While cell and gene therapies and antibody-drug conjugates (ADCs) are taking up the lion's share of headlines of late, monoclonal antibodies (mAbs) continue to be the largest class of biopharmaceutical. The market share of mAbs continues to grow and is projected to experience double-digit growth from 2023–2030. As these drug products become more complex to address a wider range of diseases, innovative purification strategies and technologies are being developed.
In March 2024, Brian O’Mara, VP Process Sciences, at Scorpius BioManufacturing was one of three mAb experts invited to contribute to a webinar entitled “Complex mAb Purification: Challenges and Solutions,” hosted by GEN and sponsored by Thermo Fisher Scientific. The other guests were Buzz Lobbezoo, senior field application specialist, Thermo Fisher Scientific, and Angela Lewandowski, senior director, Downstream Bioprocess Development, BMS. The panelists answered a series of questions about the challenges of downstream processing of mAbs.
O’Mara is ideal for this topic because of his extensive experience in downstream process development of mAbs, as well as vaccines and recombinant protein therapeutics. He came to Scorpius after spending 20+ years on the sponsor side of drug development at companies like Wyeth (acquired by Pfizer), Genencor, Bristol-Myers Squibb, and Ambrx (acquired by J&J).
O’Mara shared his perspective on everything from techniques to ensure molecule stability to assessing alternatives to protein A capture chromatography and viral clearance.
Many of the more complex antibody formats, such as bispecifics, trispecifics, and Fabs, exhibit varying degrees of complexity and heterogeneity, including truncated species and free heavy and light chains. Controlling these critical quality attributes (CQAs) during upstream production will make downstream processing predictable and, ideally, easier. This requires identifying the CQAs for each molecule, then tailoring a downstream process to those attributes. We have had enough experience with mAbs to learn which impurities and attributes we need to clear and control and implement the appropriate monitoring strategies early in process development.
Lobbezoo pointed out that the current purification techniques and technologies are not always best suited for the purification of mAbs. For this reason, mixed-mode chromatography can be used since it gives an additional layer of separation, or orthogonal approach, to remove impurities that are structurally and physiochemically similar to the target protein.
The glycosylation pattern and charge variant profile of an antibody are ideally controlled during upstream development and processing. However, some variants need to be controlled during the downstream process, which becomes a difficult challenge, especially with ion exchange chromatography. This requires tight control of key process parameters including mass loadings and pooling strategy (i.e., UV collection criteria) based on the feedstock coming from upstream.
It can be a challenge to ensure molecule stability is maintained during downstream processing, including the control of product-related variants. Stabilizing the antibody during purification relies on choosing the ideal buffer matrix during the screening process. This starts with identifying how pH, salts, and buffer species impact stability during room temperature processing. There are a lot of useful analytical tools we apply during process development and characterization to monitor the stability of process intermediates.
One of the most extreme environments a mAb will experience during purification is the low pH hold during viral inactivation. No two mAbs are the same, and monitoring CQAs such as high molecular weight and activity are critical in development.
It is an invaluable test to understand the antibody’s ability to withstand a low-pH hold, even beyond the normal target of 3.5. We evaluate this stability by testing extreme pHs (i.e., pH 3.0-3.2) for, as well as extending the time of the hold beyond the normal 60–90 minutes, to determine whether there will be any variations or excursions during antibody manufacturing.
Lewandowski added that the industry-wide push for higher protein concentrations in mAb drug products (>200 g/L) leads to the risk of forming high molecular weight species during the final TFF step.
Ultimately, identifying the CQAs or presumptive CQAs from the beginning is imperative to ensure the appropriate attributes are monitored throughout development. This allows the process and analytical team to have the appropriate assays in place to evaluate purity/impurity, activity (binding/kinetics), and stability throughout development. Additionally, understanding the key parameters in both upstream and downstream is required to remove or control each of the quality attributes.
Ideally, at least 80% of molecules are captured with a chosen platform process, such as protein A affinity capture. As powerful as protein A is, it can fail. There are three criteria that drive the need to find a non-protein A capture methodology: protein stability, construct design, and performance. For example, some of the bispecifics being developed add a lot of bulk, which impedes binding capacity on a protein A resin by as much as an order of magnitude. This reduction in productivity can have stability and cost implications.
There are specially designed ligands on the market that permit the generation of a custom affinity ligand for a specific mAb. Thermo Fisher’s CaptureSelect Technology provides ligands that bind any part of an antibody. This library of resins from the CaptureSelect portfolio provides flexibility to approach affinity capture from a different angle.
Speed and cost are factors when considering whether to move beyond the standard protein A affinity capture step.. We must also consider how quickly manufacturing volumes of a suitable resin can become available.
When protein A capture fails or is deemed inefficient from a cost or process persepctive, chromatographers need to find an alternative purification process using a non-affinity-based chromatography process. These conventional types of chromatography media include a combination of both cation and anion exchange as the first two chromatography steps.
Other processing benefits can be achieved through various methodologies. For example, operating a chromatography column in Flow-through mode improves throughput and productivity,, reduces time and buffer, increases productivity, and is relatively simple compared to a bine-and-elute step.
Additionally, evaluating the run order and harmonizing buffer species can aid in process simplification. Ideally, the transition between each unit operation should be as seamless as possible. While there are not many intermediate UF/DF steps, making the transition between each unit operation prevents or minimizes any aberrations that you might encounter with pH and conductivity adjustments of the feed stream.
This can be done by harmonizing the buffer system, leading to a reduction of raw materials and solutions used during processing.
As titers have increased over time, there has been a concomitant increase in aggregation and impurities. The mAb’s hydrophobicity can contribute to the formation of high molecular weight species and insoluble aggregates, as well as mAb-host cell protein associations, which piggyback through the downstream process. Disrupting the interactions between the mAb and HCP can be very difficult because they require the introduction of phase modulators and shifts in pH and ionic strength.
One unit operation that is often neglected in bioprocessing is the recovery step during cell culture harvest. Depth filtration needs to be developed to ensure cell lysis is minimized. This minimizes the amount of host cell proteins and other endogenous redox components that could enter your feed stream and cause antibody disulfide reduction. Since this is performed before the affinity capture step, minimizing cell lysis reduces the downstream burden during the intermediate and polishing.
Since this is mammalian manufacturing, the only source of endotoxin would be related to raw materials used in processing. Sucrose can be included as a stabilizer in the diafiltration buffer. Although the levels of endotoxin are characterized to be low, they can be concentrated throughout that Diafiltration and ultrafiltration (UFDF) process.
Preventing this contamination is essential, which is supported by the push to use closed antibody manufacturing processes and sensitive analytical techniques to release buffer. At Scorpius, our commitment to single-use technology throughout our facility maximizes flexibility while minimizing cross-contamination. The safety and reliability of single-use technologies is a game changer.
With the standard platform processes available, we are able to predict the viral clearance effectiveness. Incorporating different modalities, such as hydrophobic interaction chromatography (HIC), introduces an unknown in terms of viral clearance. We have participated in good laboratory practice (GLP) studies to enable an IND in which viral clearance capability with one resin changed when the process changed despite using similar conditions. As powerful as HIC is for HCP and HMW reduction, there is not a robust industry-wide data set about viral clearance capability to date.
Any changes in production -scale to allow further clinical trial evaluations need to be robustly studied to ensure the biomanufactured product is of comparable quality. This requires comparability studies following ICH guidelines to support regulatory filings and updates. Demonstrating comparability during the product lifecycle ensures t any changes in process, scale, or manufacturing site have not changed the drug product profile
Advanced technologies, like continuous processing, are improving biopharmaceutical manufacturing. In terms of process design, mechanistic models are being used to understand how proteins behave on chromatographic resins. Artificial intelligence (AI) is also being used for process design, as well as protein design. AI can create virtual and testable working models of a purification process.
With the growing demand for biomanufacturing capacity for mAbs, it’s imperative to find a biologics CDMO that understands both the scientific and business strategies involved in moving complex antibodies through the clinic. Contact Scorpius to discuss your next complex monoclonal antibody manufacturing project, where experts like Brian O’Mara and his process sciences team will guide you every step of the way.