Evaluation of recent Protein A stationary phase innovations for capture of biotherapeutics

Abstract

We describe a comprehensive evaluation of 12 Protein A stationary phases for capture of biotherapeutics. We first examine the morphological properties of the stationary phases using a variety of orthogonal techniques including electron microscopy, particle sizing, pressure-flow behavior, and isocratic pulse response. A panel of nine proteins spanning a wide range of structures and biochemical properties was then used to assess equilibrium uptake, mass transport, dynamic binding capacity, and elution pH. Process performance and product quality were also examined under realistic bioprocess conditions using clarified mammalian cell culture broth. Equilibrium isotherms were found to be highly favorable, with equilibrium binding capacity for monoclonal and bispecific antibodies ranging from 47-100 mg/mL packed bed across all stationary phases tested. Effective pore diffusivities, D, were obtained by fitting the chromatography general rate model to breakthrough data. The fitted D values for monoclonal antibodies ranged from 1.1-5.7 × 10 cm/s. The stationary phases had high dynamic binding capacities for the model proteins. The highest dynamic capacities for monoclonal and bispecific antibodies were seen with MabSelect SuRe pcc and MabSelect PrismA, which ranged from 58-74 mg/mL packed bed at 4 min residence times. Product capture using clarified cell culture broth as a feedstock showed high yields and elution pool volumes that ranged from 2-3 column volumes in most cases. Host cell protein, DNA, and aggregate levels in the elution pool were dependent on the specific nature of protein being purified, and levels were consistent between stationary phases. Lastly, we perform an analysis of bivariate correlations and discuss considerations for process design and optimization.

Antibody Binding Heterogeneity of Protein A Resins

“…The isotherm data denote a second increase in values for equilibrium concentrations higher than 0.3 mg mL −1 , which is more clearly seen in the TP PA isotherm. This behavior in the isotherm was also seen in a recent study by Pabst et al It is well described that the Langmuir isotherm assumes monolayer coverage and energetic equivalence of all the binding sites . Nonetheless, this isotherm has been used to model antibody adsorption to Protein A chromatography resins, known for their heterogeneous binding characteristic due to the multimeric chains, with high‐energy (also called fast) and low‐energy (also called slow) binding sites .…”

“…Therefore, antibody binding to more than one domain of the Protein A chain is possible . Thus, the slight deviation from the Langmuir model seen in the isotherm (Figure ) and in the work of Pabst et al at high concentrations could be associated with the heterogeneity of the multimeric Protein A ligand, which in the present study is more visible in the presence of TP PA resin as it has a hexamer structure compared to the tetramer of MSS.…”

“…This behavior in the isotherm was also seen in a recent study by Pabst et al It is well described that the Langmuir isotherm assumes monolayer coverage and energetic equivalence of all the binding sites . Nonetheless, this isotherm has been used to model antibody adsorption to Protein A chromatography resins, known for their heterogeneous binding characteristic due to the multimeric chains, with high‐energy (also called fast) and low‐energy (also called slow) binding sites . Therefore, antibody binding to more than one domain of the Protein A chain is possible .…”

“…MSS shows greater effective diffusivity than TP PA for both , as seen in Table , where the parameters fitted by the shrinking core model for both resins can be found. In spite of the larger particle size of MSS (Ø = 85 µm), these greater values can be related to the larger intraparticle porosity ( 0.76) compared to TP PA (Ø = 45 µm and 0.36) …”

The pearl necklace model in protein A chromatography: Molecular mechanisms at the resin interface

“…Figure 2h shows the pair density fluctuations of the MabSelect SuRe resin as assessed by the SEM image, with the typical small pores close to 80 nm in diameter(Pabst, Thai, & Hunter, 2018). Figure 2e is a magnified overlay of Figure 2c,d.…”