Real-time product attribute control to manufacture antibodies with defined N-linked glycan levels.

Pressures for cost-effective new therapies and an increased emphasis on emerging markets require technological advancements and a flexible future manufacturing network for the production of biologic medicines. The safety and efficacy of a product is crucial, and consistent product quality is an essential feature of any therapeutic manufacturing process. The active control of product quality in a typical biologic process is challenging because of measurement lags and nonlinearities present in the system.

The criticality of high-resolution N-linked carbohydrate assays and detailed characterization of antibody effector function in the context of biosimilar development.

Accurate measurement and functional characterization of antibody Fc domain N-linked glycans is critical to successful biosimilar development. Here, we describe the application of methods to accurately quantify and characterize the N-linked glycans of 2 IgG1 biosimilars with effector function activity, and show the potential pitfalls of using assays with insufficient resolution. Accurate glycan assessment was combined with glycan enrichment using lectin chromatography or production with glycosylation inhibitors to produce enriched pools of key glycan species for subsequent assessment in cell-based antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity effector function assays.

An optimized approach to the rapid assessment and detection of sequence variants in recombinant protein products.

The development of sensitive techniques to detect sequence variants (SVs), which naturally arise due to DNA mutations and errors in transcription/translation (amino acid misincorporations), has resulted in increased attention to their potential presence in protein-based biologic drugs in recent years. Often, these SVs may be below 0.1%, adding challenges for consistent and accurate detection.

Rapid identification of an antibody DNA construct rearrangement sequence variant by mass spectrometry.

During cell line development for an IgG1 antibody candidate (mAb1), a C-terminal extension was identified in 2 product candidate clones expressed in CHO-K1 cell line. The extension was initially observed as the presence of anomalous new peaks in these clones after analysis by cation exchange chromatography (CEX-HPLC) and reduced capillary electrophoresis (rCE-SDS). Reduced mass analysis of these CHO-K1 clones revealed that a larger than expected mass was present on a sub-population of the heavy chain species, which could not be explained by any known chemical or post-translational modifications.

Characterization of antibody charge heterogeneity resolved by preparative immobilized pH gradients.

A capillary isoelectric focusing (cIEF) method has been developed as an alternative to cation exchange chromatography to determine charge heterogeneity for a therapeutic antibody. Characterization of the cIEF profile is important to understand the charged isoform distribution. A variety of preparative IEF methods have been developed over the years but have had various limitations including high levels of contaminating ampholytes and complex fractionation and isolation procedures.

Asparagine-linked oligosaccharides present on a non-consensus amino acid sequence in the CH1 domain of human antibodies.

We report that N-linked oligosaccharide structures can be present on an asparagine residue not adhering to the consensus site motif NX(S/T), where X is not proline, described in the literature. We have observed oligosaccharides on a non-consensus asparaginyl residue in the C(H)1 constant domain of IgG1 and IgG2 antibodies. The initial findings were obtained from characterization of charge variant populations evident in a recombinant human antibody of the IgG2 subclass.

O-fucosylation of an antibody light chain: characterization of a modification occurring on an IgG1 molecule.

We describe the characterization of an O-fucosyl modification to a serine residue on the light chain of a recombinant, human IgG1 molecule expressed in Chinese hamster ovary (CHO) cells. Cation exchange chromatography (CEX) and hydrophobic interaction chromatography (HIC) were used to isolate a Fab population which was 146 Da heavier than the expected mass. Isolated Fab fragments were treated with a reducing agent to facilitate mass spectrometric analysis of the reduced light chain (LC) and fragment difficult (Fd).

Molecular mass analysis of antibodies by on-line SEC-MS.

Mass analysis of recombinant protein therapeutics is an important assay for product characterization. Intact mass analysis is used to provide confirmation of proper translation of the DNA sequence and to detect the presence of post-translational modifications such as amino acid processing and glycosylation. We present here a method for the rapid mass analysis of antibodies using a polyhydroxyethyl aspartamide column operated in size-exclusion mode and coupled with ESI-MS.

Characterization of nonenzymatic glycation on a monoclonal antibody.

We present here an improved analytical method for the analysis of glycation events in proteins. Nonenzymatic glycation of an IgG2 monoclonal antibody was studied using affinity chromatography, mass spectrometry, and chemical derivatization. Analysis of both forced-degraded and bulk-drug substance (BDS) samples showed the presence of glycated protein.

Fluorescent derivatization method of proteins for characterization by capillary electrophoresis-sodium dodecyl sulfate with laser-induced fluorescence detection.

A fast and improved sample preparation scheme was developed for protein analysis using capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) with laser-induced fluorescence detection. This CE-SDS method was developed as a purity assay for recombinant monoclonal antibodies (rMAbs). In this assay, rMAbs are derivatized with the fluorogenic reagent 3-(2-furoyl)-quinoline-2-carboxaldehyde (FQ) in the presence of a nucleophile (CN-), which fluoresces only upon covalent binding to the protein.