICH guidance documents (ICHQ8-11). An integrated set of risk assessments and their related elements developed at Roche/Genentech were designed to provide an overview of product and process knowledge for the production of a recombinant monoclonal antibody.

Development of a rapid RP-UHPLC-MS method for analysis of modifications in therapeutic monoclonal antibodies.

Chemical or enzymatic modifications of therapeutic monoclonal antibodies (MAbs) that have high risk to safety and efficacy are defined as critical quality attributes (CQAs). During therapeutic MAbs process development, thorough characterization and quantitative monitoring of CQAs requires a variety of analytical techniques. This paper describes the development of a rapid analytical method to assess modifications in MAbs, based on the analysis of subdomains with molecular weights of ∼25kDa.

Effect of individual Fc methionine oxidation on FcRn binding: Met252 oxidation impairs FcRn binding more profoundly than Met428 oxidation.

The long serum half-lives of mAbs are conferred by pH-dependent binding of IgG-Fc to the neonatal Fc receptor (FcRn). The Fc region of human IgG1 has three conserved methionine residues, Met252, Met358, and Met428. Recent studies showed oxidation of these Met residues impairs FcRn binding and consequently affects pharmacokinetics of therapeutic antibodies.

Characterization and stability study of polysorbate 20 in therapeutic monoclonal antibody formulation by multidimensional ultrahigh-performance liquid chromatography-charged aerosol detection-mass spectrometry.

Polysorbate 20 is a nonionic surfactant commonly used in the formulation of therapeutic monoclonal antibodies (mAb) to prevent protein denaturation and aggregation. It is critical to understand the molecular heterogeneity and stability of polysorbate 20 in mAb formulations as polysorbate can gradually degrade in aqueous solution over time by multiple pathways losing surfactant functions and leading to protein aggregation. The molecular heterogeneity of polysorbate and the interference from proteins and the excipient in the formulation matrix make it a challenge to study polysorbate in protein formulations.

High resolution reversed phase analysis of recombinant monoclonal antibodies by ultra-high pressure liquid chromatography column coupling.

Monoclonal antibodies (mAbs) represent one of the fastest growing areas of new drug development. However, their analytical characterization is complex and generally requires an array of orthogonal analytical techniques. Reversed phase liquid chromatography is a valuable strategy due to its high resolving power and straightforward compatibility to mass spectrometry.

Identification of a single base-pair mutation of TAA (Stop codon) → GAA (Glu) that causes light chain extension in a CHO cell derived IgG1.

We describe here the identification of a stop codon TAA (Stop) → GAA (Glu) = Stop221E mutation on the light chain of a recombinant IgG1 antibody expressed in a Chinese hamster ovary (CHO) cell line. The extended light chain variants, which were caused by translation beyond the mutated stop codon to the next alternative in-frame stop codon, were observed by mass spectra analysis. The abnormal peptide peaks present in tryptic and chymotryptic LC-MS peptide mapping were confirmed by N-terminal sequencing as C-terminal light chain extension peptides.

Identification and characterization of buried unpaired cysteines in a recombinant monoclonal IgG1 antibody.

The heterogeneity in therapeutic antibodies arising from buried unpaired cysteines has not been well studied. This paper describes the characterization of two unpaired cysteines in a recombinant humanized IgG1 monoclonal antibody (referred to as mAb A). The reversed-phase high-performance liquid chromatography (RP-HPLC) analysis of mAb A samples showed three distinct peaks, indicating the presence of three species.

Rapid identification of low level glycation sites in recombinant antibodies by isotopic labeling with 13C6-reducing sugars.

Recombinant antibodies exhibit low levels of glycation from exposure to reducing sugars during production. As the glycation sites are typically distributed across the entire antibody, the levels at any one site are low and it becomes difficult to detect them in the conventional peptide maps. A model antibody was subjected to forced glycation by incubating with a high concentration of a 1:1 mixture of (12)C(6)/(13)C(6) reducing sugars with the assumption that the same sites in the native antibody will be glycated but to a lower extent.

Isolation and characterization of therapeutic antibody charge variants using cation exchange displacement chromatography.

In this report, we have demonstrated the isolation and enrichment of charge variants of a monoclonal antibody IgG1 using cation exchange displacement chromatography. We successfully achieved the separation of acidic, main and basic charge variants with high recovery (>70%) and purity (>90%) by using a commercially available stationary phase in conjunction with a commercially available displacer. In addition, we have isolated and enriched a trace methionine-oxidized variant of the monoclonal antibody allowing a secondary means of identification of this variant while providing sufficient enrichment for further analysis, stability tests and potency determination.

Mixed-mode and reversed-phase liquid chromatography-tandem mass spectrometry methodologies to study composition and base hydrolysis of polysorbate 20 and 80.

Polysorbate 20 (polyoxyethylenesorbitan monolaurate) and polysorbate 80 (polyoxyethylenesorbitan monooleate) used in protein drug formulations are complex mixtures that have been difficult to characterize. Here, two HPLC methods are used with evaporative light scattering detection (ELSD) and mass spectrometry (MS) to characterize polysorbate from commercial vendors. The first HPLC method used a mixed-mode stationary phase (Waters Oasis MAX, mixed-mode anion exchange and reversed-phase sorbent) with a step gradient to quantify both the total polyoxyethylene sorbitan ester and polyoxyethylene sorbitan (POE sorbitan, a non-surfactant) in polysorbate.

Quantitation of polysorbate 20 in protein solutions using mixed-mode chromatography and evaporative light scattering detection.

An HPLC assay requiring no complex sample preparation for the measurement of polysorbate 20 in protein solutions was developed. An on-off chromatography technique was employed involving a mixed-mode stationary phase (Waters Oasis MAX, mixed-mode anion-exchange and reversed-phase sorbent) to quantify polysorbate 20 in solutions containing >100mg/mL of protein. With 2% formic acid mobile phase, proteins are typically positive charged and are not retained because of electrostatic repulsions from the quaternary amine in the mixed-mode resin.