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May 1, 2013 (Vol. 33, No. 9)

Separation of Therapeutic Biomolecules

Mixed-Mode Chromatography, Layered Bead Designs, In Silico Modeling Used at Large Scale

  • Hydrophobic-interaction chromatography and reversed-phase chromatography (HIC/RPC) for large-scale separation of proteins was the focus of a series of talks at Tosoh Bioscience’s recent “HIC/RPC Bioseparation Conference: Advancements, Applications and Theory in Downstream Processing.”

    The program provided a balance of theory, practical applications, scale-up tools, and state-of-the-art resins and media for HIC, RPC, and multimodal chromatographic purification of therapeutic biomolecules. Topics ranged from the fundamentals of chromatography; quality by design; protein structure, interactions, and stability; novel stationary phases; and industrial case studies.

    Mixed-mode chromatography has proven to be a valuable alternative to conventional ion-exchange (IEX) or hydrophobic-interaction chromatography for purifying monoclonal antibodies primarily due to the technique’s high selectivity. This technology is increasingly being applied to other challenging nonantibody therapeutic proteins for which large-scale downstream separation and purification protocols often introduce unwanted process bottlenecks and cost barriers.

  • Click Image To Enlarge +
    Bio-Rad Laboratories believes that mixed-mode chromatography has tremendous advantages in protein separation and purification.

    Xuemei He, Ph.D., senior staff scientist, process chromatography division, Bio-Rad Laboratories, described the use of Nuvia™ cPrime™, a hydrophobic cation exchange media, for mixed-mode chromatography for the purification of a variety of recombinant proteins expressed by prokaryotic or mammalian cell lines. Dr. He cites the main advantages of Nuvia cPrime as high selectivity, tolerance of load conductivity, and gentle purification conditions.

    Nuvia cPrime is composed of a functional ligand on a base matrix of macroporous hydrophilic polymers. Potential interaction modes for protein binding include weak cationic exchange, hydrophobic interaction, and hydrogen bonding. Using lysozyme isolation from an E. coli lysate as an example, Dr. He presented two different purification method development schemes and associated optimization strategies.

    In the first example, she investigated the effect of buffer salt concentration and pH on the purity and yield of final product via a design of experiment (DoE) approach. The “sweet spots” for optimal yield and purity of lysozyme were identified using a set of 11 experiments that covered the pH range of 4–8 and NaCl concentration range of 10–400 mM for binding and 10–1,000 mM for elution.

    In all of these experiments spin columns containing 50 µL of Nuvia cPrime media were used. Purification conditions obtained from these DoE screening tests were experimentally confirmed as being effective for host cell protein and double-stranded DNA removal, and were suitable for use in production scaleup.

    In the second purification method development strategy, Dr. He described an approach based on creating different types of buffer gradients on a traditional chromatographic column—pH, conductivity, or elution with additives. Each of these methods was tested for yield and purity of target protein. They were then refined and converted to a step elution protocol for larger scale production.

    Mixed-mode chromatography offers additional advantages for protein separation and purification, according to Dr. He. With options for efficient target protein elution and tolerance of a range of feed conductivity, this technique often allows easy transition between chromatography unit operations, thus eliminating the need for dilution and extensive feed manipulation.

    “Despite the fact that multiple functionalities may be employed for selective target protein binding, chromatographic methods can still be developed in a straightforward fashion by looking into the effects of buffer pH and conductivity on purification performance,” she said.

    Dr. He also presented case studies on a diverse set of therapeutic proteins, including highly glycosylated recombinant viral antigens and monoclonal IgGs and IgMs, which demonstrated the efficient removal of process and product-related impurities such as degradation fragments and high molecular weight aggregates.

    James Woo, from Rensselaer Polytechnic Institute, presented a study comparing the retention of a library of common proteins on a variety of multimodal cation-exchange ligands, including Bio-Rad’s Nuvia cPrime resin. The study explored the effect on protein retention of changes in the ligand chemistry and geometrical presentation of the functional groups—the effects of weak vs. strong charges at pH 5 in 20 mM acetate solvent, and the effects of secondary groups and hydrogen bonding.

    The results indicated that protein retention tended to decrease with increased charge and was affected by even subtle changes in ligand chemistry. Certain hydrophobic proteins, such as serum albumins, were selectively excluded from the resin, suggesting that by elucidating the role of different functional groups and modulating the design and hydrophobic nature of a multimodal ligand it is possible to cover an expanded design space and target specific selectivities for a given multimodal process step.

  • Purifying Antibody Fragments

    Egbert Müller, Ph.D., technical director at Tosoh Bioscience, discussed the need for novel purification methods to isolate mAb fragments that are increasingly being developed as drug candidates, because conventional Protein A-based separation methods that target the Fc component of the mAb may not be suitable.

    Dr. Müller’s group evaluated the use of the Tosoh Bioscience product Toyopearl MX-Trp-650M mixed-mode resin to purify a F(ab)2 fragment and other antibody-derived molecules, and compared the effects of different buffer systems on the separations.

    Dr. Müller described several advantages of mixed-mode chromatography including high selectivity, high loading capacity, and high efficiency. He discussed the mechanisms of cationic (weak acid) mixed-mode chromatography, including basic ion exchange with adsorption via electrostatic attraction at pH 4 followed by desorption by charge screening of the analyte at pH 5.0–6.5, which yields a combination of high binding capacity and good recovery. He cited examples in the literature of the use of immobilized amino acids such as lysine, histidine, tyrosine, and tryptophan as cationic mixed-mode ligands.

    The Toyopearl MX-Trp-650M resin has a mean pore size of 1,000 Å, a mean particle size of 75 µm, and a pH stability range of 3–13. It maintains a high dynamic binding capacity at elevated feedstock or buffer conductivities, and after extended clean-in-place cycles using 0.5 mol/L sodium hydroxide, and it offers fast binding and elution kinetics, according to the company.

    Dr. Müller illustrated a bind-and-elute method using immobilized tryptophan for mAb aggregate removal. The pH and conductivity gradients increased at the same time with this technique, using 1 M HCl to titrate the pH to 2.7, and the result was greater separation, he said. When they experimented with a dual salt mixture the separation improved even more, and Dr. Müller noted the greater flexibility that mixed-mode resins allow in modulating the process parameters.

    In another experiment, the group evaluated the distribution of the hydrophobic sites on IgG and the interaction of the Fc component with tryptophan on purification of a mAb pepsin digest on Toyopearl MX-Trp-650M. The mixed-mode chromatographic separation was able to isolate the Fc component, and a subsequent study demonstrated successful binding of a scFv to the immobilized tryptophan as well.

    A protocol designed for scFv polishing using MX-Trp-650M yielded two adsorption peaks and an aggregate content of 65%. The group used Protein L to optimize the gradient and reduce the aggregate content, which required dropping to a pH of 2.5. With standard resin the aggregate content did not go below 15%; with the Toyopearl resin the aggregate content was reduced to 3%.

    Dr. Müller concluded that the resolution of the different mAb-derived species can be increased significantly using mixed-mode chromatographic resin. “We think the adsorption mechanism of proteins on Toyopearl is electrostatic and hydrophobic and might be related to its salt-dependent pK value,” he said. In addition, “in contradiction to the literature, we were able to separate antibody fragments by the immobilized tryptophan ligand,” separating the F(ab)2 and Fc fragments formed on pepsin digestion.


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