![]() ![]() Materials and Methods Chromatography Media and Columnsīio-Scale™ Mini Cartridges (0.56 x 4 cm) were packed with CHT Ceramic Hydroxyapatite Type I Media, 40 µm (Bio-Rad Laboratories), Capto adhere Media (GE Healthcare Life Sciences), or Capto adhere ImpRes Media (GE Healthcare Life Sciences) to give a final bed volume of 1 ml. Our results underscore the importance of screening multiple media to develop an efficient downstream production process with the highest possible target purity and recovery. We compared the purity and recovery of monomeric mAb S purified with these chromatography media. We separated the mAb S monomers from the aggregates using three mixed-mode chromatography media, CHT, Capto adhere, and Capto adhere ImpRes, which have all been extensively researched for their applications in mAb aggregate clearance ( Gagnon 2009). Typically, a Protein A affinity chromatography eluate contains a mixture of mAb S monomers (~75%) and aggregates (dimers and other oligomers, ~25%). The molecule of interest in this present study, mAb S, was harvested from Chinese hamster ovary (CHO) cell culture. Baseline separation of these mAb species is often difficult to achieve, and extensive process development is required to balance the purity and yield of the monomeric mAbs. The separation of mAb monomers and aggregates has been a major challenge in downstream manufacturing as these molecules exhibit very similar physical and chemical properties and sometimes identical chromatographic behavior. Additional column chromatography steps are necessary for the clearance of such product-related impurities. The strong acidic elution conditions needed for Protein A affinity chromatography, commonly used for the capture of mAbs from clarified tissue culture fluid, can trigger structural changes and promote the oligomerization of pH-sensitive molecules. Elevated levels of antibody aggregates are often associated with the overproduction of mAbs due to the mispairing of disulfide bonds and the unfolding or denaturation of drug molecules at cell growth temperatures (25☌ or above) ( Rathore et al. This has had a significant impact on downstream processing. However, these advancements have often adversely affected impurity composition and concentration upon harvest. Over the years, upstream technology advancements have helped improve the titer of target antibodies, from merely 1 g/L two decades ago to 10–13 g/L in fed-batch processes and up to 25 g/L in perfusion cultures today ( Gronemeyer et al. Monoclonal antibodies (mAbs) remain a predominant class of therapeutic protein products on the market because of their wide range of applications in disease treatment and diagnosis. Comparison of CHT, Capto adhere, and Capto adhere ImpRes in the clearance of mAb S aggregates Pooled elution fractions from the Capto adhere ImpRes Column with 50 mM sodium acetate buffer at different pH.Ĭlearance of Aggregates by the Different Mixed-Mode Media Monomer Content and Recovery from Capto adhere Impres Media under Various pH Conditions Pooled elution fractions from the Capto adhere Column using 50 mM sodium acetate buffer at different pH. Monomer Content and Recovery from Capto adhere Media under Various pH Conditions ![]() Elution of mAb S monomers from a Capto adhere ImpRes Column Monomer Elution from Capto adhere ImpRes Mediaįigure 6. Purification of mAb S on a Capto adhere ImpRes Column Monomer Purification Using Capto adhere ImpRes Mediaįigure 5. Elution of mAb S monomers from a Capto adhere Column Purification of mAb S on a Capto adhere Columnįigure 4. Monomer Purification Using Capto adhere Mediaįigure 3. Elution of mAb S monomers from a CHT Column Purification of mAb S on a CHT Columnįigure 2. ![]()
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