Scaling to New Heights: Robust Scale Up to Commercial Manufacturing

Genetic therapies utilizing adeno-associated viral (AAV) vectors have evolved in recent years from proof of concept to successful treatment options for patients with previously difficult to treat diseases. AAV vectors, known for their ability to safely and efficiently deliver and introduce a correct copy of a gene into patient cells, have become essential for treating many devastating genetic diseases. However, to be successful for multiple and larger patient populations, these groundbreaking therapies must overcome the critical challenge of manufacturing at a scale that meets the growing global need.

While there has been much progress in bringing these therapies forward to the market, they still require immense resources to develop and manufacture a quality drug product at a commercial scale. Forge Biologics, an end-to-end genetic medicines contract development and manufacturing organization, has put a strategic emphasis on scaling up processes so that developers working on larger indications or patient populations can efficiently manufacture at the necessary scales. Scaling to 1,000L or industry-leading 5,000L in a single-use bioreactor provides economies of scale to gene therapy developers, reducing costs of goods (COGS) while still providing high-quality, safe, and effective drug products.

Recognizing the capacity and technical gaps in the current market needed to deliver treatments to patients, Forge has streamlined the AAV production process. By establishing a bespoke platform approach to effectively bridge the gap between small-scale laboratory experiments and commercial-scale manufacturing, Forge’s platform accelerates gene therapy programs to and through the clinic to reach patients faster. Additionally, the same process, cell line, facility, and analytics can be utilized starting at the research-grade production level. This helps reduce any need for bridging studies as manufacturing moves through preclinical, clinical, and commercial development planning.

Overcoming Challenges of Scaling AAV Vector Upstream Manufacturing Processes

Scaling up bioreactors for suspension AAV manufacturing requires a comprehensive understanding of engineering, physics, the biological properties of mammalian cells, and viral stability in culture. As processing volumes expand, bioreactor designs and associated parameters become increasingly intricate, necessitating a thorough assessment of scale-dependent factors. With numerous variables, attempting to scale without specialized expertise can be challenging and costly. Key considerations when scaling bioreactors include Mixing, Aeration and Mass Transfer, System Control Loops, and Logistics.

Mixing

Adequate mixing in the bioreactor ensures that cells, nutrients, and other reagents are uniformly distributed to support consistent growth conditions and optimal production yields. As we scale up, the dynamics of mixing change, potentially creating areas of varied temperature and concentration profiles that can be detrimental to production.

 

This challenge is compounded by the generation of shear stress from the mechanical forces of the impeller(s) and foam production, which can hinder gas exchange and disrupt cell growth. Although reagents can be introduced to mitigate these effects, excessive additives can also adversely affect the process. Achieving optimal mixing in bioreactors is imperative and requires a comprehensive strategy to effectively address these challenges and ensure the success of each batch.

 

Aeration and Mass Transfer

Through the process of aeration, optimal dissolved oxygen (DO) levels are maintained in the bioreactor, which is crucial for cell growth and vector productivity. The two main mechanisms for aeration are through direct sparging of gases into the cell culture medium, or gas overlay across the surface of the medium, often supporting the removal of off-gases. As cells grow and with increased processing scales, the Oxygen Uptake Rate (OUR) increases. To avoid the Oxygen Transfer Rate (OTR) being the metabolic rate-limiting factor, the kLa (Mass Transfer) coefficient, measuring the conversion of oxygen from a gaseous to a liquid state, must be interrogated. Since kLa is dependent on the diffusion coefficent of the liquid medium, bubble size distribution, and dispersion across the bioreactor, the most common approach is altering the sparging strategy and agitation.² With the goal of ensuring each cell gets adequate oxygen, it is important to avoid increasing gas flow and agitation to the points of shear and oxidative stress.

 

System Control Loops

The fine-tuning of process parameter control systems is a meticulous, iterative process that requires expertise in engineering and cell biology. Whether employing a closed feedback loop, cascade, or other control systems, the adjustment of Proportional - Integral - Derivative (PID) controllers – or subsets – becomes necessary as the scale of processing increases. PID controllers serve as crucial instruments for control systems, influencing the bioreactor’s response when a process parameter deviates from the set point or falls outside the established tolerance zone, known as dead band. Improper PID settings can cause the system to react too quickly or aggressively, leading to pronounced oscillations in process values. Conversely, settings can result in sluggish responses and allow values to deviate from the acceptable range, thus increasing process variability.³ The right balance of Proportional, Integral, and Derivative values is necessary to achieve stable and responsive control with minimal oscillations for optimal control performance.

 

Logistics

A critical and often underestimated challenge of scaling is adjusting methods that are straightforward at benchtop scale but become complex as volumes increase. For instance, simple additions to the bioreactor can require innovative engineering solutions, especially for time-sensitive operations. A primary example is inoculation, the addition of cells to batched media. Larger bioreactors require substantial inoculum volumes to initiate the batch. Given that HEK293 cells are sensitive to shear, developing an effective transfer strategy requires careful consideration, such as exploring various pump systems and evaluating flow rate and transfer time to identify proven acceptance ranges (PARs) to maintain cell health.

Equally critical is the process of transient transfection, which relies on the precise complexation of three plasmids: pGOI, pHelper, and pRep/Cap, with a cationic polymer like polyethyleneimine (PEI). While optimizing transfection at a smaller scale is necessary, scaling up introduces a host of variables–from the operating equipment and the timing of complexation to the method of transfection cocktail delivery. Each of these variables demands investigation and engineering controls. The goal is to ensure consistent, efficient transfection, crucial for high-yield AAV processes.

 

Scaling To New Heights at Forge

The experts at Forge have devoted significant time and effort to overcoming scaling challenges to support AAV manufacturing from 250mL to 1,000L. Forge has established a platform process utilizing Ignition Cells that drives consistency informed by the following:

• Incorporation of bioreactor design metrics to predict optimal scaling parameters

• Extensive kLa studies to optimize sparge profiles and agitation rates

• Fine-tuned PID controllers at various bioreactor scales to maintain robust engineering control of key process parameters

• Pioneered innovative processing methods for time-sensitive operations at scale, overcoming the logistical challenges between small-scale and commercial-scale

As our data supports, Forge’s platform process achieves comparable vector productivity across scales, as seen in Figure 1.

 

Figure 1: Consistent Yields from 1L to 500L

Figure 1: Consistent Yield from 1L to 500L Scale. Platform development results where Ignition Cells were cultured in F17 media up to production-scale (1L SF, 5L, 50L, 250L, 500L SUB). Once cells reached the target VCD, the culture underwent triple plasmid transfection using AAV9 Rep/Cap, eGFP Transgene, and pEMBRTM Ad Helper Plasmids. The AAV9.eGFP product was then harvested on day 4 through lysis, nuclease treatment, and depth filtration. Samples were taken and submitted for GOI-specific ddPCR titer analysis. All samples were analyzed for titer and absence of matrix inhibition.

 

Ensuring consistency demands numerous development and characterization experiments of the platform. From the onset, Forge’s process development team emphasized scalability, tailoring the platform to address scale-dependent factors. For example, evaluating power per unit volume (W/m3) can be tested to determine the impact of mixing on cell growth, product yield, and product quality.

During the agitation characterization study, shown in Figure 2, growth profiles of the Ignition Cells were examined in 5L SUBs at preset agitation rates varied based on power per unit volume. Power per unit volume is one of several metrics, such as tip speed and KLa3, that can be used to maintain consistency during scale-up. The characterization can identify optimal agitation rates and/or inform PARs if dynamic agitation is required.

 

Figure 2: Impact of Bioreactor Agitation on Viable Cell Density Over Time

Figure 3: Achieving Consistent Transfection Efficiency Across Scale through Method Optimization. Ignition Cells were cultured in 50L SUBs, 500L SUB, and a 1L shake flask using equivalent expansion strategies. Each bioreactor or shake flask was transfected by either the research-based approach (control) or Forge’s optimized transfection method. The product was harvested at the same duration post-trans- fection. Clarified Lysate samples were pulled and submitted to compare titer (vg/mL) by a GOI-specific ddPCR method. Titer results were normalized to the shake flask control.

Following detailed planning and development, shake-down, and engineering runs are essential to evaluate platform models. These models account for scale-dependent factors and deploy scale-specific control strategies. Maintaining a consistent growth as well as metabolite profile, as depicted in Figure 4, is crucial for reliable manufacturing and is a primary metric for gauging upstream success.