Fc-containing proteins, including monoclonal antibodies (mAbs), are widely used in therapeutic applications, and Protein A affinity chromatography is the most common technique for their purification due to its high specificity. During the purification process, the elution of target molecules is typically triggered by lowering the pH to values around 3.0 to 3.5. However, this acidic environment may destabilize sensitive molecules and may not be ideal for removing impurities such as aggregates, host cell proteins (HCP), and DNA. The introduction of a new resin, Praesto™ Jetted A50 HipH, aims to address these challenges by providing alkaline stability and enabling elution at higher pH values.
Praesto™ Jetted A50 HipH Resin
The Praesto™ Jetted A50 HipH resin is designed to handle pH-sensitive mAbs and Fc-containing proteins, which are prone to instability at the lower pH levels typically used in elution with other Protein A resins. It offers a higher stability at alkaline pH, enabling elution at pH 5.0 (compared to the typical pH 3.0-3.5 range used by traditional Protein A resins). The resin has a high capacity of 60 g/L for polyclonal human IgG, making it suitable for large-scale production of therapeutic proteins.
Study Design
The study evaluates the impact of milder elution pH on three types of Fc-containing molecules:
- IgG1 Fc-fusion protein (Fc-Fusion)
- IgG4 monoclonal antibody (mAb)
- IgG1 bispecific antibody (BsAb)
The experiments used a 6-minute residence time and a 45 g/L loading for all resins. An intermediate wash was used to remove impurities before elution. The elution pH for the Praesto™ Jetted A50 HipH was pH 4.5 for the Fc-fusion and mAb, and pH 4.8 for the BsAb, while for the traditional Protein A resins (Competitor A and B), the elution pH was 3.5 for all three molecules.
Key Findings
- Milder Elution pH Benefits: The use of Praesto™ Jetted A50 HipH with milder elution pH (4.5-4.8) was shown to be advantageous in terms of maintaining the stability of the proteins being purified. Elution at a lower pH (such as 3.5) could lead to protein degradation or aggregation, while milder pH helped preserve product quality.
- Purity and Impurity Removal: The milder pH also helped in reducing the levels of residual host cell proteins (HCP) and DNA in the eluted products. This is particularly significant for the overall product quality and therapeutic efficacy, as these impurities can cause adverse immune responses.
- Comparison with Competitor Resins: The study compared Praesto™ Jetted A50 HipH to traditional Protein A resins (Competitor A and B), which eluted at lower pH (3.5). The results indicated that the milder elution conditions with Praesto™ Jetted A50 HipH resulted in better purity and protein stability.
DurA Cycle A50 Resin
The DurA Cycle A50 resin is another innovation discussed in the study, designed to provide even greater stability and flexibility during the purification of challenging proteins, including those requiring higher concentrations of sodium hydroxide or facing protease cleavage. Like Praesto™ Jetted A50 HipH, it supports milder pH elution, further enhancing the ability to reduce impurities and increase product stability.
This article is posted at purolite.com
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