Recombinant adeno-associated viral vectors (rAAV) are currently the preferred mode of delivery for in-vivo gene therapy treatments [1]Alliance for Regenerative Medicine. Project A-Gene A case study-based approach to integrating QbD principles in Gene Therapy CMC programs. 2021.. Over 200 rAAV-based gene therapy clinical trials are currently ongoing, and five products have been approved and commercialized, including Luxturna (AAV2), Zolgensma (AAV9), Hemgenix (AAV5), Upstaza (AAV2), and Roctavian (AAV5) [2]Shen W , Liu S, Ou L. rAAV immunogenicity, toxicity, and durability in 255 clinical trials: A meta-analysis. Front. Immunol. 2022; 13, 1001263.. However, large-scale manufacturing of rAAV to support late-stage clinical trials and commercial production remains a significant challenge due to low production yields, limited scalability, and high facility and consumable costs [3]Capra E, Gennari A, Loche A, Temps C. Viral-vector therapies at scale: Today’s challenges and future opportunities. Mar 29 2022. . The average cost-of-goods (COGs) for a gene therapy treatment is estimated to be in the range of US $ 0.5-1 million per patient, and the treatments themselves range from USD 0.8–3.0 million per patient [4]Macdonald GJ. Vector Production Processes Play Key Role in High Gene Therapy Prices. Genet. Eng. Biotechnol. News. Nov 22 2022. . Thus, it is evident that COGs are a significant contributor to the current price of rAAV treatments, that is likely to be out of reach for most patients that can benefit from treatment. This challenge must be addressed before rAAV products can move beyond ultra-rare indications and become accessible to larger patient populations, particularly in lesser developed economies.

Shifting from batch to continuous processing is a promising way to significantly lower manufacturing costs. Continuous processing has been extensively explored in the context of monoclonal antibody (mAb) manufacturing and has been estimated to result in 68% and 35% reduction of COGs per gram of mAb produced for clinical and commercial scales, respectively [5]Gupta P, Kateja N, Mishra S, Kaur H, Rathore AS. Economic assessment of continuous processing for manufacturing of biotherapeutics. Biotechnol. Prog., 2021; 37(2), e3108.. In a continuous process, there is a constant flow of material across all manufacturing unit operations running simultaneously [6]Konstantinov KB, Cooney CL. White paper on continuous bioprocessing May 20–21 2014 continuous manufacturing symposium. J. Pharm. Sci. 2015; 104(3), 813–820.. This contrasts with batch-mode manufacturing processes, in which the material is first produced in a bioreactor and then processed sequentially through each batch unit operation, including harvest, clarification, tangential flow filtration, capture chromatography, viral inactivation, polishing chromatography, viral retentive filtration, and final formulation. Figure 1Setup and scheduling for batch processing of rAAV products.  and Figure 2Setup and scheduling for continuous processing of rAAV products. illustrates a typical process for rAAV in terms of setup, instrumentation, and scheduling for batch and continuous modes of operation.

Both batch and continuous processing use the same sequence of unit operations, and the difference in COGs and productivity is driven by reduced downtime, smaller operating scales, higher equipment utilization rates, and higher volumetric productivity. Batch processing requires large-scale equipment sufficient to process an entire batch in one go, unlike continuous processing where smaller scales can be used as the material volume is spread out across all unit operations and across time. Furthermore, batch processing results in significant equipment down-time as all operations are idle during manufacturing except for the currently ongoing step, whereas all units operate non-stop in a continuous process. Options exist for intensification of batch operations such as via ‘six-pack’ manufacturing facilities in which six 12,000 L bioreactors are used to feed a single purification train [7]Sinclair A. Disposable Bioreactors: The Next Generation. BioPharm Int. 2008; 21(4). (Accessed Apr 13 2023). . In this case, equipment utilization rate is midway between the options shown in Figures 3A and 3B below. Batch mode operations can be converted to continuous mode by adapting the unit operations to continuously accept material inflow and outflow, and several technology enablers are available to achieve this, including perfusion or parallel batch bioreactor systems, single-pass filtration modules, multi-column chromatography systems, and twin-tank viral inactivation and formulation units [8]Arnold L, Lee K, Rucker-Pezzini J, Lee JH. Implementation of fully integrated continuous antibody processing: effects on productivity and COGm. Biotechnol. J. 2019; 14(2), 1800061. .

Though this enabling technology has been developed in the context of mAb processes, it is are highly applicable to rAAV production, as the unit operations in both processes are based on the same principles of bioreactor production, packed bed chromatographic purification, depth filtration and membrane operations [9]Adams B, Bak H, Tustian AD. Moving from the bench towards a large scale, industrial platform process for adeno-associated viral vector purification. Biotechnol. Bioeng. 2020; 117(10), 3199–3211.. Moreover, there are several characteristics of rAAV processes, including low titers, short bioreactor production times, and multiple ultrafiltration steps, which are uniquely well-suited to continuous processing. Also, the economic driving force to reduce COGs and increase production volumes while avoiding scalability issues is much stronger in the case of rAAV compared to mAb processes due to the high costs of these therapies and the scale-up challenges which have not yet been resolved for many expression systems. Finally, as the rAAV production landscape is less established, the barrier to entry for new manufacturing approaches is lower than it is for mAbs. There are few existing commercial manufacturing facilities, particularly as it is challenging to convert existing mAb facilities to produce rAAV. This facility fit issue arises due to viral cross-contamination risks as well as the different scales involved, especially in the latter stages of operation where bioreactor production volumes are reduced 100- to 1000-fold for rAAVs compared to 10-fold for mAbs [10]Hebben M. Downstream bioprocessing of AAV vectors: Industrial challenges \& regulatory requirements. Cell Gene Ther. Insights 2018; 4(2), 131–146.. Thus, continuous processing can be realistically considered for future rAAV manufacturing facilities, unlike in the mAbs space where there is less of an economic driving force and more legacy infrastructure available, limiting the need to build new facilities or retrofit existing
ones [11]Rathore AS, Thakur G, Kateja N. Continuous integrated manufacturing for biopharmaceuticals: A new paradigm or an empty promise? Biotechnol. Bioeng. 2023; 120(2), 333–351.  .

However, end-to-end continuous operation has not been implemented at scale for any biotherapeutics till date. Furthermore, only one product manufactured on a fully continuous platform has entered clinical trials, namely a biosimilar antibody produced by Australia-based company BiosanaPharma B. V. which reported successful Phase 1 results for the drug in March 2020 [12]Vuksanaj K. First mAb Produced via Fully Continuous Biomanufacturing. Genet. Eng. Biotechnol. News (Sep 24, 2019). (Accessed Apr 14 2023). . One reason for the lack of adoption of continuous processing till date is the need for high levels of automation, monitoring, and control across all unit operations simultaneously to compensate for any process deviations or drifts in input material to ensure that the critical quality attributes (CQA) of the final product are maintained within the established specifications. This requires specialized equipment, high levels of process digitalization, and rapid analytics [13]The National Institute for Innovation in Manufacturing Biopharmaceuticals. N-mAb: A case study to support development and adoption of integrated continuous bioprocesses for monoclonal antibodies. 2022, 1–256. . However, the principles and key enablers of continuous processing, including perfusion cell culture, continuous multi-column chromatography, and single-pass tangential flow filtration, have been implemented by biopharmaceutical companies to intensify different sub-units of their manufacturing processes. In this article, we explore recent developments in continuous processing for rAAV, as well as provide an overview of some of the major opportunities for intensification of rAAV processes by applying continuous processing tools, many of which were originally developed for mAbs but can be equally well or better suited to rAAV production.

Continuous upstream processing

Currently, the three most common rAAV manufacturing systems are transient transfection (TT) of HEK293 cells, baculovirus expression vector (BEV) systems used in Sf9 insect cells, and stable producer cell lines (PCL) [14]Shupe J, Zhang A, Odenwelder DC, Dobrowsky T. Gene therapy: challenges in cell culture scale-up. Curr. Opin. Biotechnol. 2022; 75, 102721. . TT systems are most common as they can be used to produce a wide range of rAAV constructs using different plasmids, and adherent TT (aTT) systems are commonly used in research laboratories due to their versatility and ease of use. However, aTT is challenging to scale up, driving up the popularity of suspension TT (sTT) systems that allow for volumetric scale-up [15]Dobrowsky T, Gianni D, Pieracci J, Suh J. AAV manufacturing for clinical use: insights on current challenges from the upstream process perspective. Curr. Opin. Biomed. Eng. 2021; 20, 100353. . BEV systems have been reported to be easier to scale-up than sTT systems, and their volumetric productivity in terms of viral particles produced can be greater than for mammalian cell-based systems [16]Wu Y, Mei T, Jiang L, Han Z, Dong R, Yang T, Xu F. Development of Versatile and Flexible Sf9 Packaging Cell Line-Dependent OneBac System for Large-Scale Recombinant Adeno-Associated Virus Production. Hum. Gene Ther. Methods 2019; 30(5), 172–183.. However, due to inherent differences between mammalian and insect cells, there are structural differences in the product, including in the ratios of the three viral proteins and post-translational modifications [17]Kondratov O, Marsic D, Crosson SM et al. Direct Head-to-Head Evaluation of Recombinant Adeno-associated Viral Vectors Manufactured in Human versus Insect Cells. Mol Ther. 2017; 25(12), 2661–2675.. Finally, suspension PCL systems are ideal in their ease of scalability and batch-to-batch consistency, and 2,000 L batches have been recently demonstrated. However, it is a significant challenge to stably incorporate all the required genes in the correct configuration needed to produce viral proteins along with the required gene of interest while also ensuring correct assembly and packaging of the final viral particle, particularly as some of the required genes are cytotoxic, which limits the rapid development of these systems [18]Escandell JM, Pais DA, Carvalho SB, Vincent K, Gomes-Alves P, Alves PM. Leveraging rAAV bioprocess understanding and next generation bioanalytics development. Curr. Opin. Biotechnol. 2022; 74, 271–277. .

Scheduled multi-batch production integrated with continuous downstream operations

To integrate upstream operations into an end-to-end continuous process, there must either be continuous production of material, or multiple parallel batch bioreactors that are scheduled such that the remaining unit operations can be operated continuously with a constant inflow of material [19]Subramanian G, Bisschops M, Schofield M, Grace J. (2017). Two Mutually Enabling Trends: Continuous Bioprocessing and Single-Use Technologies. In Continuous Biomanufacturing - Innovative Technologies and Methods. (Editor: Subramanian G).  . As seen in Figure 3Setup and scheduling for batch (A) and continuous (B) processing of rAAV productsA and 3B, the key requirement is an elimination of down-time between steps with continuous flow of upstream material through the downstream purification train. Complete continuity in upstream processes is not achievable, as cells by their nature require time to grow and expand before they can begin to produce the product. Moreover, unlike mAb or lentiviral production in which the product is secreted from the cells and can be removed from the bioreactor using a media perfusion system, rAAV is produced intracellularly except in limited cases, requiring cell lysis prior to further processing [20]Vandenberghe LH, Xiao R, Lock M, Lin J, Korn M, Wilson JM. Efficient serotype-dependent release of functional vector into the culture medium during adeno-associated virus manufacturing. Hum. Gene Ther. 2010; 21(10), 1251–1257 . Another key challenge with rAAV bioreactors is that the cells only remain viable for 3–5 days post-transfection, and thus cannot be kept alive for extended durations such as 30+ days as in the case of mAb therapeutics. Thus, a scheduled multi-batch approach is well-suited to continuous processing for rAAV, though advances have also been made towards fully continuous operations, as discussed in the next section.

With regards to scheduling, rAAV processes have an advantage over mAb processes as rAAV bioreactors are typically harvested
2–3 days post-transfection, unlike mAb bioreactors which are harvested on day 10–16 [21]Grieger JC, Soltys SM, Samulski RJ. Production of Recombinant Adeno-associated Virus Vectors Using Suspension HEK293 Cells and Continuous Harvest of Vector From the Culture Media for GMP FIX and FLT1 Clinical Vector. Mol. Ther. 2016; 24(2), 287–297. . Thus, there is an opportunity to have faster cycling time with a lesser number of total mid-scale bioreactor systems, while still achieving constant supply of feed material to downstream. For example, mAb manufacturing facilities are able to eliminate the need for scale-up to 20,000 L for commercial manufacturing by having six parallel 2,000 L bioreactors operating with staggered harvest schedules. This can be considered continuous processing if the downstream train is receiving a continuous flow of material into continuous capture chromatography and subsequent purification steps, and these are able to operate without pause between bioreactor harvests [22]Enright M. Amgen’s new $200M next-gen biologic plant a first in U.S. Providence Business News. 2020.. Similar throughputs could likely be achieved for rAAV processes with only two to four parallel bioreactors due to the more frequent harvests.

Recent advancements in TT bioreactor technologies can help to overcome the challenges of large-scale operations including reductions in cell-specific productivities and difficulty in addition of time-sensitive transfection complexes. For aTT systems, a recent advancement is the development of the iCELLis fixed-bed bioreactor system which has been shown to lower plasmid DNA requirements by 20% and increase transfection efficiency by 20% compared to sTT systems at the 1,000 L scale [23]Pall Corporation. Reducing Cost of Gene Therapy Commercialization. Pall Corporation. (Accessed Apr 03 2023).. Moreover, it is suggested to use more than one iCELLis system to increase throughput rather than scaling up beyond 1,000 L, which is ideal for continuous processing. There have also been several advances towards automated and integrated upstream production systems, such as the NevoLineTM system which integrates all upstream and midstream steps, including inoculation, upstream production, clarification, concentration and diafiltration to deliver a concentrated, clarified bulk ready for downstream processing [24]Univercells Technologies. NevoLine^TM platform. (Accessed Apr 03 2023).. The system is adaptable to aTT, sTT, and PCL systems and enables hands-off operation with integrated process steps in a single closed unit.

Overall, operating multiple automated bioreactor systems in parallel can be a key component of continuous rAAV manufacturing facilities by providing a steady stream of well-controlled material to the downstream train. Improvements in titer and productivity of rAAV bioreactors are also key enablers of continuous processing as they allow more material to be produced per unit time, allowing downstream operations to operate continuously without an upstream material bottleneck. Novel transfection reagents have been recently developed which have been shown to increase genomic and capsid titer 10- to 20-fold compared to the traditional polyethyleneimine (PEI) transfection regent [25]Hebben M, Nyamay’antu A. FectoVIR-AAV Transfection Reagent. BioProcess Int. (Oct 29 2021). (Accessed Apr 06 2023).. Novel plasmids have also been engineered with optimized transfection ability in HEK293 cells, along with minimum generation of replication-competent rAAV [26]Yin J. Achieving rAAV Bioreactor Titer Of >1E15 vg/L With Novel Plasmid Transient Transfection Platform. Bioprocess online. (Accessed Apr 06 2023).. Significant research is also ongoing on optimizing bioreactor parameters for higher titers and yield, including using different ratios of the transgene and packaging plasmids, and cultivating the bioreactor to higher cell densities [27]Zhao H, Lee KJ, Daris M et al. Creation of a High-Yield AAV Vector Production Platform in Suspension Cells Using a Design-of-Experiment Approach. Mol. Ther. Methods Clin. Dev. 2020; 18, 312–320..

Finally, it is important to note that PCL systems are particularly well-suited to parallel-batch continuous processing as they demonstrate the highest batch-to-batch consistency and do not require transfection. One such example is the a stable PCL system called ELEVECTA was recently developed in which a human amniocyte cell line (CAP^® cells) are stably integrated with all components necessary for rAAV production, namely adenovirus helper functions E1A, E1B, E2A, E40RF6, VA RNA, as well as AAV replicase, rAAV8 capsid sequences and a gene of interest flanked by the AAV inverted terminal repeats [28]Coronel J, Patil A, Al-Dali A, Braβ T, Faust N, Wissing S. Efficient Production of rAAV in a Perfusion Bioreactor Using an ELEVECTA^® Stable Producer Cell Line. Genet. Eng. Biotechnol. News 2021. . As some of the integrated components are cytotoxic, expression is regulated by a doxycycline inducible promoter. The ELEVECTA system is well suited for continuous facilities as it does not rely on continuous availability of cGMP plasmids, a potential supply bottleneck which can put continuous operations at risk. The system is also scalable up to 2,000 L scale and has high yield in the order of 5e13 viral genomes/L. However, due to the scientific and technical challenges of developing PCL systems, it remains to be seen whether such approaches will become the norm for rAAV manufacturing as they are for mAbs.

Fully continuous upstream systems

Fully continuous upstream production systems require constant outflow of material from the production bioreactor prior to lysis, in contrast to the former approach in which staggered scheduled batch or fed-batch harvests are used to feed a continuous downstream purification train. This has been achieved for mAbs in the form of perfusion cell culture systems in which cells are grown to high densities and supernatant containing the mAb product as well as spent media is continuously removed from the bioreactor while cells are retained [29]Bielser JM, Wolf M, Souquet J, Broly H, Morbidelli M. Perfusion mammalian cell culture for recombinant protein manufacturing-A critical review. Biotechnol. Adv. 2018; 36(4), 1328–1340.. This is accomplished using alternating tangential-flow filtration (ATF) combined with continuous feed of fresh media in a ‘feed and bleed’ approach [30]Clincke MF, Mölleryd C, Samani PK, Lindskog E, Fäldt E, Walsh K, Chotteau V. Very high density of Chinese hamster ovary cells in perfusion by alternating tangential flow or tangential flow filtration in WAVE Bioreactor™-part II: Applications for antibody production and cryopreservation. Biotechnol. Prog. 2013; 29(3), 768–777.. Such systems are only applicable to rAAV processes in which the viral particles are predominantly or exclusively secreted extracellularly, which are relatively rare. Furthermore, as mentioned previously, the key challenge with rAAV bioreactors is that the cells only remain viable for 3–5 days post-transfection, and thus cannot be kept alive for extended durations such as 30+ days as in the case of mAb therapeutics.

A recent example of perfusion in rAAV production is the ELEVECTA system discussed in the preceding section, in which quantification of viral genome concentration from 2–6 days post-induction showed that over 90% of the total viral particles were in the supernatant from day four onwards [28]Coronel J, Patil A, Al-Dali A, Braβ T, Faust N, Wissing S. Efficient Production of rAAV in a Perfusion Bioreactor Using an ELEVECTA^® Stable Producer Cell Line. Genet. Eng. Biotechnol. News 2021. Coronel J, Patil A, Al-Dali A, Braβ T, Faust N, Wissing S. Efficient Production of rAAV in a Perfusion Bioreactor Using an ELEVECTA^® Stable Producer Cell Line. Genet. Eng. Biotechnol. News 2021. . The product concentration was in the order of 5e13 viral genomes/L and was consistent across scales from 10 L to 200 L. This provides an opportunity for a 3-day window of continuous harvest via a perfusion ATF system, like the 10-day window in mAb processing, with material flowing continuously out of the bioreactor and into downstream processing train. The ELEVECTA system was also demonstrated with N-1 perfusion, which is an approach utilizing continuous expansion of cell lines by attaching a cell retention device to the N-1 bioreactor to attain high cell density and viability, allowing the N bioreactor to be seeded at a higher starting cell density and shortening the production time [31]Dobrowsky T, Shupe JW. Seed culture process for aav production WO2020154607A1. (Jul 30 2020). (Accessed: Apr 06 2023). . The ELEVECTA cells were initially grown in batch mode, after which perfusion was initiated to enable growth to high cell density, which was 5-fold higher at the time of induction than in the batch-mode process. Furthermore, overall volumetric production for rAAV was 40-fold higher in perfusion yield, leading to 8-fold higher cell-specific yield with 30–40% full particles [28]Coronel J, Patil A, Al-Dali A, Braβ T, Faust N, Wissing S. Efficient Production of rAAV in a Perfusion Bioreactor Using an ELEVECTA^® Stable Producer Cell Line. Genet. Eng. Biotechnol. News 2021. Coronel J, Patil A, Al-Dali A, Braβ T, Faust N, Wissing S. Efficient Production of rAAV in a Perfusion Bioreactor Using an ELEVECTA^® Stable Producer Cell Line. Genet. Eng. Biotechnol. News 2021. . Another benefit of N-1 perfusion is that a single bioreactor could provide a continuous supply of seed to multiple N bioreactors, facilitating robust parallel-batch continuous processing.

Another example of perfusion was demonstrated for rAAV8 and rAAV9 production in a 2 L WAVE bioreactor, in which a 1 L cell culture was transfected and 80% of the media was harvested through a perfusion filter and replaced with fresh media every
24 h from 48- to 144-h post-transfection [21]Grieger JC, Soltys SM, Samulski RJ. Production of Recombinant Adeno-associated Virus Vectors Using Suspension HEK293 Cells and Continuous Harvest of Vector From the Culture Media for GMP FIX and FLT1 Clinical Vector. Mol. Ther. 2016; 24(2), 287–297. . The viral genome yield recovered from the harvested media across all time-points was 92% of the total process yield, with the yield from the lysed pellet at 144 h comprising only 8%. Overall, the approach resulted in a 6.5-fold and 4.8-fold increase in the yield of rAAV8 and rAAV9, respectively. Though this has only been demonstrated at lab-scale till date, the approach is promising for continuous processing. Such perfusion production systems also have other advantages as there is no need for cell lysis which introduces additional host cell proteins and host cell DNA into the feed stream, burdening downstream processing and requiring additional processing steps including endonuclease treatment and depth filtration, all of which increase COGs. Overall, this approach represents the ideal situation for continuous processing, as the perfusate can be fed directly into downstream unit operations with overall enhanced process yields even without lysis, though it is not yet realistic to expect such high yields of fully packaged rAAV directly in the bioreactor supernatant for all serotypes or even for rAAV8 and rAAV9 processes.

Finally, a recent proof-of-concept explored the potential for using continuous processing principles in the bioreactor transfection step to overcome the variability seen with manual transfections at scale [32]Ladd B, Bowes K, Lundgren M, Gräslund T, Chotteau V. Proof-of-concept of continuous transfection for adeno-associated virus production in microcarrier-based culture. Processes, 2022; 10(3), 515. . It was demonstrated that a 2,000 L suspension bioreactor would require 500 L of PEI:DNA complex, and that there were uneven mixing effects in the transfection tank prior to addition, which led to a wide distribution in the size of transfection complexes and adversely affected transfection efficiency. An automated continuous transfection system was developed in which cells were grown in a stirred-tank suspension bioreactor, after which they were anchored on microcarriers prior to automated addition of transfection complexes. The continuous system used automated mixing of microcarriers with HEK293 cells as well as parallel mixing of transfection reagent and plasmid DNA, prior to automated mixing of all four components after fixed incubation time to control the growth of the transfection complex. This operation was repeated seven times over 20 days, and the continuous transfection approach was shown to effectively achieve a narrow size distribution of transfection complex across the upstream process.

Continuous downstream processing

The downstream continuous processing landscape is rich in tools that have been developed for mAbs, enzymes, and microbial biotherapeutics. For continuous clarification, options used include ATF, continuous depth filtration using multi-filter skids, and continuous centrifugation [33]Gupta A, Amara JP, Gousseinov E, Cacace B. Recent advances in harvest clarification for antibodies and related products. Approaches Purif. Anal. Charact. Antib.-Based Ther. 2020,117–136. . For continuous chromatography, versatile equipment setups have been commercialized with in-built pumps, valves, and sensors enabling integrated multi-column continuous operation, including the BioSMB (Sartorius), Akta PCC (Cytiva), Octave SMB (Tarpon), Contichrom CUBE (Chromacon), and BioSC (Novasep) systems [34]Kateja N, Tiwari A, Thakur G, Rathore AS. Complete or periodic continuity in continuous manufacturing platforms for production of monoclonal antibodies? Biotechnol. J. 2021; 16(7), e2000524.. Single-use, column free systems have also been proposed, such as Continuous Counter-Current Tangential Chromatography (CCTC) [35]Dutta AK, Fedorenko D, Tan J, Costanzo JA, Kahn DS, Zydney AL, Shinkazh O. Continuous countercurrent tangential chromatography for mixed mode post-capture operations in monoclonal antibody purification. J. Chromatogr A. 2017; 1511, 37–44.  . Continuous viral clearance has been shown via continuous viral retentive filtration as well as via continuous pH or detergent-based viral inactivation using twin-tank systems or coiled flow reactors [36]Thakur G, Ghumade P, Rathore AS. Process analytical technology in continuous processing: Model-based real time control of pH between capture chromatography and viral inactivation for monoclonal antibody production. J. Chromatogr. A, 2021; 1658, 462614.. Finally, continuous formulation has been achieved using single-pass tangential flow filtration modules for in-line concentration and in-line diafiltration of the process material [37]Casey C, Gallos T, Alekseev Y, Ayturk E, Pearl S. Protein concentration with single-pass tangential flow filtration (SPTFF). J. Membr. Sci. 2011; 384(1–2), 82–88..

Converting these unit operations from batch to continuous mode for mAbs has been shown to result in smaller operating scales, improved utilization of consumables including resins and membranes, savings in buffer volumes, lower manpower requirements, lower residence time of the biotherapeutic in-process, and the ability to achieve steady-state operation across long continuous campaigns without batch-to-batch variability in critical process parameters (CPP) and critical quality attributes (CQA) [13]The National Institute for Innovation in Manufacturing Biopharmaceuticals. N-mAb: A case study to support development and adoption of integrated continuous bioprocesses for monoclonal antibodies. 2022, 1–256. . These advantages can also apply to rAAV production, as the rAAV process comprises similar unit operations including clarification, initial concentration, affinity chromatography, polishing chromatography, viral retentive filtration, and formulation. Different enabling technology is used at each step in the process to convert the operation from batch to continuous mode, as illustrated in Figure 1 and 2. Of these, continuous ultrafiltration and continuous chromatography are the most critical and are discussed further below.

Continuous single-pass tangential flow ultrafiltration

Single-pass tangential flow filtration (SPTFF) is a key approach used for converting ultrafiltration and diafiltration (UF-DF) steps from batch to continuous mode [37]Casey C, Gallos T, Alekseev Y, Ayturk E, Pearl S. Protein concentration with single-pass tangential flow filtration (SPTFF). J. Membr. Sci. 2011; 384(1–2), 82–88.. In batch mode, UF-DF is carried out by storing the process material in a large tank and recirculating it across an ultrafiltration membrane such that the biotherapeutic molecule is retained on the retentate side and the buffer passes into the permeate stream, facilitating concentration and/or buffer exchange. This operation is difficult to integrate into a continuous setup due to long processing times and the need to concentrate inside a retentate vessel, which does not allow continuous inflow or outflow of material from the unit operation. SPTFF overcomes this problem by essentially replacing the single membrane module with an internally staged series of membranes with overall several-fold higher membrane area, enabling high volumetric concentration factors in a single pass of the process material across the SPTFF module without the need for recirculation [38]Arunkumar A, Singh N, Peck M, Borys MC, Li ZJ. Investigation of single-pass tangential flow filtration (SPTFF) as an inline concentration step for cell culture harvest. J. Membr. Sci. 2017; 524, 20–32.. Diafiltration using SPTFF is also possible with multi-inlet SPTFF modules available in which process material and diafiltration buffer can be fed simultaneously, achieving >99% buffer exchange in a single pass [39]Pall-Biotech. Performance of the Cadence^TM Inline Concentrator Modules for the Concentration of Human IgG. Appl. NoteUSD 3309, 2015..

Downstream rAAV processes stand to benefit significantly from SPTFF technology. Firstly, low titers from upstream typically necessitate 10-fold concentration prior to affinity chromatography to avoid long loading times. Additionally, diafiltration of the harvest material prior to capture has been shown to improve removal of host cell proteins and potentially oncogenic host cell DNA fragments which would otherwise be loaded onto the affinity column along with the target rAAV. Converting this step from batch to continuous mode would allow clarified material to be loaded directly onto an affinity chromatography column after a single pass across an SPTFF module, effectively integrating these two unit operations into a single step and reducing processing time, complexity, and footprint. Similar pre-affinity concentration steps have been demonstrated for mAb processes and have been shown to result in significant productivity enhancements for the affinity capture step due to the more concentrated load [40]Casey C, Rogler K, Gjoka X, Gantier R, Ayturk E. Cadence^TM Single-pass TFF Coupled with Chromatography Steps Enables Continuous Bioprocessing while Reducing Processing Times and Volumes. Appl. Note, 1–7, 2015.. This step is non-essential in the case of mAb processing due to high bioreactor titers with no need for further concentration prior to capture. Thus, adding SPTFF at this point in the process is an optional extra enhancement for mAb process, but more significant for rAAV where the operation is carried out even in the base process.

Another advantage of using SPTFF for concentration of low-titer rAAV process streams is that high volumetric concentration factors of 10–50× can be readily achieved, as showcased in a recent case study for 50× concentration of rAAV9 using SPTFF post-harvest [41]Purification of adeno-associated virus by single-pass tangential flow filtration (SPTFF) (Aug 22 2021). (Accessed Apr 03 2023).. For example, an rAAV titer of 1×10¹⁵ capsids/L is equivalent to only
0.065 g/L of protein, in stark contrast to mAb processes where titers range from 1–10 g/L and final doses can exceed 200 g/L. Operating at low concentrations leads to exceptionally high fluxes in SPTFF processes as there is low viscosity and little to no concentration polarization effects reducing the permeate flux [42]Thakur G, Thori S, Rathore AS. Implementing PAT for single-pass tangential flow ultrafiltration for continuous manufacturing of monoclonal antibodies, J. Membr. Sci. 2020; 613, 118492. . Thus, SPTFF can be readily deployed for both in-process UF-DF as well as in the final formulation step without the need for complex flux control systems with permeate and retentate pumps and/or valves, which are typically required in mAb processing to achieve high concentrations. Furthermore, modular SPTFF kits are available in which the internally staged configuration can be adjusted as needed, with increasing serial stages used for achieving higher concentration factors, while increasing parallel stages facilitate higher volumetric throughput. Thus, customized assemblies can be configured to suit both the initial large-scale volume reduction in rAAV processes post-harvest and the low-volume final formulation step.

Continuous chromatography

Chromatography steps can be considered continuous if there is either continuous loading of input material onto the columns, continuous outflow of eluted material to the next unit operation, or both [43]Coffman J, Lin H, Wang SB et al. Balancing continuous, integrated, and batch processing. 2017.. As conventional chromatography is by nature a periodic unit operation, with a single column cycling through equilibration, loading, wash, elution, and cleaning phases, the most common way to achieve continuity is by increasing the number of columns operating in parallel, with one or more columns in the ‘loading’ phase while the others cycle through ‘non-loading’ steps of the same total duration [44]Holzer M, Osuna-Sanchez H, David L. Multicolumn chromatography. BioProcess Int. 2008; 6(8), 74–84.. Alternative approaches to continuous chromatography include re-engineering the chromatography setup such as by continuously recirculating resin beads across a flow path in continuous counter-current tangential chromatography, or using continuous annular chromatography in which mobile and stationary phases move across each other concurrently by spinning [35]Dutta AK, Fedorenko D, Tan J, Costanzo JA, Kahn DS, Zydney AL, Shinkazh O. Continuous countercurrent tangential chromatography for mixed mode post-capture operations in monoclonal antibody purification. J. Chromatogr A. 2017; 1511, 37–44.  . However, despite advantages in productivity and the true continuous nature of these systems, these are not widely accepted in the industry, unlike multi-column chromatography which has already been implemented at manufacturing scale by several large biopharmaceutical manufacturers for the affinity chromatography step in mAb processing.

Continuous affinity capture chromatography

The main benefit of continuous multi-column affinity chromatography is reduced column sizes and lower resin requirements, along with increased productivity in terms of both g protein/day as well as g protein/mL resin [5]Gupta P, Kateja N, Mishra S, Kaur H, Rathore AS. Economic assessment of continuous processing for manufacturing of biotherapeutics. Biotechnol. Prog., 2021; 37(2), e3108.. The driving factors behind these improvements are twofold. Firstly, there is no need to process the entire batch in one or two chromatography cycles, as the material inflow is spread out continuously across time and across all other unit operations. Thus, columns can be much smaller and are sized according to the overall continuous process flow rate. Secondly, in batch mode, columns are loaded until 2–5% breakthrough to prevent loss of material. However, a well-established technique in continuous processing is to connect two columns in series during the loading step, thus enabling the first column to be loaded up to 70–80% breakthrough as the second column is in place to capture any breakthrough material. Using this technique, binding capacities of mAb affinity capture resins have been shown to increase significantly, for example from 35 g/L at 1% breakthrough to 50–60 g/L at 70% breakthrough for mAbSelect SuRe resin in recent case studies [45]Warikoo V, Godawat R, Brower K et al. Integrated continuous production of recombinant therapeutic proteins. Biotechnol. Bioeng. 2012; 109(12), 3018–3029.  [46]Thakur G, Bansode V, Rathore AS. Continuous manufacturing of monoclonal antibodies: Automated downstream control strategy for dynamic handling of titer variations. J. Chromatogr. A. 2022; 1682, 463496.  .

Similar or better COGs reduction can be expected for continuous capture in rAAV processes, as affinity resins including Capto AVB and Poros CaptureSelect AAV resins are 30-200% more expensive than mAb affinity ligands. Furthermore, in rAAV processes, column volumes are driven down by the concentration step prior to capture, lowering the process flow rate and requiring less volume to achieve the desired residence time for loading. For example, a 200 L upstream bioreactor with titer in the order of
1×10¹⁵ capsids/L harvested daily would result in a flow rate of only 14 mL/min into capture chromatography post-10× concentration, requiring a column volume of 35 mL to achieve the required 2.5-minute loading residence time. The total loading time would be 25 minutes given a resin binding capacity of 1×10¹⁷ capsids/L resin, and a total of two to three columns in parallel would result in a total resin requirement of 70–105 mL and would be sufficient to process the entire
200 L of material over the course of 24 h of continuous operation. In contrast, if this material was to be processed in batch mode in a single cycle, the required resin volume considering the same binding capacity of
1×10¹⁷ capsids/L resin would be 2 L. This is a >90% reduction in resin requirements, even if the smaller columns are periodically replaced to limit the total number of cycles per column. Further COGs reduction can be driven by using three rather than two affinity columns to enable higher binding capacities to be achieved by using two columns in series during loading.

Continuous polishing chromatography

Though continuous chromatography has been mainly implemented for affinity capture, several groups have demonstrated continuous polishing chromatography for mAb processes including anion exchange, cation exchange, mixed-mode, and hydrophobic interaction chromatography steps. Two key approaches are parallel multi-column operation on systems such as the Akta PCC or BioSMB, and multi-column counter-current solvent gradient purification (MCSGP) systems. In the former approach, two or more columns are operated in parallel as discussed in the preceding section, with a single elution step and/or gradient running on the system at any given time with volume-, conductivity-, or UV-based fractionation [34]Kateja N, Tiwari A, Thakur G, Rathore AS. Complete or periodic continuity in continuous manufacturing platforms for production of monoclonal antibodies? Biotechnol. J. 2021; 16(7), e2000524. [47]Feidl F, Vogg S, Wolf M et al. Process-wide control and automation of an integrated continuous manufacturing platform for antibodies. Biotechnol. Bioeng. 2020; 117(5), 1367–1380.  . In the latter approach, elution is integrated with automated side-cut recycling and in-line dilution between two or more identical columns to enrich for the target species and enable removal of the unwanted variants across multiple columns with overall higher yield and purity than possible in a single-column process [48]Krättli M, Müller-Späth T, Morbidelli M. Multifraction separation in countercurrent chromatography (MCSGP). Biotechnol. Bioeng. 2013; 110(9), 2436–2444. . In the context of rAAV purification, both approaches are promising for separations of full and empty capsids.

The parallel multi-column approach is operationally simpler and enables batch operations to be directly converted to continuous mode without any adjustments required in the process method, simply by increasing the number of columns to two or more such that loading of the incoming material from the previous unit operation can be handled continuously. Thus, existing full/empty separation methods using anion exchange chromatography on resins such as Capto Q and POROS HQ, as well as on monoliths such as CIM QA, can be directly transferred to the continuous multi-column system [49]Joshi PRH, Bernier A, Moço PD, Schrag J, Chahal PS, Kamen A. Development of a scalable and robust AEX method for enriched rAAV preparations in genome-containing VCs of serotypes 5, 6, 8, and 9. Mol. Ther. Methods Clin. Dev. 2021; 21, 341–356. . BioSMB systems are particularly advantageous as they have a unique manifold configuration with 240 valves, seven pumps, and 16 column positions, and can carry out both capture and polishing chromatography steps on the same system as has been demonstrated in several case studies for mAbs [50]Thakur G, Nikita S, Tiwari A, Rathore AS. Control of surge tanks for continuous manufacturing of monoclonal antibodies. Biotechnol. Bioeng. 2021; 118(5), 1913–1931. . A typical rAAV chromatography process consisting of affinity capture followed by full/empty separation in step or linear gradient can thus be executed on a single BioSMB system in continuous mode. Alternatively, MCSGP-based polishing strategies can also be explored to potentially achieve higher enrichment of full capsids, though independent chromatography systems would be required for continuous capture and polishing in this case.

Need for improved process analytical technology (PAT) tools

One of the challenges in continuous operation which need to be addressed in the context of rAAV production is the lack of rapid and reliable analytical tools that can be deployed automatically over long continuous campaigns. As in the case of mAb processes, there is a need for rapid analytics executed in real time or near-real time that can track the critical process parameters (CPP) and critical quality attributes (CQA) across all continuous processing steps to monitor for deviations and respond by triggering operator alarms or executing predetermined control strategies [13]The National Institute for Innovation in Manufacturing Biopharmaceuticals. N-mAb: A case study to support development and adoption of integrated continuous bioprocesses for monoclonal antibodies. 2022, 1–256. . Process analytical technology (PAT) monitoring and tools have been developed for mAb processes, including spectroscopic sensors based on Raman/IR spectroscopy [51]Bakeev KA. Process analytical technology: spectroscopic tools and implementation strategies for the chemical and pharmaceutical industries. John Wiley & Sons 2010., rapid at-line HPLC methods [52]Tiwari A, Kateja N, Chanana S, Rathore AS. Use of HPLC as an Enabler of Process Analytical Technology in Process Chromatography. Anal. Chem. 2018; 90(13), 7824–7829. , and statistical process controls [53]Manser B, Bisschops M. Technical Regulatory Topic-Automation and Control in Continuous Bioprocesses. 2021. for tracking CPP and ensuring that they remain within the design space established for steady-state continuous operation in line with the principles of Quality by Design [54]Rathore AS. Roadmap for implementation of quality by design (QbD) for biotechnology products. Trends Biotechnol. 2009; 27(9), 546–553. . Additionally, work has been done on end-to-end process integration using digital control systems combined with level-controlled surge tanks that act as potential breakpoints between unit operation for additional process robustness [50]Thakur G, Nikita S, Tiwari A, Rathore AS. Control of surge tanks for continuous manufacturing of monoclonal antibodies. Biotechnol. Bioeng. 2021; 118(5), 1913–1931.  [55]Chen Y, Yang O, Sampat C, Bhalode P, Ramachandran R, Ierapetritou M. Digital twins in pharmaceutical and biopharmaceutical manufacturing: a literature review. Processes 2020; 8(9), 1088.  [56]Johnson MD, May SA, Mc Clary Groh et al. Understanding residence time, residence time distribution, and impact of surge vessels in Continuous pharmaceutical processing, Springer, 2020, 51–85.. However, implementation of these techniques is limited to lab or pilot scale in the mAbs space, and such development is largely absent for rAAV processes [57]Cashen P, Manser B. Quality by Design (QbD) for Adeno-Associated Virus (AAV) A Framework for a QbD Assessment for AAV Products Within the Chemistry Manufacturing and Controls (CMC) Documentation. Pall Biotech White Pap. 2021, 1–33. .

It is important to note not all methods used for final product release are required for in-process control during manufacturing, and thus limited measurements of key CQA including capsid titer, empty/full capsid ratio, genomic titer, and % of aggregates and fragments are the primary CQA measurements likely required in continuous operation, with other CQA including infectivity, replication competency, and residuals including HCP, HCDNA, plasmid DNA, endonuclease, transfection reagent, and/or affinity ligand required only at the final DS stage [58]Tustian AD, Bak, H. Assessment of quality attributes for adeno-associated viral vectors. Biotechnol. Bioeng. 2021; 118, 4186–4203. . Automated approaches for sampling, measurement and data analysis of ddPCR [59]Brink BG, Meskas J, Brinkman RR. ddPCRclust: an R package and Shiny app for automated analysis of multiplexed ddPCR data. Bioinformatics 2018; 34(15), 2687–2689. and ELISA [60]BPI contributer/ Automated AAV8 Capsid Titer Assay Development - BioProcess InternationalBioProcess International. (Dec 10 2021). (Accessed Apr 03 2023).  assays for quantification of genomic and capsid titer are a critical first step. Additionally, assays for quantification of genomic and capsid titer are a critical first step. Both are a current focus in the industry. Furthermore, mass photometry has emerged as a rapid tool for quantification of full/empty ratio with measurement times in the range of 2–5 minutes, and this has high potential to be deployed in continuous processing if the manual sample prep step can be automated [61]Wu D, Hwang P, Li T, Piszczek G. Rapid characterization of adeno-associated virus (AAV) gene therapy vectors by mass photometry. Gene Ther. 2022; 29(12), 691–697.. Finally, in-line dynamic light scattering, multi-angle light scattering, and fluorescence measurements have been shown to be effective for monitoring rAAV CQA and can be deployed for real-time analytics in a continuous process, particularly when combined with at-line HPLC methods [62]Gagnon P, Goricar B, Mencin N, Zvanut T, Peljhan S, Leskovec M, Strancar A. Multiple-Monitor HPLC Assays for Rapid Process Development, In-Process Monitoring, and Validation of AAV Production and Purification. Pharmaceutics 2021; 13(1), 113.  [63]Gimpel AL, Katsikis G, Sha S et al. Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies. Mol. Ther. Methods Clin. Dev. 2021; 20, 740–754..

Lastly, a key requirement of continuous processing is the need for concentration sensors to monitor the rAAV in the input and output process streams from each unit operation in near-real time [53]Manser B, Bisschops M. Technical Regulatory Topic-Automation and Control in Continuous Bioprocesses. 2021.. These concentration measurements are critical for many downstream unit operations as the loading onto columns, membranes and filters is a CPP affecting performance, resolution, and/or yield. The need for this measurement is exacerbated in case of titer variability in the upstream process, or variability in the in-process streams due to deviations or fluctuations in any of the preceding unit operations [64]Thakur G, Bansode V, Rathore AS. Continuous manufacturing of monoclonal antibodies: Automated downstream control strategy for dynamic handling of titer variations. J. Chromatogr. A. 2022; 1682, 463496. . Additionally, concentration-based feedback control is critical in SPTFF processes, particularly if SPTFF is used for the final formulation step, as it is critical to track and control the concentration of rAAV in the final lot. Several tools have been developed for in-line concentration measurements in mAb processes and have been integrated into feedback control loops for chromatography and SPTFF, including in-line single- and multi-wavelength UV [65]Rüdt M, Brestrich N, Rolinger L, Hubbuch J. Real-time monitoring and control of the load phase of a protein A capture step. Biotechnol. Bioeng. 2017; 114(2), 368–373., and infrared spectroscopy [66]Thakur G, Hebbi V, Rathore AS. An NIR-based PAT approach for real-time control of loading in Protein A chromatography in continuous manufacturing of monoclonal antibodies. Biotechnol. Bioeng. 2020; 117(3), 673–686.. Similar sensors and tools need to be developed for rAAV processes to facilitate monitoring and control of continuous processes. Overall, though there have been promising advances in rAAV analytics that have the potential to be converted into PAT tools, more work is required to establish these technologies in the context of continuous processing [18]Escandell JM, Pais DA, Carvalho SB, Vincent K, Gomes-Alves P, Alves PM. Leveraging rAAV bioprocess understanding and next generation bioanalytics development. Curr. Opin. Biotechnol. 2022; 74, 271–277.  [67]Iglesias Jr CF, Ristovski M, Bolic M, Cuperlovic-Culf M. rAAV Manufacturing: The Challenges of Soft Sensing during Upstream Processing. Bioeng. 2023; 10(2), 229. .

Conclusion

In conclusion, continuous processing offers significant opportunities for process improvement and intensification in rAAV manufacturing. Though most continuous processing developments are in the mAb space, essential process similarities between mAb and rAAV processes enable direct transfer of many of these tools and techniques including parallel-batch bioreactors or perfusion systems, single-pass tangential flow ultrafiltration for in-line concentration, diafiltration, and final formulation, and multi-column chromatography for affinity capture and full/empty separations. Moreover, many of the unique challenges of rAAV processes, including shorter bioreactor production times, low titers, and the need for multiple concentration steps, can be particularly well-handled by continuous processing. Furthermore, continuous processing can reduce the scale-up challenges in upstream bioreactors by enabling processes to be scaled-out across multiple bioreactors at 500–2,000 L scale, instead of scaled-up to 20,000 L where cell growth and transfection kinetics are difficult to control. Continuous processing also reduces the scale imbalance between the initial and final unit operations when the material is concentrated 100- or 1000-fold to reach the target concentration required for reasonable dosage volumes.

The business case for continuous processing is perhaps more compelling for rAAV than for mAb processes for two key reasons: The first is the cost of rAAV production, translating into list prices of millions of dollars for the patient. This not only limits the availability and affordability of rAAV products, but also attracts censure especially in the case of life-saving therapies. Thus, the reduction in COGs afforded by continuous processing is a major advantage. The second reason is the paucity of existing rAAV manufacturing plants and the difficulty of converting mAb facilities to rAAV due to the differences in scale and the risks of viral cross-contamination. Thus, there is a need to implement modular facilities adaptable to both clinical and commercial production of rAAV. This allows biopharmaceutical companies to genuinely consider the pros and cons of integrated continuous operation, without being held back by the need to utilize existing legacy infrastructure or the challenges of redefining well-established batch processing norms, as is common in the mAbs space.

Affiliation

Garima Thakur
Preclinical Manufacturing and Process
Development,
Regeneron Pharmaceuticals Inc.

Sheldon Mink
Preclinical Manufacturing and Process
Development,
Regeneron Pharmaceuticals Inc.

Hanne Bak
Preclinical Manufacturing and Process
Development,
Regeneron Pharmaceuticals Inc.

Andrew D Tustian
Preclinical Manufacturing and Process
Development,
Regeneron Pharmaceuticals Inc.

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Authorship & Conflict of Interest  

Contributions: All named authors take responsibility for the integrity of the work as a whole, and have given their approval for this version to be published.  

Acknowledgements: None.  

Disclosure and potential conflicts of interest: Thakur G, Mink S, Bak H and Tustian AD are employees of Regeneron Pharmaceuticals Inc. and have stocks/stock options within the company.  

Funding declaration: Thakur G, Mink S, Bak H and Tustian AD received financial support for the research, authorship and/or publication of this article from Regeneron Pharmaceuticals Inc.  

Article & copyright information  

Copyright: Published by Cell and Gene Therapy Insights under Creative Commons License Deed CC BY NC ND 4.0 which allows anyone to copy, distribute, and transmit the article provided it is properly attributed in the manner specified below. No commercial use without permission.  

Attribution: Copyright © 2023 Regeneron Pharmaceuticals. Published by Cell and Gene Therapy Insights under Creative Commons License Deed CC BY NC ND 4.0.  

Article source: Invited.  

Revised manuscript received: May 22 2023; Publication date: June 2 2023