Integrated Process Model Applications Linking Bioprocess Development to Quality by Design Milestones.

Maximizing the value of each available data point in bioprocess development is essential in order to reduce the time-to-market, lower the number of expensive wet-lab experiments, and maximize process understanding. Advanced in silico methods are increasingly being investigated to accomplish these goals. Within this contribution, we propose a novel integrated process model procedure to maximize the use of development data to optimize the Stage 1 process validation work flow. We generate an integrated process model based on available data and apply two innovative Monte Carlo simulation-based parameter sensitivity analysis linearization techniques to automate two quality by design activities: determining risk assessment severity rankings and establishing preliminary control strategies for critical process parameters. These procedures are assessed in a case study for proof of concept on a candidate monoclonal antibody bioprocess after process development, but prior to process characterization. The evaluation was successful in returning results that were used to support Stage I process validation milestones and demonstrated the potential to reduce the investigated parameters by up to 24% in process characterization, while simultaneously setting up a strategy for iterative updates of risk assessments and process controls throughout the process life-cycle to ensure a robust and efficient drug supply.

Size distribution analysis of influenza virus particles using size exclusion chromatography.

Size exclusion chromatography is a standard method in quality control of biopharmaceutical proteins. In contrast, vaccine analysis is often based on activity assays. The hemagglutination assay is a widely accepted influenza quantification method, providing no insight in the size distribution of virus particles. Capabilities of size exclusion chromatography to complement the hemagglutination assay are investigated. The presented method is comparatively robust regarding different buffer systems, ionic strength and additive concentrations. Addition of 200mM arginine or sodium chloride is necessary to obtain complete virus particle recovery. 0.5 and 1.0M arginine increase the hydrodynamic radius of the whole virus particles by 5nm. Sodium citrate induces virus particle aggregation. Results are confirmed by dynamic light scattering. Retention of a H1N1v strain correlates with DNA contents between 5ng/mL and 670ng/mL. Quantitative elution of the virus preparations is verified on basis of hemagglutination activity. Elution of hemagglutination inducing compounds starts at a flow channel diameter of 7000nm. The universal applicability is demonstrated with three different influenza virus samples, including an industrially produced, pandemic vaccine strain. Size distribution of the pandemic H1N1v 5258, H1N1 PR/8/34, and H3N2 Aichi/2/68 preparations spreads across inter- and intra-particle volume and extends to the secondary interaction dominated range. Thus, virus particle debris seems to induce hemagglutination. Fragments generated by 0.5% Triton™ X-100 treatment increase overall hemagglutination activity.

Mono- and polyprotic buffer systems in anion exchange chromatography of influenza virus particles.

Different ions typically used in downstream processing of biologicals are evaluated for their potential in anion exchange chromatography of an industrially produced, pandemic influenza H1N1 virus. Capacity, selectivity and recovery are investigated based on single step elution parallel chromatography experiments. The inactivated H1N1 feedstream is produced in Madin-Darby Bovine Kidney cells. Interesting effects are found for sodium phosphate and sodium citrate. Both anions are triprotic kosmotropes. Anion exchange chromatography generally offers high scalability to satisfy sudden demands for vaccines, which may occur in case of an emerging influenza outbreak. Appropriate pH conditions for H1N1 adsorption are determined by Zeta potential measurements. The dynamic binding capacity of a salt tolerant polyamine-type resin is up to 6.4 times greater than the capacity of a grafted Q-type resin. Pseudo-affinity interactions of polyamines with the M2 protein of influenza may contribute to the obtained capacity increase. Both resins achieve greater capacity in sodium phosphate buffer compared to Tris/HCl. A recovery of 67% and DNA clearance close to 100% without DNAse treatment are achieved for the Q-type resin. Recovery of the virus from the salt tolerant resin requires the use of polyprotic acids in the elution buffer. 85% of the DNA and 60% of the proteins can be removed by the salt tolerant resin. The presence of sodium phosphate during anion exchange chromatography seems to support stability of the H1N1 particles in presence of hydrophobic cations.

Routes to improve binding capacities of affinity resins demonstrated for Protein A chromatography.

Protein A chromatography is a well-established platform in downstream purification of monoclonal antibodies. Dynamic binding capacities are continuously increasing with almost every newly launched Protein A resin. Nevertheless, binding capacities of affinity chromatography resins cannot compete with binding capacities obtained with modern ion exchange media. Capacities of affinity resins are roughly 50% lower. High binding capacities of ion exchange media are supported by spacer technologies. In this article, we review existing spacer technologies of affinity chromatography resins. A yet known effective approach to increase the dynamic binding capacity of Protein A resins is oligomerization of the particular Protein A motifs. This resembles the tentacle technology used in ion exchange chromatography. Dynamic binding capacities of a hexameric ligand are roughly twice as high compared to capacities obtained with a tetrameric ligand. Further capacity increases up to 130mg/ml can be realized with the hexamer ligand, if the sodium phosphate buffer concentration is increased from 20 to 100mM. Equilibrium isotherms revealed a BET shape for the hexamer ligand at monoclonal antibody liquid phase concentrations higher than 9mg/ml. The apparent multilayer formation may be due to hydrophobic forces. Other quality attributes such as recovery, aggregate content, and overall purity of the captured monoclonal antibody are not affected.

Kinetic plots in aqueous size exclusion chromatography of monoclonal antibodies and virus particles.

The growing importance of monoclonal antibodies and virus particles has led to a pressure for faster size exclusion chromatography. In recent years, numerous small particle columns for size exclusion chromatography of biologicals have been introduced. Small particles are a strategy to reduce analysis time. In the following study, opportunities of small particles in size exclusion chromatography of large biomolecules are investigated. Poppe plots reveal that the lower particle size limit depends on the size of the sample molecule. Hydrodynamic radii of monoclonal antibody monomer, aggregates and H1N1 as well as the diffusion coefficients were determined. Considering this sample compound dependency, kinetic plots referring to the resolution of a distinct compound pair instead of the plate number of a single analyte are more meaningful. Plate times were found to be equivalent with 4 and 2µm particles for a monoclonal antibody aggregate separation at resolutions smaller than 1.8. Quantification of a H1N1 in clarified cell culture can be accomplished with 17µm and 13µm particles at equal plate times at resolutions smaller than 2.5. Virus polydispersity is likely to be affected by run times of several hours at room temperature and shear forces resulting from particles smaller than 10µm. Comparatively high flow rates should be applied in size exclusion chromatography of the 100nm H1N1 virions.

Dual salt mixtures in mixed mode chromatography with an immobilized tryptophan ligand influence the removal of aggregated monoclonal antibodies.

In downstream processing of monoclonal antibodies, proper aggregate removal is crucial. Mixed mode ligands such as immobilized tryptophan have been developed to satisfy the need for efficient removal of antibody aggregates. However, method development for mixed mode applications is complicated, since protein binding and elution can be modulated by an increased set of parameters. In the current study, we investigate the effect of different dual salt mixtures on mixed mode chromatography using TOYOPEARL MX-Trp-650M resin, with respect to the dynamic binding capacity, resolution and monomer purity of two different humanized immunoglobulins. Binding capacities varying by more than 50% were observed for different salt mixtures. Furthermore, antibody monomer and aggregate resolution deviated by 30% for different salt mixtures and linear gradient elution. Similar trends were obtained using an immobilized carboxymethyl ligand for the same set of experiments, but the overall resolution was lower. Less kosmotropic salt systems emphasize the electrostatic binding of the relatively hydrophobic mAbs and reduce hydrophobic attraction to a selectivity-determining constraint. Kosmotropic salts such as citrate appear to cause dominating hydrophobic interactions in protein adsorption that hinder electrostatic protein-ligand interactions. This effect may depend on the ionic and hydrophobic site distribution of a protein. The data presented here are important for the further improvement of downstream processing of therapeutic monoclonal antibodies.

Mixed electrolytes in hydrophobic interaction chromatography.

An essential part of the modulation of protein-binding capacity in hydrophobic interaction chromatography is the buffer-salt system. Besides using "single" electrolytes, multicomponent electrolyte mixtures may be used as an additional tool. Both the protein solubility and the binding capacity depend on the position of a salt in the so-called Hofmeister series. Specific interactions are observed for an individual protein-salt combination. For salt mixtures, selectivity, recovery, and binding capacity do not behave like for the single salts that are positioned in between the two mixed components in the Hofmeister series, as the continuous correlation would suggest. Thus, finding strategies for mixed salts could potentially lead to improved capacities in hydrophobic interaction chromatography. Mixtures of ammonium sulfate, sodium citrate, sodium sulfate, sodium chloride, sodium acetate, and glycine were used to investigate the binding capacities for lysozyme and a monoclonal antibody on various hydrophobic resins. Resin capacity for two investigated proteins increases when mixtures consisting of a chaotropic and a kosmotropic salt are applied. It seems to be related to the rather basic isoelectric points of the proteins.