An Intra-Company Analysis of Inherent Particles in Biologicals Shapes the Protein Particle Mitigation Strategy Across Development Stages.

To better understand protein aggregation and inherent particle formation in the biologics pipeline at Novartis, a cross-functional team collected and analyzed historical protein particle issues. Inherent particle occurrences from the past 10 years were systematically captured in a protein particle database. Where the root cause was identified, a number of product attributes (such as development stage, process step, or protein format) were trended. Several key themes were revealed: 1) there was a higher propensity for inherent particle formation with non-mAbs than with mAbs; 2) the majority of particles were detected following manufacturing at scale, and were not predicted by the small-scale studies; 3) most issues were related to visible particles, followed by subvisible particles; 4) 50% of the issues were manufacturing related. These learnings became the foundation of a particle mitigation strategy across development and technical transfer, and resulted in a set of preventive actions. Overall, this study provides further insight into a recognized industry challenge and hopes to inspire the biopharmaceutical industry to transparently share their experiences with inherent particles formation.

Protein-protein interactions and reduced excluded volume increase dynamic binding capacity of dual salt systems in hydrophobic interaction chromatography.

Deploying two salts in hydrophobic interaction chromatography can significantly increase dynamic binding capacities. Nevertheless, the mechanistic understanding of this phenomenon is lacking. Here, we investigate whether surface tension or ionic strength govern dynamic binding capacities of the chromatographic resin Toyopearl Butyl-650 M in dual salt systems. Small-angle X-ray scattering was employed to analyze the model proteins and the protein-resin adduct in the respective dual salt systems. The dual salt systems incorporate sodium citrate and a secondary sodium salt (acetate, sulfate, or phosphate). As model proteins, we used lysozyme, GFP, and a monoclonal antibody (adalimumab). Moreover, for the protein-resin adduct, we determined the model parameters of a self-avoiding random walk model fitted into the pair density distribution function of the SAXS data. Ionic strength is more predictive for dynamic binding capacities in HIC dual salt systems than surface tension. However, dynamic binding capacities still differ by up to 30 % between the investigated dual salt systems. The proteins exhibit extensive protein-protein interactions in the studied dual salt HIC buffers. We found a correlation of protein-protein interactions with the well-known Hofmeister series. For systems with elevated protein-protein interactions, adsorption isotherms deviate from Langmuirian behavior. This highlights the importance of lateral protein-protein interactions in protein adsorption, where monomolecular protein layers are usually assumed. SAXS analysis of the protein-resin adduct indicates an inverse correlation of the binding capacity and the excluded volume parameter. This is indicative of the deposition of proteins in the cavities of the stationary phase. We hypothesize that increasing protein-protein interactions allow the formation of attractive clusters and multilayers in the cavities, respectively.

Calorimetry for studying the adsorption of proteins in hydrophobic interaction chromatography.

Hydrophobic interaction chromatography is a very popular chromatography method for purification of proteins and plasmids in all scales from analytical to industrial manufacturing. Despite this frequent use, the complex interaction mechanism and the thermodynamic aspects of adsorption in hydrophobic interaction chromatography are still not well understood. Calorimetric methods such as isothermal titration calorimetry and flow calorimetry can help to gain a deeper understanding of the adsorption strength, the influence of salt type and temperature. They can be used to study conformational changes of proteins, which are often associated with the adsorption in hydrophobic interaction chromatography. This review offers a detailed introduction into the thermodynamic fundamentals of adsorption in hydrophobic interaction chromatography with a special focus on the potential applications of isothermal titration calorimetry and flow calorimetry for studying specific problems and relationships of the adsorption behavior of proteins and its various influencing factors. Models for characterizing conformational changes upon adsorption are presented together with methods for assessing this problem for different proteins and stationary phases. All of this knowledge can contribute greatly to forming a sound basis for method development, process optimization and finding modelling strategies in hydrophobic interaction chromatography.

How Similar Is Biosimilar? A Comparison of Infliximab Therapeutics in Regard to Charge Variant Profile and Antigen Binding Affinity.

Biosimilars are increasing in economic importance. Just how similar a biosimilar needs to be to gain market approval is currently still decided on a per case basis. The authors try to shed light on one often cited critical quality attribute of monoclonal antibodies, namely charge heterogeneity. Using high resolution electrophoretic and chromatographic methods, the authors are able to separate and quantify the charge variant content of infliximab originator and three biosimilars. Additionally the authors quantified and compared the antigen binding affinity in an SPR based binding assay and analyzed the glycosylation pattern of all four of these infliximab biosimilar products. Even though the analytical methods did not show full similarity between originator and some biosimilars, all of the biosimilars have gained approval based on their clinical comparability. The authors would therefore argue, that analytical comparison is not always a good predictor for clinical interchangeability. Any future regulatory framework for the approval of biosimilars should reflect that the parameters chosen for analytical comparability have to be chosen carefully.

Hydrophobic interaction chromatography of proteins: Studies of unfolding upon adsorption by isothermal titration calorimetry.

Heat of adsorption is an excellent measure for adsorption strength and, therefore, very useful to study the influence of salt and temperature in hydrophobic interaction chromatography. The adsorption of bovine serum albumin and ß-lactoglobulin to Toyopearl Butyl-650 M was studied with isothermal titration calorimetry to follow the unfolding of proteins on hydrophobic surfaces. Isothermal titration calorimetry is established as an experimental method to track conformational changes of proteins on stationary phases. Experiments were carried out at two different salt concentrations and five different temperatures. Protein unfolding, as indicated by large changes of molar enthalpy of adsorption Δhads , was observed to be dependent on temperature and salt concentration. Δhads were significantly higher for bovine serum albumin and ranged from 578 (288 K) to 811 (308 K) kJ/mol for 1.2 mol/kg ammonium sulfate. Δhads for ß-lactoglobulin ranged from 129 kJ/mol (288 K) to 186 kJ/mol (308 K). For both proteins, Δhads increased with increasing temperature. The influence of salt concentration on Δhads was also more pronounced for bovine serum albumin than for ß-lactoglobulin. The comparison of retention analysis evaluated by the van't Hoff algorithm shows that beyond adsorption other processes occur simultaneously. Further interpretation such as unfolding upon adsorption needs other in situ techniques.

Conformational changes of antibodies upon adsorption onto hydrophobic interaction chromatography surfaces.

Differential scanning calorimetry was established for in-situ measurement of the transition temperatures of antibodies when adsorbed on hydrophobic interaction chromatography media. This method is also suitable for non-transparent media, which is an advantage over spectroscopic methods that cannot be used in many cases due to large background signals. The three transition temperatures of an antibody were lowered when the molecule was adsorbed onto Phenyl and Butyl functionalized Toyopearl particles as well as on Phenyl Sepharose 6 Fast Flow when bound at moderate to high salt concentration compared to the values in free solution. The first two melting points, representing the CH2 domain and the Fab fragment, are more affected than the highest melting point, which corresponds to the CH3 domain. It is obvious that domains which are less stable are more likely to undergo conformational change upon adsorption. It could be shown that the conformational changes occurring in antibodies upon adsorption to HIC media are directly proportional to the hydrophobicity of the stationary phase and that they are reversible. Upon elution, the protein returns to its original conformation. For all four tested resins, a negative value for both ΔH as well as ΔS was calculated, leading to opposing contributions to ΔG.

Temperature dependence of antibody adsorption in protein A affinity chromatography.

Staphylococcal protein A affinity chromatography is a well-established platform for purification of clinical-grade antibodies. The wild type ligand has been mutated to improve caustic stability, elution behavior, and/or to increase binding capacity. Several modified protein A ligands are nowadays commercially available, one of them being the thermosensitive chromatography medium Byzen Pro from Nomadic Bioscience Co., Ltd. According to the manufacturer, Byzen Pro has the ability to release IgG upon a change in temperature. It is based on a thermosensitive mutant of protein A which should allow elution at neutral pH by changing the temperature from binding at 5 °C to elution conditions at 40 °C. We determined equilibrium binding capacities of the thermosensitive protein A medium (Byzen Pro), MabSelect SuRe (GE Healthcare), and TOYOPEARL AF-rProtein A HC-650F (Tosoh Bioscience LLC) for antibodies of the subclass IgG1 and IgG2 at five different temperatures from 4 °C to 40 °C to elucidate the temperature effect. We also observed a temperature dependence of the dynamic binding capacities which were determined for the subclass IgG2 at three temperatures from 4 °C to 40 °C. However, for Byzen Pro, the temperature dependence was only present at a low flow rate and vanished at high flow rates indicating that pore diffusion is the rate-limiting step. Binding of the antibody to MabSelect SuRe and TOYOPEARL AF-rProtein A HC-650F stabilized the conformations as shown by an increase in melting temperature in differential scanning calorimetry measurements. The antibody conformation was slightly destabilized upon binding to the thermosensitive ligand. The conformation change upon binding was fully reversible as shown by circular dichroism, differential scanning calorimetry and size exclusion chromatography. Isothermal titration calorimetry was used to measure the raw heat of adsorption for the IgG2 molecule. The thermosensitive ligand can also be used for antibodies with low stability, because elution can also be effected by salt.

Microheterogeneity of Recombinant Antibodies: Analytics and Functional Impact.

Antibodies are typical examples of biopharmaceuticals which are composed of numerous, almost infinite numbers of potential molecular entities called variants or isoforms, which constitute the microheterogeneity of these molecules. These variants are generated during biosynthesis by so-called posttranslational modification, during purification or upon storage. The variants differ in biological properties such as pharmacodynamic properties, for example, Antibody Dependent Cellular Cytotoxicity, complement activation, and pharmacokinetic properties, for example, serum half-life and safety. Recent progress in analytical technologies such as various modes of liquid chromatography and mass spectrometry has helped to elucidate the structure of a lot of these variants and their biological properties. In this review the most important modifications (glycosylation, terminal modifications, amino acid side chain modifications, glycation, disulfide bond variants and aggregation) are reviewed and an attempt is made to give an overview on the biological properties, for which the reports are often contradictory. Even though there is a deep understanding of cellular and molecular mechanism of antibody modification and their consequences, the clinical proof of the effects observed in vitro and in vivo is still not fully rendered. For some modifications such as core-fucosylation of the N-glycan and aggregation the effects are clear and should be monitored, but with others such as C-terminal lysine clipping the reports are contradictory. As a consequence it seems too early to tell if any modification can be safely ignored.