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    Features

    Single-Use Technologies Enable Biologic Scaling

    Discussing the use of single-use technologies to improve the biologics manufacturing process.

    Single-Use Technologies Enable Biologic Scaling
    Dr. Kai Lipinski , Chief Scientific Officer, Vibalogics, a Recipharm company05.03.23
    As technologies advance, treatments are becoming increasingly innovative. Breakthroughs in the biopharmaceutical space have led to an upsurge in the number of biologics available—and demand from patients and healthcare systems has followed. Biologics are complex pharmaceutical products that may present challenges when production is scaled up to meet increased demand. One way to overcome these obstacles is to integrate recent improvements in single-use technologies (SUTs) into increasingly complex production processes to provide manufacturers with additional flexibility and capacity. This article explores the rise of biologics production in the biotech space and discusses how the pharmaceutical industry can use SUTs to improve the manufacturing process and bring advanced treatments to patients.

    A rise in the prevalence of rare, chronic, or age-related diseases has led the biopharma industry to seek novel treatment strategies in the form of biologics. Increasing demand for biologics is directly affecting the market, with significant implications for both the industry and patients. Valued at approximately $366.4 billion in 2021, the biologics market is expected to reach $719.8 billion by 2030 at a compound annual growth rate of 7.8%.1

    The increasing demand for biologics is also being driven by technological advances. The relatively new ability of biopharmaceutical developers to produce these complex medicines has enabled the probing of diverse therapeutic mechanisms to treat diseases—resulting in the development of a range of novel therapeutics:

    •  Gene therapy viral vectors (mainly lentivirus and adeno-associated virus)
    •  Cell therapies (e.g., chimeric antigen receptor T-cell therapy and modified stem cells)
    •  Oncolytic viruses (e.g., adenoviruses, herpes viruses, and vaccinia virus)
    •  Messenger RNA (mRNA) vaccines
    •  Prophylactic vaccines
    •  Proteins and monoclonal antibodies (mAbs)

    With healthcare systems now benefiting from approvals in many of these classes of therapeutics, developers are left trying to meet the needs of a growing and aging patient population. Meeting these needs requires bioreactor vendors and contract development and manufacturing organizations (CDMOs) to facilitate scaling up the production of biologics while considering safety, infrastructure, costs, manual operator handling, engineering, and biological processing itself.

    The advantages of SUTs

    Fortunately, advances in SUTs are allowing the scale-up of high-quality biologics for clinical development and commercial supply. SUTs are being integrated into upstream and downstream biological manufacturing operations to enhance the production power of biologics.
    Their increased adoption is primarily driven by the following advantages they offer:

    •  Shortening campaign turnover times
    •  Decreasing operating costs
    •  Minimizing cross-contamination risk
    •  Enabling simple installation compared with fixed technologies
    •  Offering a design that is beneficial for closed processing operations
    •  Avoiding costly, laborious, and time-consuming cleaning-in-place and sterilization-in-place validation
    •  Increasing manufacturing and operational flexibility with modular design
    •  Minimizing energy and water usage
    •  Reducing early capital investment

    SUTs are primarily used in biological production to enhance the processes of bioreactors, media and buffer preparations, in-process mixing, and liquid storage, among others. In 2018, more than 66% of pharmaceutical companies preferred SUTs over permanent technologies.2

    SUT suitability for advanced therapy medicinal products

    The preference for SUTs is likely to increase with personalized medicine, orphan drugs, and cell and gene therapy modalities gaining momentum, partly due to the SUTs’ role in reducing the risk of cross-contamination during the manufacture of these treatments.
    Biological treatments that concern cell and gene therapy and tissue-engineered product therapeutics can primarily be classed as ATMPs. These medicines have an active therapeutic substance based on at least one of the following specifications:3

    •  Tissue-engineered products (apart from skin transplants for burn treatment)
    •  Patient genome modifying technology
    •  Recombinant nucleic acids or genes
    •  Cells that are substantially manipulated
    •  Cells intended to serve a different essential function in the recipient compared to the donor

    As biologically produced products, ATMPs and other biologics require unique manufacturing and processing requirements to ensure quality when scaling production. The flexibility offered by SUTs when scaling will help facilitate the commercialization of ATMPs as personalized medicines become more common. The increase in ATMPs is shown in the number of clinical trials for these therapeutics, with more than 2,000 ATMP clinical trials ongoing globally in mid-2022.4

    Assessing process suitability to scaling

    Previously, single-use (SU) bioreactor volumes were limited by pressure challenges from the weight of the liquid medium as well as handling issues. Limitations in SUTs were the rationale behind the preferential selection of stainless-steel bioreactors. The capacity of stainless-steel bioreactors used to be significantly larger than the SU alternatives available, with volumes commonly reaching 10,000–20,000 L against the 2,000-2,500 L scale for SUTs.

    Advances over the past five years have allowed SU bioreactors to become commercially available at higher working volumes of 5,000 L (ThermoFisher, HyPerforma DynaDrive)5 and even 6,000 L (ABEC Inc.). However, some situations require scaling up to volumes above the limits of newer SU bioreactors. This scenario necessitates the consideration of scaling out (increasing the number of bioreactors used in parallel) or process intensification (typically, a high cell density perfusion process). Not all cell lines and processes are suitable or adaptable to perfusion processes, which can lead to additional challenges.

    A key regulatory requirement and safeguard of both product quality and patient safety is to know and qualify the processes involved in biologics manufacturing at various scales. Qualifying processes at the commercial scale are particularly important due to the large number of patients that products will reach.

    Developing a suitable process development, characterization, scale-up, and validation are important exercises. When assessing the scalability of a manufacturing process, many important factors must be considered—particularly for upstream processes. These factors include:

    •  Features and properties of the growing organism (e.g., mammalian cell lines such as HEK or CHO cells cultures)
    • Inherent scalability of the engineered system (e.g., critical engineering parameters and bioreactor design)
    • Potential to adapt manufacturing and processing steps
    •  Resulting product (e.g., live virus titer or monoclonal antibody (mAb))

    A comprehensive list of additional aspects to be considered and the potential factors they will impact is shown in Figure 1. These encompass input considerations such as choosing between an SU or stainless-steel system and output considerations including defining critical scaling parameters. Resourcing time and effort in considering these factors is essential to avoid a project quickly becoming very costly due to delays in the time to market.


    Figure 1. The input considerations and potential outputs for determining process scalability.

    Utilizing scale-down models

    Although scaling up is important for development, the capacity for effective scale-down models is critical for reducing risks and costs while shortening timelines. Employing the design of experiment (DoE) and multi-variant data analysis approaches can achieve higher-throughput process modeling. Examples include the Sartorius Ambr 250 and Ambr 15 systems, which enable upstream scale-down modeling of up to 24 or 48 parallelized and miniaturized bioreactors, respectively.
    Scale-down DoE work enables the definition of certain critical parameters:

    •  Operation, process, and material parameters
    •  Controlling parameters and set points
    •  Operating and acceptable range parameters and their influence on a product’s critical quality attributes

    Additional supportive software is available to simulate risk based on the scale down or scale up of projects (Sartorius, BioPat Process Insights). Such software can significantly reduce workloads to shorten timelines for successful scale-up operations and reduce the risk of expensive late-stage failures.

    Scalability of upstream processes

    Scalability challenges predominantly concern upstream bioreactor scale-up, as this is often more complex than the mostly linear scale-up of downstream processing (DSP) steps. Further complexity arises from challenges associated with the chosen host cell line, as its characteristics and performance indicators will need to be accounted for when scaling up and down. Whether the cell line grows as adherent cultures or in suspension will drastically impact the processes and technologies required during scaling. Many SUT bioreactors are designed to accommodate scaling operations to ease potential upstream development difficulties involved with either adherent or suspension cell lines.

    The upstream processes for scaling suspension cell lines
    All commonly used suspension cell lines (e.g., CHO-S, HEK293/T, HeLa S3) can be linearly scaled up using non-baffled shake flasks at scales between 125 mL and 5 L. The working volume between scales is normally kept constant, with typical values of 10-30%. When suspension cultures are ready to move forward to the bioreactor scale, several important parameters must be considered—a number of these parameters are detailed in Table 1.

    Streamlining the scale-up and scale-down processes requires careful selection of a bioreactor system designed to allow a transfer of process between all available scales.

    Consistent geometrical designs allow key output parameters to stay consistent when using increasingly large SUT bioreactors to scale-up production. The geometry of bioreactors can particularly affect output parameters like specific power input, kLa, and mixing time. Vendors will often develop their SUT bioreactor scales to be cylindrical (although there are exceptions – PALL has a cubical shape) with constant ratios for bag height and diameter, and d/D (impeller diameter/bag diameter).

    The upstream processes for scaling adherent cell lines
    Traditionally, the scaled growth of adherent cell cultures was more difficult than suspension cell cultures, as the process would rely on both scale-out and the development of scalable microcarrier-based processes. Scale-out involves increasing the number of cultivation units (e.g., T-flasks, roller bottles, CellSTACKs (Corning)), which is operator-intensive and carries an inherently high risk of contamination compared with scale-up processes. Operational challenges also often arise as certain processing parameters must be carefully considered and controlled – these parameters are mainly cell-related. Scaling up microcarrier-based processes is also not an easy task and is not suitable for all cell lines and product combinations (e.g., it is difficult to produce some viruses; cell aggregation can also be an issue).

    These difficulties have been overcome by advances in SUT for bioreactors. Closed-operation and linearly scalable fixed-bed bioreactor SU systems are now on the market, with vendors including PALL (iCELLis),7 Univercells Technologies (scale-X),8 and Corning (ASCENT).9 The design of these bioreactors considers factors such as shear stress, power input, oxygen supply capacities, and medium volumes per growth surface area to enable a seamless scale-up.

    In addition to scale-up capabilities, all three bioreactors mentioned offer scale-down development and modeling systems for proof-of-concept, establishment, and characterization purposes. The largest GMP production system has adherent growth capacity scales of 500 square meters (PALL), 600 square meters (Univercells Technologies), and 1000 square meters (Corning). Another fixed-bed bioreactor SU system from India-based company OmniBRx Technologies offers a maximum scale of 1,500 square meters.10

    Scalability of downstream processing steps

    DSP steps are typically well suited to linear scale-up and scale-down, with many processes reliant on SU components, including filters and chromatography resins. Manufacturers must be aware of the challenges during the scaling of DSP steps when using SUTs, including identifying vendors offering technologies across a range of scales (particularly for scale-down models). Consideration must be given when determining if some SUTs are better suited to scale-out as opposed to scale-up, particularly for chromatography, normal flow filtration (NFF), and cross or tangential-flow filtration (CFF/TFF).

    Chromatography
    In most biological production processes, a chromatography step is commonly used to enrich the product and remove process-related residuals, either through a bind/wash/elute or flow-through modus. The most utilized modalities are affinity, ion exchange, size exclusion, hydrophobic interaction, and mixed-mode resins.

    During scaling, flow velocity (cm/h) or product residence time are the critical linear scaling parameters. To maintain constant flow velocity between scales, column volume should be increased linearly with the volume to be loaded, while keeping the column height constant and increasing the column diameter. Buffer volumes should also be scaled linearly while flux rates are maintained constant (column volumes/hour).

    However, linear scale-up of column volumes can be challenging, as columns, membrane adsorbers, or monolith units are only available in discrete sizes. Options to mitigate this problem include running multiple product cycles or parallelization.

    An alternative approach to scaling up is to use constant column volumes/hour, which may become the industry standard for certain modalities such as affinity and ion exchange. This approach helps avoid running processes beyond column capacity.

    Normal flow filtration 
    NFF is typically employed in a DSP operation either at the point of harvest for clarification or at the end of the process for bioburden reduction and terminal sterile filtration purposes. These filters come in either a single pore size cut-off membrane or a range of cut-offs with a large-to-small funneling function to improve filter capacity.

    NFF can be operated at a constant pressure or constant flux (liters/m2/hr (LMH)), and potential shear stress must always be considered to avoid damaging the final product. When scaling, it is important to keep the vendor, membrane chemistry, and pore sizes constant to ensure final product consistency and avoid compatibility issues. Additionally, membrane area and conditioning volumes should be scaled linearly to process feed volume; contaminants are regarded as comparable between different process runs and production scales.

    In addition, stackable (i.e., parallelizable) filter elements should be considered as they offer more flexibility in usable membrane area and can cover larger scales than self-contained filter capsules.

    Particular care and widened filter screening should be performed for sterile filtration operations, if the product (mostly larger viruses) is close to the sterile filter’s 0.2 µm cut-off size.

    Cross or tangential-flow filtration
    CFF/TFF involves feeding the material in a horizontal flow over the membrane, which reduces the likelihood of filter membrane fouling. The key optimization parameter for CFF/TFF is the flux (LMH) as a function of transmembrane pressure (TMP) (bar) and commonly used formats are either hollow-fiber modules or membrane cassettes.

    The molecular weight cut-off (MWCO) is an essential parameter that considers the molecular weight of contaminants to be removed, and the size of the product particle itself. MWCO must be maintained as a constant across all scales.

    CFF/TFF can be controlled using constant trans-membrane pressure or permeate flow flux. While the filter load (volume per area) is kept constant during scale-up, the crossflow rate is often scaled up by maintaining a constant shear rate. This approach is particularly useful for shear-sensitive products (e.g., enveloped viruses).

    Scale-out versus scale-up
    Scaling up can require alterations to an existing facility and can present significant challenges. Adjustments such as changing a facility’s ceiling height, autoclave capacities, floor weight loads, gas supply capacities, liquid handling size units, and seed train changes can be associated with significant delays and additional costs. Consequently, scale-out is sometimes seen as a more viable alternative to scale-up, providing manufacturing flexibility and better adaptation to market fluctuations. SUTs are particularly well-suited for this strategy.

    Process validation is also more straightforward when scaling out; when a bracket validation design is used to test extreme case scenarios, no additional validation is necessarily needed. Scale-out manufacture also means that in the event of failure (dependent on the root cause), only one unit is lost rather than the entire batch. With more options now available for successful process intensification (e.g., using ATF or TFDF systems from Repligen), scale-out has become an increasingly attractive option to biologics manufacturers.

    Key lessons

    There are several important considerations to be made when scaling processes for biologics production. With advancements in SUTs and their availability in varying sizes, the adoption of these technologies is likely to increase—especially with the safer development of personalized medicines. However, manufacturers will still face challenges when scaling with SUTs. To tackle these challenges, manufacturers will need to understand and consider all process parameters that may be affected at different scales of production and decide whether scaling out or scaling up offers the biggest advantages.

    References
    1. https://www.precedenceresearch.com/biologics-market
    2. Boyd Biomedical, “The Rise of Single-use Bioreactors: Why Make the Switch?”, www.boydtech.com, June 21, 2018.
    3. https://atmpsweden.se/about-atmps/what-are-atmps/#:~:text=ATMPs%20are%20'Biologics'%2C%20medicines,recombinant%20nucleic%20acid%20or%20cells
    4. Alliance for Regenerative Medicine State of the Industry Briefing September 2022 (http://alliancerm.org/wp-content/uploads/2022/10/ARM-H1-2022-R13.pdf).
    5. D. Stanton, “Update: Thermo Fisher Scaling Single-use Bioreactors Up to 5,000 L,” www.bioprocessintl.com, September 25, 2019.
    6. BioPhorum Operations Group Ltd., “Justification of Small-Scale Models: An Industry Perspective,” www.biophorum.com, May 5, 2021.
    7. PALL Corporation, “Cell Culture,” www.pall.com, accessed March 2022 (www.pall.com/en/biotech/technologies/cell-culture.html).
    8. Corning, “Corning® Ascent® FBR System,” https://www.corning.com, accessed May 2022 (https://www.corning.com/worldwide/en/products/life-sciences/products/bioprocess/ascent-fbr-system.html).
    9. Corning, “Corning® Ascent®: Fixed Bed reactor System: Scaling from Process Development to Production with an Attachment-dependent Cell Growth Platform,” accessed March 2022.
    10. www.omnibrx.com



    Kai Lipinski joined Vibalogics in 2010 as head of cell culture and virus production. Kai was named head of process development in 2013, then promoted to chief scientific officer (CSO) in 2020. Kai has a wealth of experience in viral vector manufacturing stemming from a career working within industry. Prior to Vibalogics, he was principal scientist at Cobra Biologics, focusing on upstream process development for virus and mammalian protein expression projects. Prior to Cobra, Kai worked as senior principal scientist at ML Laboratories, where he was responsible for the development of targeted adenoviral vectors for gene therapy approaches to cancer. At Vibalogics, Kai is central to the establishment of process development and manufacturing capabilities, technology evaluations, and the oversight of client technical relationships.
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