May 17, 2023
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Monoclonal antibodies (MAbs) are the fastest-growing modality in clinical trials and more than 100 such therapies have been approved to treat diseases. But despite the modality’s success, MAb manufacturing is often contaminated by host-cell protein (HCP) by-products. Tom Valorose, senior product manager at Astrea Bioseparations, discussed how companies can reduce HCPs dramatically during MAb purification.
Valorose’s Presentation
Removing HCPs is an important part of MAb downstream processing. After cell harvest, scientists perform a primary capture using affinity chromatography with protein A or L resin. Anion-exchange chromatography then removes the majority of contaminating HCPs. However, at large batch sizes and process scales, the differing salt conditions required for protein elution and ion exchange necessitate a potentially disruptive dilution step.
Additionally, removing high– molecular-weight (HMW) aggregates requires hydrophobic-interaction chromatography (HIC), which separates molecules based on their hydrophobicity. As with protein A elution, HIC requires high salt concentrations that can cause target precipitation and yield reduction.
A mixed-mode adsorbent increases separation power and selectivity by using two interactions — with no manipulations between steps. The Astrea Bioseparations HCPure mixed-mode resin is designed to remove HCPs from starting feedstocks. The resin’s binding interactions eliminate the need to dilute protein A eluate and provides higher specificity than HIC polishing by removing HMW aggregates without decreasing yield. The HCPure ligand is cross-linked to the Astrea Bioseparations Purabead product. Purabead 6XL beads can achieve flow rates >600 cm/hr.
Valorose’s team performed two studies using HCPure resin. The first study sought to determine the optimal conditions for polishing an Escherichia coli feedstock in a single step. The team evaluated both HCP reduction and antibody yields in flowthrough material. Feedstocks that expressed light kappa chain–variable chain (Vκ) antibodies were prepared and passed through a protein L affinity resin for primary capture. Then the team applied HCPure resin for the polishing step. To optimize purification parameters, they evaluated the resin’s performance across a range of pH and conductivity values, to determine HCP removal and Vκ yields.
To evaluate the HCPure resin’s scaling capabilities, Valorose’s team loaded antibody samples (pH 6,6 mS/cm) onto small-volume prepacked columns and connected them to an ÄKTA chromatography system (Cytiva). The load and nonbound fractions were analyzed by an HCP enzyme-linked immunosorbent assay (ELISA), results from which showed a 96% reduction in HCPs and Vκ yields >80%.
Samples were spiked with HCPs to overwhelm the adsorbent’s binding capacity and test its clearance capabilities. Samples with HCP concentrations of ~30,000 ppm were passed through a column containing HCPure resin. Analysis revealed that HCP levels dropped to ~200 ppm.
In a second case study, Astrea Bioseparations tested HCPure resin with a more common feedstock and workflow. Chinese hamster ovary (CHO) cells were used to prepare feedstocks expressing two different forms of an immunoglobulin G (IgG) antibody. Protein A affinity resin was used for the primary capture step. The team sought to determine the purification performance of HCPure resin from CHO feedstocks when it was used as a single polishing step. After protein A purification, host-cell DNA (hcDNA) levels were quantified using a fluorochrome-based assay.
After determining baseline values, Valorose’s team loaded protein A eluate onto a column of HCPure resin in flowthrough mode at pH 8 and 3 mS/cm conductivity. After column passage, contaminant concentrations in the flowthrough material were compared with those from an initial sample load, showing 79% HCP reduction and 93% hcDNA reduction. HCPure resin maintained yields of 75%.
To determine the efficacy of HCPure resin for removing aggregates, column material was manipulated to produce aggregates, raising the starting concentration from 0.9% to 6.7%. Passing the high-aggregate load through the HCPure column reduced that concentration to 2.9%.
For bringing HCP levels below the limits of quantitation, HCPure resin can work in a multistep polishing process. When the resin is combined with a cation exchanger, HCPs can be reduced below the limit of quantitation.
Questions and Answers
How does HCPure resin differ from other mixed-mode products? HCPure resin uses HIC and hydrogen bonding, providing the benefits of a HIC–ion-exchange product but with a more robust platform technology.
Are HCPs a concern in other application areas? Yes, HCPs are a concern with every biologic production system, including cell and gene therapies (CGTs). HCPure resin has been shown to remove impurities in CGT workflows with high rates of recovery.
Find the full webinar online at www.bioprocessintl.com/category/webinars.
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