Using enhanced development tools offered by analytical Quality by Design to support switching of a quality control method.

Quality by Design (QbD) principles play an increasingly important role in the pharmaceutical industry. Here, we used an analytical QbD (AQbD) approach to develop a capillary electrophoresis sodium dodecyl sulfate under reducing conditions (rCE-SDS), with the aim of replacing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as release and stability test method for a commercialized monoclonal antibody product. Method development started with defining analytical method performance requirements as part of an analytical target profile, followed by a systematic risk assessment of method input parameters and their relation to defined method outputs. Based on this, design of experiments studies were performed to identify a method operable design region (MODR). The MODR could be leveraged to improve method robustness. In a bridging study, it was demonstrated that the rCE-SDS method is more sensitive than the legacy SDS-PAGE method, and a conversion factor could be established to compensate for an off-set due to the higher sensitivity, without losing the correlation to the historical data acquired with the former method. Overall, systematic application of analytical Quality by Design principles for designing and developing a new analytical method helped to elucidate the complex dependency of method outputs on its input parameters. The link of the method to product quality attributes and the definition of method performance requirements were found to be most relevant for derisking the analytical method switch, regarding impact on the control strategy.

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.

Solution structure, dynamics, and hydrodynamics of the calcium-bound cross-reactive birch pollen allergen Bet v 4 reveal a canonical monomeric two EF-hand assembly with a regulatory function.

Birch pollinosis is one of the prevailing allergic diseases. In all, 5-20% of birch pollinotics mount IgE antibodies against the minor birch pollen allergen Bet v 4, a Ca2+-binding polcalcin. Due to IgE cross-reactivity among the polcalcins these patients are polysensitized to various plant pollens. Determination of the high-resolution structure of holo Bet v 4 by heteronuclear NMR spectroscopy reveals a canonical two EF-hand assembly in the open conformation with interhelical angles closely resembling holo calmodulin. The polcalcin-specific amphipathic COOH-terminal alpha-helix covers only a part of the hydrophobic groove on the molecular surface. Unlike the polcalcin Phl p 7 from timothy grass, which was recently shown to form a domain-swapped dimer, the hydrodynamic parameters from NMR relaxation, NMR translational diffusion, and analytical ultracentrifugation indicate that both apo and holo Bet v 4 are predominantly monomeric, raising the question of the physiological and immunological significance of the dimeric form of these polcalcins, whose physiological function is still unknown. The reduced helicity and heat stability in the CD spectra, the poor chemical shift dispersion of the NMR spectra, and the slightly increased hydrodynamic radius of apo Bet v 4 indicate a reversible structural transition upon Ca2+ binding, which explains the reduced IgE binding capacity of apo Bet v 4. The remarkable structural similarity of holo Bet v 4 and holo Phl p 7 in spite of different oligomerization states explains the IgE cross-reactivity and indicates that canonical monomers and domain-swapped dimers may be of similar allergenicity. Together with the close structural homology to calmodulin and the hydrophobic ligand binding groove this transition suggests a regulatory function for Bet v 4.

Mutational epitope analysis of Pru av 1 and Api g 1, the major allergens of cherry (Prunus avium) and celery (Apium graveolens): correlating IgE reactivity with three-dimensional structure.

Birch pollinosis is often accompanied by adverse reactions to food due to pollen-allergen specific IgE cross-reacting with homologous food allergens. The tertiary structure of Pru av 1, the major cherry (Prunus avium) allergen, for example, is nearly identical with Bet v 1, the major birch (Betula verrucosa) pollen allergen. In order to define cross-reactive IgE epitopes, we generated and analysed mutants of Pru av 1 and Api g 1.0101, the major celery (Apium graveolens) allergen, by immunoblotting, EAST (enzyme allergosorbent test), CD and NMR spectroscopy. The mutation of Glu45 to Trp45 in the P-loop region, a known IgE epitope of Bet v 1, significantly reduced IgE binding to Pru av 1 in a subgroup of cherry-allergic patients. The backbone conformation of Pru av 1 wild-type is conserved in the three-dimensional structure of Pru av 1 Trp45, demonstrating that the side chain of Glu45 is involved in a cross-reactive IgE epitope. Accordingly, for a subgroup of celery-allergic patients, IgE binding to the homologous celery allergen Api g 1.0101 was enhanced by the mutation of Lys44 to Glu. The almost complete loss of IgE reactivity to the Pru av 1 Pro112 mutant is due to disruption of its tertiary structure. Neither the mutation Ala112 nor deletion of the C-terminal residues 155-159 influenced IgE binding to Pru av 1. In conclusion, the structure of the P-loop partially explains the cross-reactivity pattern, and modulation of IgE-binding by site-directed mutagenesis is a promising approach to develop hypo-allergenic variants for patient-tailored specific immunotherapy.

Maillard reaction and enzymatic browning affect the allergenicity of Pru av 1, the major allergen from cherry (Prunus avium).

The influence of thermal processing and nonenymatic as well as polyphenoloxidase-catalyzed browning reaction on the allergenicity of the major cherry allergen Pru av 1 was investigated. After thermal treatment of the recombinant protein rPru av 1 in the absence or presence of carbohydrates, SDS-PAGE, enzyme allergosorbent tests, and inhibition assays revealed that thermal treatment of rPru av 1 alone did not show any influence on the IgE-binding activity of the protein at least for 30 min, thus correlating well with the refolding of the allergen in buffer solution as demonstrated by CD spectroscopic experiments. Incubation of the protein with starch and maltose also showed no effect on IgE-binding activity, whereas reaction with glucose and ribose and, even more pronounced, with the carbohydrate breakdown products glyceraldehyde and glyoxal induced a strong decrease of the IgE-binding capacity of rPru av 1. In the second part of the study, the effect of polyphenoloxidase-catalyzed oxidation of polyphenols on food allergen activity was investigated. Incubation of rPru av 1 with epicatechin in the presence of tyrosinase led to a drastic decrease in IgE-binding activity of the protein. Variations of the phenolic compound revealed caffeic acid and epicatechin as the most active inhibitors of the IgE-binding activity of rPru av 1, followed by catechin and gallic acid, and, finally, by quercetin and rutin, showing significantly lower activity. On the basis of these data, reactive intermediates formed during thermal carbohydrate degradation as well as during enzymatic polyphenol oxidation are suggested as the active chemical species responsible for modifying nucleophilic amino acid side chains of proteins, thus inducing an irreversible change in the tertiary structure of the protein and resulting in a loss of conformational epitopes of the allergen.