https://www.onlinelibrary.wiley.com/doi/pdf/10.1002/jps.20727

Antibody structure, instability, and formulation

Effects of glycosylation on the stability of protein pharmaceuticals

Future of antibody purification.

A biotechnological perspective on the application of iron oxide magnetic colloids modified with polysaccharides

Antibodies, also known as immunoglobulins, have emerged as one of the most promising classes of therapeutics in the biopharmaceutical industry The need for complete characterization of the quality attributes of these molecules requires sophisticated techniques Mass spectrometry (MS) has become an essential analytical tool for the structural characterization of therapeutic antibodies, due to its superior resolution over other analytical techniques It has been widely used in virtually all phases of antibody development Structural features determined by MS include amino acid sequence, disulfide linkages, carbohydrate structure and profile, and many different post-translational, in-process, and in-storage modifications In this review, we will discuss various MS-based techniques for the structural characterization of monoclonal antibodies These techniques are categorized as mass determination of intact antibodies, and as middle-up, bottom-up, top-down, and middle-down structural characterizations Each of these techniques has its advantages and disadvantages in terms of structural resolution, sequence coverage, sample consumption, and effort required for analyses The role of MS in glycan structural characterization and profiling will also be discussed

Mass spectrometry for structural characterization of therapeutic antibodies

Antibodies and antibody derivatives constitute 20 % of biopharmaceutical products currently in development, and despite early failures of murine products, chimeric and humanized monoclonal antibodies are now viable therapeutics. A number of genetically engineered antibody constructions have emerged, including molecular hybrids or chimeras that can deliver a powerful toxin to a target such as a tumor cell. However, the general use in clinical practice of antibody therapeutics is dependent not only on the availability of products with required efficacy but also on the costs of therapy. As a rule, a significant percentage (50-80%) of the total manufacturing cost of a therapeutic antibody is incurred during downstream processing. The critical challenges posed by the production of novel antibody therapeutics include improving process economics and efficiency, to reduce costs, and fulfilling increasingly demanding quality criteria for Food and Drug Administration (FDA) approval. It is anticipated that novel affinity-based separations will emerge from the development of synthetic ligands tailored to specific biotechnological needs. These synthetic affinity ligands include peptides obtained by synthesis and screening of peptide combinatorial libraries and artificial non-peptidic ligands generated by a de novo process design and synthesis. The exceptional stability, improved selectivity, and low cost of these ligands can lead to more efficient, less expensive, and safer procedures for antibody purification at manufacturing scales. This review aims to highlight the current trends in the design and construction of genetically engineered antibodies and related molecules, the recombinant systems used for their production, and the development of novel affinity-based strategies for antibody recovery and purification.

Antibodies and Genetically Engineered Related Molecules: Production and Purification

An artificial protein L for the purification of immunoglobulins and fab fragments by affinity chromatography.

Advances and applications of de novo designed affinity ligands in proteomics.

A new methodology for the rapid assessment of affinity ligands synthesized by combinatorial solid-phase chemistry is reported. This screening strategy utilizes the target protein conjugated to FITC, and represents an almost universal technique for the preliminary screening of solid-phase combinatorial libraries. The assessment of a triazine-scaffolded solid-phase combinatorial library of ligands, designed to bind to human IgG, was performed with FITC-human IgG, and the results compared with those obtained by conventional affinity chromatographic screening assays. The effect of different molar conjugation ratios of FITC–IgG (F/P) was evaluated. Independently of the F/P ratio, no false negative results were observed, although lower F/P ratios diminished non-specific interactions and the number of false positives. The nature of the substituents on the triazine scaffold was not related to the number of false positive IgG-binding ligands. The reproducibility of the FITC technique, using FITC–human IgG conjugates with low F/P ratio (F/P=2), was also evaluated. The FITC-based technique proved to be efficient and accurate in the identification of strongly binding ligands (binding >50% of loaded protein, by standard affinity chromatographic assays), and is envisaged as a versatile and cost-effective method to screen other systems, and evaluate several binding/elution conditions at small-scale, prior to scale-up to standard affinity chromatography. Copyright © 2004 John Wiley & Sons, Ltd.

A new method for the screening of solid-phase combinatorial libraries for affinity chromatography.

The reversible phosphorylation of proteins regulates many biological processes. Despite the technological advances in the enrichment and detection of phosphorylated proteins, the currently available techniques still struggle with the complexity of the human proteome. The aim of this review is to highlight the molecular recognition elements of the interaction between phosphorylated proteins and peptides and pTyr or pSer/Thr-binding domains. The identification of the recognition features of the naturally occurring pTyr- and pSer/Thr-binding domains can contribute to an understanding of the molecular aspects of the affinity and specificity for phosphorylated residues. This might inspire the design of small “biomimetic” molecules with potential applications in assessing the extent of the phosphoproteome using affinity-based strategies. © 2005 Wiley Periodicals, Inc.

Christopher R. Lowe

Papers

Antibodies and Genetically Engineered Related Molecules: Production and Purification

An artificial protein L for the purification of immunoglobulins and fab fragments by affinity chromatography.

Advances and applications of de novo designed affinity ligands in proteomics.

A new method for the screening of solid-phase combinatorial libraries for affinity chromatography.

Lessons from nature: On the molecular recognition elements of the phosphoprotein binding‐domains