Evaluation of mild pH elution protein A resins for antibodies and Fc-fusion proteins.

Protein A affinity chromatography is widely used as a capture step for monoclonal antibodies (mAb) and molecules that possess an Fc-domain, such as fusion proteins and bispecific antibodies. However, the use of low pH (3.0-4.0) to elute the molecule and achieve acceptable yield (>85 %) can lead to product degradation (e.g. fragmentation, aggregation) for molecules sensitive to low pH. In this paper, we describe a comprehensive evaluation of two protein A resins with ligands designed to elute at a milder pH as a result of modified sequences in their Fc and VH3 binding regions. One of the evaluated resins has been made commercially available by Purolite and named Praesto Jetted A50 HipH. Results demonstrated that Jetted A50 HipH could elute the Fc-fusion protein and most mAbs evaluated with an elution pH at or above 4.6. Elution and wash optimization determined run conditions for high recovery (>90 % monomer yield), reduction of high molecular weight (HMW) species (>50 %), and significant host cell protein (HCP) clearance at the mildest elution pH possible. For a pH-stable mAb and a pH-sensitive fusion protein, cell culture material was purified with optimized conditions and demonstrated the mild elution pH resins' ability to purify product with acceptable yield, comparable or better impurity clearance, and significantly milder native eluate pH compared to traditional resins. The benefits of the mild elution pH resins were clearly exemplified for the pH-sensitive protein, where a milder elution buffer and native eluate pH resulted in only 2 % HMW in the eluate that remained stable over 48 h. In contrast, a traditional protein A resin requiring low pH elution led to eluate HMW levels of 8 %, which increased to 16 % over the same hold time. Additionally, these resins have high dynamic binding capacity and allow the use of traditional HCP washes. Therefore, Jetted A50 HipH is an ideal candidate for a platform protein A resin and provides flexibility for pH-sensitive proteins and stable mAbs, while preserving product quality, recovery, and seamless integration into a downstream process.

Affinity chromatography for vaccines manufacturing: Finally ready for prime time?

Affinity chromatography is among the most powerful separation techniques, achieving the finest separation with high yields even in the most challenging feed streams. Incorporating affinity chromatography in vaccine purification has long been attempted by researchers to improve unit yield and purity with the secondary goal of reducing the number of downstream process operations. Despite the success in laboratory-scale proof of concept, implementation of this technique in pilot or cGMP manufacturing has rarely been realised due to technical and economic challenges in design and manufacturing of ideal ligands as well as availability of high-productivity chromatography media. This paper reviews evolving technologies in engineered ligands and chromatography media that are encouraging companies to re-visit the possible use of affinity chromatography in larger scale vaccine purification. It is postulated that commercial-scale implementation of high throughput single-use affinity chromatography can significantly simplify process architecture, improve productivity and flexibility, and reduce cost of goods.

Mabfilin and Fabfilin - New antibody-scaffold fusion formats for multispecific targeting concepts.

Protein based binding molecules have a broad applicability from therapeutic to technical use. Monoclonal antibodies represent the major class of this type of agents complemented by innovative approaches using scaffold proteins with tailor-made properties. Various concepts for new formats combining antibody chains or antibody fragments and fusions with other entities have been developed recently. This strategy opens up options to design molecules with biophysical, biochemical and pharmacological characteristics in a broad range while simultaneously addressing several targets or epitopes. The demand for such compounds is still growing as reflected by the literature and further new ideas are expected. In this context we developed so called Mabfilin and Fabfilin molecules. The formats synergistically bring together the classical antibody or fragments thereof supplemented with additional binding moieties, the Affilin® molecules. These are based on the scaffold ubiquitin endowed with novel targeting properties by local randomization and selection from combinatorial libraries. Mab-/Fabfilin variants show advantageous biochemical properties and open a new route for the development of multispecific compounds for flexible applications.

Ubiquitin is a versatile scaffold protein for the generation of molecules with de novo binding and advantageous drug-like properties.

In the search for effective therapeutic strategies, protein-based biologicals are under intense development. While monoclonal antibodies represent the majority of these drugs, other innovative approaches are exploring the use of scaffold proteins for the creation of binding molecules with tailor-made properties. Ubiquitin is especially suited for this strategy due to several key characteristics. Ubiquitin is a natural serum protein, 100% conserved across the mammalian class and possesses high thermal, structural and proteolytic stability. Because of its small size and lack of posttranslational modifications, it can be easily produced in Escherichia coli. In this work we provide evidence that ubiquitin is safe as tested experimentally in vivo. In contrast to previously published results, we show that, in our hands, ubiquitin does not act as a functional ligand of the chemokine receptor CXCR4. Cellular assays based on different signaling pathways of the receptor were conducted with the natural agonist SDF-1 as a benchmark. In none of the assays could a response to ubiquitin treatment be elicited. Furthermore, intravenous application to mice at high concentrations did not induce any detectable effect on cytokine levels or hematological parameters.

Engineering neprilysin activity and specificity to create a novel therapeutic for Alzheimer's disease.

Neprilysin is a transmembrane zinc metallopeptidase that degrades a wide range of peptide substrates. It has received attention as a potential therapy for Alzheimer's disease due to its ability to degrade the peptide amyloid beta. However, its broad range of peptide substrates has the potential to limit its therapeutic use due to degradation of additional peptides substrates that tightly regulate many physiological processes. We sought to generate a soluble version of the ectodomain of neprilysin with improved activity and specificity towards amyloid beta as a potential therapeutic for Alzheimer's disease. Extensive amino acid substitutions were performed at positions surrounding the active site and inner surface of the enzyme and variants screened for activity on amyloid beta 1-40, 1-42 and a variety of other physiologically relevant peptides. We identified several mutations that modulated and improved both enzyme selectivity and intrinsic activity. Neprilysin variant G399V/G714K displayed an approximately 20-fold improved activity on amyloid beta 1-40 and up to a 3,200-fold reduction in activity on other peptides. Along with the altered peptide substrate specificity, the mutant enzyme produced a markedly altered series of amyloid beta cleavage products compared to the wild-type enzyme. Crystallisation of the mutant enzyme revealed that the amino acid substitutions result in alteration of the shape and size of the pocket containing the active site compared to the wild-type enzyme. The mutant enzyme offers the potential for the more efficient degradation of amyloid beta in vivo as a therapeutic for the treatment of Alzheimer's disease.

Novel ubiquitin-derived high affinity binding proteins with tumor targeting properties.

Targeting effector molecules to tumor cells is a promising mode of action for cancer therapy and diagnostics. Binding proteins with high affinity and specificity for a tumor target that carry effector molecules such as toxins, cytokines, or radiolabels to their intended site of action are required for these applications. In order to yield high tumor accumulation while maintaining low levels in healthy tissues and blood, the half-life of such conjugates needs to be in an optimal range. Scaffold-based binding molecules are small proteins with high affinity and short systemic circulation. Due to their low molecular complexity, they are well suited for combination with effector molecules as well as half-life extension technologies yielding therapeutics with half-lives adapted to the specific therapy. We have identified ubiquitin as an ideal scaffold protein due to its outstanding biophysical and biochemical properties. Based on a dimeric ubiquitin library, high affinity and specific binding molecules, so-called Affilin® molecules, have been selected against the extradomain B of fibronectin, a target almost exclusively expressed in tumor tissues. Extradomain B-binding molecules feature high thermal and serum stability as well as strong in vitro target binding and in vivo tumor accumulation. Application of several half-life extension technologies results in molecules of largely unaffected affinity but significantly prolonged in vivo half-life and tumor retention. Our results demonstrate the utility of ubiquitin as a scaffold for the generation of high affinity binders in a modular fashion, which can be combined with effector molecules and half-life extension technologies.

Sustained peripheral depletion of amyloid-ß with a novel form of neprilysin does not affect central levels of amyloid-ß.

Alzheimer's disease is characterized by the accumulation of amyloid deposits in the brain and the progressive loss of cognitive functions. Although the precise role of amyloid-ß in disease progression remains somewhat controversial, many efforts to halt or reverse disease progression have focussed on reducing its synthesis or enhancing its removal. It is believed that brain and peripheral soluble amyloid-ß are in equilibrium and it has previously been hypothesized that a reduction in peripheral amyloid-ß can lower brain amyloid-ß, thereby reducing formation of plaques predominantly composed of insoluble amyloid-ß; the so-called peripheral sink hypothesis. Here we describe the use of an amyloid-ß degrading enzyme, the endogenous metallopeptidase neprilysin, which is fused to albumin to extend plasma half-life and has been engineered to confer increased amyloid-ß degradation activity. We used this molecule to investigate the effect of degradation of peripheral amyloid-ß on amyloid-ß levels in the brain and cerebrospinal fluid after repeated intravenous dosing for up to 4 months in Tg2576 transgenic mice, and 1 month in rats and monkeys. This molecule proved highly effective at degradation of amyloid-ß in the periphery but did not alter brain or cerebrospinal fluid amyloid-ß levels, suggesting that the peripheral sink hypothesis is not valid and is the first time that this has been demonstrated in non-human primates.

Femtomolar Fab binding affinities to a protein target by alternative CDR residue co-optimization strategies without phage or cell surface display.

In therapeutic or diagnostic antibody discovery, affinity maturation is frequently required to optimize binding properties. In some cases, achieving very high affinity is challenging using the display-based optimization technologies. Here we present an approach that begins with the creation and clonal, quantitative analysis of soluble Fab libraries with complete diversification in adjacent residue pairs encompassing every complementarity-determining region position. This was followed by alternative recombination approaches and high throughput screening to co-optimize large sets of the found improving mutations. We applied this approach to the affinity maturation of the anti-tumor necrosis factor antibody adalimumab and achieved ~500-fold affinity improvement, resulting in femtomolar binding. To our knowledge, this is the first report of the in vitro engineering of a femtomolar affinity antibody against a protein target without display screening. We compare our findings to a previous report that employed extensive mutagenesis and recombination libraries with yeast display screening. The present approach is widely applicable to the most challenging of affinity maturation efforts.

Single-molecule detection technologies in miniaturized high-throughput screening: fluorescence intensity distribution analysis.

Single-molecule detection technologies are becoming a powerful readout format to support ultra-high-throughput screening. These methods are based on the analysis of fluorescence intensity fluctuations detected from a small confocal volume element. The fluctuating signal contains information about the mass and brightness of the different species in a mixture. The authors demonstrate a number of applications of fluorescence intensity distribution analysis (FIDA), which discriminates molecules by their specific brightness. Examples for assays based on brightness changes induced by quenching/dequenching of fluorescence, fluorescence energy transfer, and multiple-binding stoichiometry are given for important drug targets such as kinases and proteases. FIDA also provides a powerful method to extract correct biological data in the presence of compound fluorescence.