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The development of an innovative purification process simplifies downstream processing for biologics.
The increasing pace and the diversification of modalities in the biologics entering the development stage of the industry pipeline have a direct impact on the process development and manufacturing landscapes. To deal with the acceleration of the early stage good manufacturing practice (GMP) campaigns, Sanofi developed an innovative purification process—the EASY purification process—which is a downstream sequence composed of three steps using only versatile, disposable, and easy-to-operate technologies.
Challenging the status quo regarding the use of the capture Protein A resin, EASY steps run in full continuous flowthrough. First, chelating agents, cast into filters and embedded with the clarification step, are used to trap impurities coming from the cell culture. Then, a washing/exchanging step made of ultrafiltration membranes performs in-line concentration and in-line diafiltration to replace the feed components inherited from the cell culture by a single process buffer. Finally, the feed characteristics are made compatible with the polishing step, composed of cutting-edge membrane adsorbers able to clear the last contaminants. The technologies used, along with the removal of all inter-step adjustments and intermediate storages, reduce the process duration and cost of goods (COGs).
A comprehensive simulation of a downstream processing (DSP) campaign of 1000-L bioreactor-scale batches has been performed with Ypso-Proxima software to estimate the COGs of the EASY process and compare it to a standard monoclonal antibody (mAb) process.
Incrementally, biopharmaceutical companies are experiencing a diversification of their project portfolio. Where standards immunoglobulins G (IgGs) represented most incoming research programs, the new generation of biologics products is now composed of more complex and various modalities such as fusion proteins, multispecific antibodies, nanobodies, and antibody drug conjugates (ADCs). In addition, as the biologics research area thrives, more and more candidates enter the development stage every year (1). These new molecules often show high potency and targeted indications, so the first clinical studies need relatively low amounts of drug substance. The early stage GMP campaigns that produce this drug substance tend to be shorter, with fast turnaround of projects requiring higher plant agility to reduce inter-campaign duration. From this perspective, technologies and materials that become cost-effective only when reused over many manufacturing batches are no longer economically optimal.
Focusing on downstream process operations, the standard mAb process composed of several chromatography columns may soon be challenged. Indeed, columns need significant upfront investments because of costly resins as well as numerous preparation operations, including packing, testing, cleaning, and storage, not to mention the time-consuming effort to validate their lifetime and cleaning procedure to properly demonstrate the absence of protein carry-over. Re-using a same-packed resin for multiple distinct products is extremely challenged from a quality assurance (QA) perspective. Therefore, if the first clinical campaign for a given program only needs a few batches, the resins are used only for a small number of runs, which only represent a fraction of their full potential lifetime. This process represents an economical loss and requires storage space. It comes down to the developers to transfer to processes that use less expensive, more versatile, disposable, and easier-to-operate technologies.
From a complementary aspect, the media used in downstream processes has been roughly the same for decades. The Protein A resin is immutable and remains the core of the mAb process. The Protein A capture step is the most selective step, thus positioned first in the purification train to capture the mAb while removing a maximum of impurities as early as possible. Additional chromatographic steps are positioned after Protein A capture to meet the drug substance quality specifications, clearing the remaining aggregates and host cell proteins as well as a few other impurities. These polishing resins, based on ion-exchange, hydrophobic, or mixed-mode ligands, are less selective—and, therefore, less expensive—than Protein A. To match the decreasing gradient of contaminants down the process stream, process developers choose a decreasing gradient of media selectivity. This rationale makes sense from a process perspective but is economically questionable. Indeed, large volumes of costly Protein A resin are used to cope with the harvest coming out of a bioreactor, and the ligand binding capacity is only improved incrementally by the successive generations of Protein A.
Inverting this gradient of media selectivity could substantially decrease the overall COGs in the downstream process. By placing, first, cheap purification media in large volume to deal with the bulk of easy-to-clear impurities, then putting the most selective technologies late in the stream to remove the remaining small amount of process specific contaminants, the most expensive technologies would be used at smaller scale.
Considering all these drivers of process economics, the Sanofi Purification Process Development team designed the EASY purification process to be a simple purification sequence composed of three main steps without Protein A.
In the first step, chelating agents are cast into filters and embedded with the clarification step. The filters trap impurities through size exclusion and chemical interactions. The second step combines in-line concentration and in-line diafiltration, both working in continuous mode. This improved exchanging step not only washes out the feed components inherited from the cell culture, but also sets an optimal target molecule titer and process buffer for the polishing step. The third step is composed of at least two cutting-edge membrane adsorbers that clear the remaining contaminants.
For a downstream process to be efficient without any capture step, it was critical to optimize the purpose and distribute the clearance contribution of each step. Chelating agents used in this process are cheap, efficient technology, and they are well-known because they are routinely used in the flood, blood, and plasma industries. Chelating agents now provide all the requirements needed to be used in GMP conditions. The ultrafiltration step, usually time-consuming and relegated to the end of mAb processes, gain a pivotal role here, which combines washing and exchanging purposes in a fully continuous mode. Based on the work over the past decade on the use of membrane technologies in DSP (2), the polishing step here leverages a new concept linking two membrane adsorbers—orthogonal in terms of mechanism of action—as a sole integrated step.
Compared to a standard mAb process, this innovative process architecture shows advantages (Figure 1). Fully continuous, it minimizes the overall process duration. It also ensures a closed processing, which secures product stability and microbiological risk, thus removing the need of intermediates storage and frequent 0.2-µm filtration. EASY simplifies the process by promoting a plug-and-play approach (3): all the technologies rely on disposable and ready-to-use filters; the number of process buffers and intermediate adjustments is drastically reduced; and there is no investment in expensive columns. Finally, the three purification steps work in flowthrough mode, so it focuses on contaminants and not specifically on the product molecule. Therefore, this process is less dependent on the modality and could fit a broad range of biologics.
All these changes result in a significant decrease in the process COGs. To illustrate this, a simulation has been performed with Ypso-Proxima software. EASY is matched against a standard mAb DSP to calculate the cost of each process when applied to a campaign of GMP batches. The process steps included in the scope of the simulation are all those that differ between the two process architectures (i.e., all those between bulk harvest clarification and viral filtration).
Several hypotheses have been chosen to perform this simulation (Table I). The clinical manufacturing campaign is composed of a variable number of equivalent batches. The scale of these batches is the same for both processes (i.e., 1000 L bioreactors producing 4 kg of product to purify). It is also assumed that the same direct labor force is needed to pilot and operate both processes. All the indicated values of prices are based on materials and consumables commonly used in Sanofi.
Some relevant information can help better understand the hypotheses related to EASY process steps. Chelating filters are used as single-use filters, continuously loaded up to 800 grams per liter of media and directly discarded at the end of each batch. In-line diafiltration and concentration cassettes are also loaded once, but a clean-in-place (CIP) operation is performed at the end of the batch to reuse the cassettes for up to five consecutive batches. The polishing membranes are two single-use capsules of 800 mL each and of orthogonal ligand chemistries. The high flowrates that can be achieved on these technologies favor a sub-division of the loading into dozens of small cycles separated by a regeneration sequence, which clears the contaminants stacked onto the membranes after each cycle. Overall, all the steps run simultaneously and continuously for eight hours. Flowrates are adapted based on the volume of the devices used.
The three resins used in the standard process all share the advantage of being reusable for up to 200 cycles, which corresponds to a lifetime of more than 30 batches. However, relatively large volumes of resins must be packed in order to limit the total duration of the chromatography steps to 24 hours. A volume of 20 liters per resin was chosen, which is typical for manufacturing batches performed at this scale. In addition, the amount allocated to common process consumables, such as sterilizing filters, single-use storage bags, and transfer lines, is significantly reduced in EASY.
All these data and hypotheses were filled in Ypso-Proxima software, and a cost-per-kg of product was calculated for a varying number of batches in the campaign and for each process.
For a single-batch campaign, COGs for EASY are estimated to be $24,000 (EUR 19,800) per kg of processed product, almost entirely imputed to the cost of the consumables (Figure 2). This cost represents an 86% cut when matched against the standard process. This significant gap decreases with an increasing number of batches (Figure 3) because the investment made on the new set of resins eventually pays off with the resin reusability. EASY cost is almost constant when iterating the number of batches and remains cost-effective for a 10-batch campaign, which is already relatively extended for an early clinical project.
EASY is a full flowthrough, continuous process based on disposable elements, working without resins and Protein A. The process simplifies architecture and contaminant-focused steps to allow for a significant range of applications for biologics. Based on the promising results of the simulation work performed with Ypso-Proxima software, the application of this process for large-scale clinical batches can considerably reduce the COGs related to the DSP part of the production. The purification is performed in a few hours and with small volumes of media, increasing the productivity. Moreover, the preparation and validation tasks are significantly simplified, allowing for fast turnaround between projects.
The authors would like to thank the Ypso-Facto company, and especially Lucrèce Nicoud and Agnès Corbin, for the early access to their new software, Ypso-Proxima, and their work on the simulations.
1. BioPlan Associates, Top 15 Trends in Biopharmaceutical Manufacturing, 2019 (April 2019).
2. B. Mothes, “EASY Process: A Full Flowthrough & Disposable mAb Purification Process,” presentation at the 2019 BioProcess International Conference (Boston, MA, Sept. 9–12, 2019).
3. F. Mirasol, “Single-Use for Downstream Chromatography: Benefit or Hindrance?” BioPharm International 32 (5) 34–37 (2019).
Thomas Prouzeau is a purification process development scientist, Jerome Pezzini*, Jerome.email@example.com, is purification process and technology leader, and Benoit Mothes is purification process development group head; all are at Sanofi.
*To whom all correspondence should be addressed.
Vol. 34, No. 1
When referring to this article, please cite is as T. Prouzeau, et al., “EASY: a Disruptive mAb Purification Process to Reduce Cost of Goods,” BioPharm International 34 (1) 2021.