How To Produce Biopharmaceuticals Through Single-Use Continuous Production And Process Intensification

The high cost of medicines has repeatedly become a major bottleneck in the healthcare industry as large populations cannot afford prescription treatments. At the molecular level, many innovations are underway, including various recombinant monoclonal antibodies (mABs), antibody drug conjugates (ADCs), and gene therapies. In the case of targeted gene therapy, it is crucial and challenging that the targeted drug should be personalized and effective for various individuals. Thanks to innovations and improvements in drugs known as biobetters, the healthcare industry is targeting specific patient populations and providing a continuous supply when needed.

In addition, we had to be able to deal with the dynamic environment of the product pipeline in the healthcare industry to manage capacity uncertainty due to competition among companies, and capacity planning became very difficult when we entered the market.

In order to solve the problem of expensive production infrastructure, it is better to choose a contract manufacturing organization (CMO), which can save the cost of facility construction, because regardless of the production of the product, there will be facility running costs and depreciation costs. At the same time, however, regulatory and other challenges associated with CMOs deserve careful consideration.

Therefore, it is economically advantageous to produce products through process intensification, whereby repeated batches can be run to efficiently mass-produce materials using the same facilities. Even a few percent increase in overall yield through process intensification compared to traditional production design is complementary to overall profitability.

As upstream processes become more refined, downstream processes are adopting a new generation of more cost-effective and efficient chromatography media. To match the continuous upstream, the downstream is also working on continuous processes to reduce intermediate hold times in the process.

Furthermore, the addition of single-use technology to the process revolutionizes the production of therapeutics and offers greater advantages over traditional stainless steel technology as it reduces cleaning, sterilization-in-place (SIP) and maintenance costs of the system , also saving potential time.

Process intensification and single-use technologies are more helpful in reducing the risk of capacity uncertainty when the production facility has a smaller footprint. For example, in order to meet the demand and reduce the capital risk in the initial stage, it is recommended to expand horizontally rather than vertically. In scale-out, smaller facilities will strive to produce larger and larger doses. If the producer is sure of the market demand and supply, then building a traditional large-scale production facility is a good choice. However, it also has the disadvantage of large investment. On the other hand, if the target is uncertain demand, short-term supply, or a specialty product with limited demand, then a single-use technology with a smaller footprint is a better choice.

To succeed in an ever-changing market, single-use technologies provide the flexibility to handle operations in a short period of time with less capital investment.

As the population increases, some medicines become expensive for many people. Therefore, in order to reduce prices, biopharmaceutical companies are taking cost-effective measures.

Automation, process integration and digitalization are essential tools for transitioning from batch process mode to continuous mode, as these are the basis for future facility design, and process intensification is an important part of continuous production, they remove difficulties and provide savings cost and time advantages.

Part 1 Status of Upstream And Downstream Processes

In response to today’s highly competitive biosimilars/biologics market, industries must develop advanced technologies that can be integrated into most biopharmaceutical product pipelines. Now is the time to establish a good manufacturing practice (cGMP) facility capable of handling small-scale production that can handle multiple products. Today, every function in the biotech industry is going through a progressive step, whether it is equipment, upstream process media, or downstream media/media. Thanks to continuous advances in equipment, technology, and process design, large bioreactors can be replaced by smaller and fully disposable technologies. When we compare this to the range of productivity of products from 1982 to 2004, there is an 8- to 10-fold increase in productivity/titer, and many biosimilars and biologics have achieved over 5 g/L. Perfusion bioreactors have shown remarkable results in terms of productivity, more than 25 times higher than batch culture. Furthermore, to cope with such high titers, downstream processes have begun to include emerging tools such as replacing chromatography media with high-throughput membrane chromatography. Now, even for affinity chromatography like Protein A media, a membrane-based format is available, which will have the added advantage of high flow rates, high productivity and purity compared to commonly used Protein A media. In this way, affordable biotherapeutics can be produced using novel and advanced process technologies.

Some operations in the downstream production process are complementary to the upstream, which gives an “illusion” of continuity, but this is not the case. Due to the downstream inclusion of multiple chromatography steps, operation in continuous mode is rare and usually limited to one or two steps. Practical continuous bioprocessing requires process intensification rather than repetitive cyclical movements between batches. Compared with the previous mode, only when the complexity is reduced and the process becomes “easy”, can it be called a continuous process.

Part 2 Adjust The Upstream Process

Upstream, when a fed-batch process is scaled up, there are two ways to increase productivity. One of these is to use larger bioreactor tanks, which will require expanding the plant, and another is to use multiple bioreactors with periodic harvests. However, this will eventually lead to high pressure downstream. To match upstream will require the use of multiple columns and sets of filtration equipment to match the frequency of multiple bioreactor harvests. For example, for commercial-scale mAb production, traditional fed-batch bioreactors typically culture cells in 10,000-25,000 L stainless steel tanks for 7-21 days with product yields of 2-6 g/L.

New interest is growing in the upstream process area to achieve higher titers with shorter incubation times. To achieve this goal, ongoing progress is being made in the development of high-yielding and stable cell lines that deliver high titers that can significantly reduce the facility footprint.

Perfusion bioreactors typically run for longer periods of time and can reach cell densities 10-30 times higher than fed-batch reactors. At regular intervals, a portion of the harvest is continuously removed and provided with fresh medium. In this way unnecessary by-products are removed and new growth-promoting substances are regularly added. In this way, a >4-fold increase in productivity (in mg/l/d) can be achieved using a perfusion bioreactor compared to a fed-batch setup with the same reactor volume. Therefore, within the limited space and capital cost, the same quantity of products can be produced continuously.

Perfusion reactor implementations have been successfully commercialized from large biopharmaceutical companies such as Pfizer, Genentech, Shire, and Genzyme/Sanofi to smaller companies and innovative vaccine producers such as CMC Biologics and Crucell.

While challenges remain with this technology, such as the need for extensive mid- to high-level operator training due to the complexity and intensity of operations, the economic gains from smaller tanks and facilities have a critical impact on process considerations.

Part 3 Adjust Downstream Process

The downstream process is the second complex and expensive step after the upstream process. Continuous operation in purification/downstream is now possible. Major purification steps such as clarification, affinity chromatography, and intermediate and polishing steps can be combined and designed to include single-use techniques for continuous operation.

The use of centrifuges in clarification is difficult to scale up and complex to operate on a larger or commercial scale. As an alternative to centrifugation, single-use continuous processes have been successfully demonstrated for large-scale commercial processes (Humira®) and use flexible and versatile single-use depth filtration systems such as Stax (Pall). Millipore’s two-stage depth filters (Clarisolve, D0HC and X0HC adsorption depth filters) can also be used directly in bioreactors or with the addition of flocculants to precipitate impurities present in cultures or cell harvests.

These depth filters maximize sample loading on subsequent columns by reducing host cell protein (HCP) and DNA impurities and removing cellular debris.

In some processes, diatomaceous earth can be added to the cell culture fluid to prevent clogging of depth filters, allowing for maximum efficiency in clarification of larger batch volumes in a single-use format, as described by Sartoclear Dynamics (Sartorius Stedim Biotech) Show. Various column chromatography techniques such as affinity chromatography (AFC), ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), multimodal chromatography (MMC), etc. are used to purify molecules.

Typically, columns are sized based on volumetric flow rate rather than throughput to increase productivity. This resulted in oversized columns requiring investment in large columns, associated equipment and space. Larger columns can suffer from scale-related packing issues, including hysteresis, edge effects, and packing compression.

As an alternative, the industry is using cost-effective expanded bed adsorption (EBA), simulated moving bed and membrane chromatography. In EBA, all three steps are integrated into one, such as clarification, filtration and capture, which is advantageous in terms of cost, time and space requirements.

In EBA, the crude harvest is applied to the expanded bed in an upflow. The protein of interest binds to the column, while cells and other particulate contaminants pass through the column. Loosely bound material, such as cells and other particulate contamination, is washed away. Subsequently, the captured protein is eluted by reverse flow. The output obtained will be clarified, concentrated and partially purified, allowing for further purification. Although EBA has the advantages of lower capital expenditure, buffer consumption, time and space requirements, its main disadvantages are the need for recirculation and non-specific adsorption, which affects bed stability and overall purification performance.

The second generation EBA Robust Technology is an advanced EBA. According to data presented at the BioProcess conference, Rhobust MabDirect Protein A requires only a third of the process time, provides 12% better yields, and uses only half the buffer volume compared to packed bed Protein A columns , with no compromises in purity and enhanced DNA clearance. EBA is a promising option to increase yield even with increased cell density and productivity of the bioreactor. The use of EBA eliminates the need for large filter areas which are critical to dealing with contamination from coarse feed liquids.

Simulated moving bed (SMB) is another promising purification technique that offers a fully continuous method for performing chromatography. The BioSMB system provides continuous sample loading and continuous elution as multiple Pro A columns cycle through the stages of loading, rinsing and elution at different times

The Accelerated Seamless Antibody Purification (ASAP) process is a continuous and fully single-use mAb DSP based on AKTA Periodic Counter-Current Chromatography (PCC) comprising Protein A, anion-exchange and mixed-mode columns that circulate simultaneously. Another advantage of SMB mode is that the columns can be connected in series, and any breakthrough of the first column is directly loaded onto the second subsequent column, thereby utilizing the full capacity of the main column without loss of any valuable product. This allows the use of shorter bed height columns that can run at faster retention times but may have shallower breakthrough curves, increasing overall productivity. The use of SMB offers several advantages over batch chromatography, including a 30% increase in productivity, a 40% increase in loading capacity, and a 27% reduction in buffer consumption. Executing procedures in SMB continuous mode not only reduces the high risk of contamination due to human error and process interruptions, but also reduces operating costs by reducing the volume of media and buffers.

Another alternative to packed columns is nanofiber adsorption, Puridifye’s FibroSelect platform, which, while having a low binding capacity (10 mg/ml), can be operated at very high process while achieving high productivity.

Monolithic column platforms can also prove convenient for mAb purification, as their high-porosity structure provides efficient mass transfer of antibody-sized targets. In addition, production costs are also reduced due to the ease of material preparation. Monolithic column operations have not translated well from analytical to industrial scale. These platforms are commercially available from various manufacturers including Millipore, Sepragen, BIA Separations. Another alternative to packed chromatography is membrane, now popular for its high throughput and ease of use. Mass transfer is dominated by advection, allowing operation at higher flow rates compared to diffusion-dependent packing-based spheres. Several suppliers, including Pall (Mustang) and Sartorius (Sartobind), offer traditional ion exchange membranes well suited for flow-through (FT) applications. However, bind and elute (B&E) applications are limited due to low ligand density. Natrix’s HD membrane technology consists of a high density of binding ligands in a porous polymer hydrogel that dramatically improves flow characteristics without compromising binding capacity. Based on mAb process simulations, cycling smaller devices with high binding capacity results in significantly lower CapEx because the hardware required for larger columns is expensive and, given the high cost of media (especially Protein A), its Can reduce OpEx. Higher throughput can be achieved with this new chromatography medium without increasing device size, and medium volume can be reduced by utilizing rapid circulation. This will lead to increased productivity and greater flexibility at a much lower capital expenditure.