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Highlights • Structure of CHT Type I and II characterized at the nanoscale. • Adsorption of antibody monomer-dimer mixtures controlled by CEX mechanism. • Antibody monomer and dimer adsorption kinetics is mass transfer controlled. • Mass transfer model predicts two-component binding kinetics. Abstract The internal structure of ceramic hydroxyapatite CHT Type I and Type II and the adsorption behavior of a monoclonal antibody in monomeric and dimeric forms are determined. Both CHT types contain elongated nanocrystals with size about 20 x 100 nm for Type I and about 50 x 200 nm for Type II. Internal porosities and apparent pore radii based on inverse size exclusion chromatography are 0.73 and 30 nm for Type I and 0.70 and 49 nm for Type II. Adsorption isotherms show maximum capacities of 95 and 110 mg/mL of particle for monomer and dimer on Type I and of 55 and 67 mg/mL of particle on Type II, in approximate agreement with the ratio of surface areas. The isotherms are dependent on the Na+ concentration consistent with an electrostatically driven mechanism. Mixture adsorption shows selectivity toward the dimer, with mean α-values of 4.3 and 5.8 for Type I and II, respectively. Effective pore diffusivities for non-binding conditions are 0.54 × 10−7 and 0.33 × 10−7 cm2/s for monomer and dimer in CHT Type I increasing to 0.94 × 10−7 and 0.66 × 10−7 cm2/s as a result of the larger pore size of Type II. Effective pore diffusivities for strong binding conditions, obtained by confocal microscopy, are much smaller than the non-binding values for Type I but essentially the same for Type II, indicating that diffusional hindrance by the bound protein is greater in the smaller pores of Type I. Mixture confocal adsorption experiments show that the competitive binding kinetics is largely controlled by pore diffusion in both CHT types with the dimer readily displacing the monomer even for strong binging conditions, but is much faster on Type II. [ABSTRACT FROM AUTHOR]

Abstract: A simple, low-cost, and scalable protein purification method was developed by using a biodegradable regenerated amorphous cellulose (RAC) with a binding capacity of up to 365mgprotein per gram of RAC. The recombinant protein with a cellulose-binding module (CBM) tag can be specifically adsorbed by RAC. In order to avoid using costly protease and simplify purification process, a self-cleavage intein was introduced between CBM and target protein. The cleaved target protein can be liberated from the surface of RAC by intein self-cleavage occurring through a pH change from 8.0 to 6.5. Four recombinant proteins (green fluorescence protein, phosphoglucomutase, cellobiose phosphorylase, and glucan phosphorylase) have been purified successfully. [Copyright &y& Elsevier]

• mAb structural stability decreases when lowering the solution pH from 6.0 to 4.5 • mAb structural stability further decreases when bound on CEX surfaces • mAb-accessible hydrophobicity of CEX resins affects structural stability of bound mAb • A fluorescence-based method is used to quantitatively characterize CEX resin hydrophobicity • Overall mAb/resin interactions and inherent resin hydrophobicity dictate mAb aggregation in CEX step High molecular weight (HMW) aggregate formation of therapeutic monoclonal antibodies (mAbs) during cation-exchange chromatography (CEX) has been frequently observed, and can be a challenge for downstream purification. To gain mechanistic understanding of this phenomenon, aggregate formation in bind-elute CEX for two therapeutic mAbs (IgG1 and IgG4) was examined on three CEX resins (Capto SP ImpRes, Fractogel EMD SE Hicap, and POROS XS). First, mAb structural stability was studied in solutions under CEX load conditions. Using differential scanning fluorimetry (DSF), the measured melting temperature (Tm DSF (Unbound)) decreased from 60.7 to 52.4°C for mAb1 and 51.5 to 45.2°C for mAb2 when lowering pH from 6.0 to 4.5. Then, mAb structural stability was further investigated in the bound state on CEX surfaces. Using differential scanning calorimetry (DSC), the measured melting temperature of the bound mAbs (Tm DSC (Bound)) was 4.5 - 6.5°C lower than that for the unbound mAbs (Tm DSC (Unbound)) in the same solutions. The Tm differences (∆Tm DSC (Unbound minus Bound)) between the two states correlated with the severity of mAb aggregation in CEX operations, indicating the importance of both intrinsic mAb stability and resin properties. In particular, resin hydrophobicity was shown to have a critical impact. The interplay among these protein- and resin-related factors, together with solution conditions, ultimately dictates the aggregate formation observed. Finally, the hydrophobicity of the CEX resins (Capto SP ImpRes < Fractogel EMD SE Hicap < POROS XS) was measured using a fluorescence-based method to quantitatively characterize this resin property. Results suggest that the mAb-accessible hydrophobic regions of the CEX resins affect the structural stability of the bound mAbs to various degrees, leading to differences in aggregate formation upon mAb elution. In summary, this study offers insight into the mechanism of mAb aggregation in bind-elute CEX operations, and the in-depth understanding facilitates the development of robust CEX conditions for mAb purification. [ABSTRACT FROM AUTHOR]

• Antibody monomer-dimer separation achieved by gradient elution with hydroxyapatite. • 99% antibody monomer purity and 90% recovery obtained starting with 30% dimer. • Hybrid model predicts performance as a function of protein load and gradient slope. • Optimized protein loads lead to high productivity. Modeling the chromatographic separations of proteins at manufacturing scale is important since downstream processing costs are often dominant. At such scales, the columns are highly overloaded heightening the challenge of predicting performance. In this work, the separation of a monoclonal antibody monomer-dimer mixture is conducted by gradient elution chromatography with ceramic hydroxyapatite (CHT) columns Type I and Type II under overloaded conditions. Phosphate gradients are shown to be preferable over sodium chloride gradients since the latter result in undesirable pH transitions generated within the column itself. Using sodium phosphate gradients separation is obtained with both CHT types, achieving approximately 90% recovery at 99% monomer purity starting with a mixture containing 30% dimer at total protein loads up to 30 mg/mL. Because of its higher binding capacity, even higher loadings can be obtained with CHT Type I without monomer breakthrough. A hybrid model is developed to describe the separation. The model, based on an empirical description of two-component, competitive isotherms at low sodium phosphate concentration coupled with the stoichiometric displacement model at higher sodium phosphate concentrations, is in good agreement with the experiments using the linear driving force (LDF) approximation to describe adsorption/desorption kinetics. The same LDF rate coefficient predicts the separation at loadings between 0.8 and 30 mg/mL. The model developed in this work can be used as a general tool to optimize operating conditions, understand what factors limit performance, and compare different operating modes. [ABSTRACT FROM AUTHOR]

• Frontal analysis separation of antibody monomer/dimer mixtures on CHT columns. • Adsorbent structure and mobile phase effects on transport and adsorption kinetics. • Mechanistic model accounting for surface spreading describes column dynamics. This work examines the separation dynamics of monoclonal antibody monomer/dimer mixtures by frontal analysis using ceramic hydroxyapatite CHT Type I and Type II columns. The binding capacity and selectivity are dependent on the CHT type and salt concentration. While the rate of protein adsorption on CHT Type I is slow and controlled largely by pore diffusion resulting in relatively poor separation, adsorption on CHT Type II is much faster and better separation is obtained than with Type I. However, comparison with predictions based on pore diffusion alone, reveals the presence of additional resistances associated with adsorption and displacement kinetics. A spreading kinetics model assuming multiple binding configurations coupled with pore diffusion was developed to describe these effects and found to be in quantitative agreement with the frontal analysis results and able to predict the separation achieved for conditions outside the range of the experiments. To help validate the assumed mechanism, isocratic elution experiments were also conducted at low protein loads. The chromatograms could be described by the solution of the spreading model coupled with pore diffusion in the linear region of the isotherm with parameters determined from the analytical expressions for the peak moments. This confirms that there is an increasing tendency to spread with slower kinetics as the salt concentration is decreased and binding strength is increased. [ABSTRACT FROM AUTHOR]