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Overcoming time and cost constraints can help enable seed train intensification efforts to maximize product yield.
The use of seed trains to increase product yield is common to cell culture processes; however, the process can be costly and time-consuming. Seed train intensification technologies have been useful, however, in achieving the desired cell density for inoculation into a bioreactor while shortening the seed train duration, which can optimize biological product output.
“The seed train is the scaling of the culture from a small volume of cells in a cell bank vial to a larger volume of cells that are used to inoculate the N, or main production reactor at the required cell density,” explains Christine Gebski, senior vice-president, Filtration and Chromatography, Repligen. The seed train process aims to ensure cell viability and the attainment of the desired or required final cell density.
“The purpose of the seed train is to reach a sufficient cell mass to inoculate the production bioreactor in order to start the protein production in an optimal way,” agrees Darren Verlenden, head of BioProcessing at MilliporeSigma. Verlenden notes that, from the working cell bank vial to the production bioreactor, the cell number has to increase approximately 6–8 logs. “This [increase] is typically achieved by running cells through many cultivation systems (e.g., spin tubes, shake flasks, rocking motion bioreactors, stirred tank bioreactors), which become larger with each passage.”
While each process is different, typical seed trains are time consuming and labor intensive and have relatively low value-add, Verlenden says. In addition, the risk of failure remains significant because of the open nature of the early stages of cell expansion when working cell bank vials are thawed and cells are manually transferred into spin tubes and shake flasks under a laminar-flow hood.
Incorporating seed train intensification can benefit the overall manufacturing strategy. As Verlenden explains it, seed train intensification aims to lower manufacturing costs, achieve higher process throughput, and increase flexibility.
Gebski adds that increasing the cell density of the cell bank or the N-1 reactor enables a shorter timeframe to attain the higher cell densities needed in the “N” or main production bioreactor. “Reducing process time drives meaningful efficiency gains, with a larger number of processes being completed in a given amount of time. Attainment of a higher cell density directly correlates to an increase in productivity—the amount of biologic that can be produced in a set amount of time.”
Two main options are currently being assessed or are already implemented in the biopharmaceutical industry to intensify seed trains, says Verlenden. These options are based on perfusion: N-1 perfusion and high cell density cryopreservation (HCDC). Verlenden explains that perfusion operation is based on the introduction of fresh cell-culture media at a constant rate in the bioreactor, while the spent media is removed and cells are returned to the bioreactor vessel using a cell retention device. “This way the nutrients and waste-product content remain stable over time, which allows greater cell mass levels in the bioreactor,” he says.
Improved and simplified process controls and cell retention technologies have allowed greater adoption of perfusion, he notes. Verlenden also notes that drug manufacturers are assessing the implementation of perfusion in the N-1 bioreactor to reach high cell densities. “N-1 perfusion can be used in two different ways. The first and the most impactful is to use N-1 perfusion in order to seed the production bioreactor with higher initial cell density, known as high seed fed-batch. The other way to use N-1 perfusion is to take advantage of the higher cell density to use a smaller bioreactor and seed the production bioreactor,” he says. In both cases, the advantage of using N-1 perfusion is to keep the production bioreactor operation in fed-batch mode, which inherently eases the process validation, especially post-approval changes, Verlenden says.
Meanwhile, to reduce the duration of the cell expansion period, drug manufacturers have developed higher-density cell banking approaches in larger vials (5 mL), resulting in some improvements but without alleviating the contamination risk due to manual transfers of cells, Verlenden states. “This concept has evolved with the use of single-use 2D [two-dimensional] bags of 100 mL–250 mL volumes with high cell densities (100–150 x106 cells/mL) generated by perfusion processes in HCDC settings. From a 250-mL seeding single-use bag at 100 x106 cells/mL, you can directly spike a 20-L rocking motion bioreactor or a 50-L stirred-tank bioreactor to start the cell expansion—reducing the number of steps and accelerating speed,” he says.
Verlenden also points out another benefit of using HCDC bags, which is to make the whole seed-train functionally closed. “After thawing the bag, you can weld it to the bioreactor and transfer cells aseptically, allowing drug manufacturers to start the seed train in a less controlled area to save time on environmental testing,” he says.
Using perfusion in the seed train, whether in a N-1 perfusion scenario or HCDC for cell banking, is a first step into upstream intensification—from a process control and cell retention technology standpoint. Perfusion also provides opportunities for cost-of-goods improvements and inherently increases product safety, Verlenden adds.
With the use of seed train intensification in the manufacturing process, bioreactor design has been adapted to keep pace and innovative technologies have helped with that adaptation.
There are obvious challenges to enable high cell densities from a bioreactor perspective, says Verlenden. Sparging and mixing capabilities, for instance, are essential to achieving the right balance between maximizing the oxygen transfer rate and minimizing the shear stress imposed on the cells in suspension.
“Today, kLa [volumetric mass transfer coefficient for oxygen] up to 50/h and power input per volume up to 100 W/m3 at 2000-L scale are becoming the norm in single-use bioreactors. Bioreactor vessels geometry improvements, design optimization of impellers, and the diversification of drilled hole spargers have been instrumental in achieving these specifications,” Verlenden says.
Meanwhile, he continues, from a single-use bioreactor perspective, the emergence of new films for 3D bags that are more resistant to mechanical constrains are alleviating the roadblock to running a single-use bioreactor in perfusion for extended duration. And from a seed train intensification perspective, perfusion is being made easier with new technology that is based on tangential flow filtration (TFF). This new TFF-based technology (Cellicon Solution, MilliporeSigma) is specially designed for seed train intensification. “Coupled with modern bioreactor designs, this is currently making perfusion more attractive, especially for N-1 perfusion and HCDC cell bank manufacturing,” says Verlenden.
Proven and scalable cell retention devices (e.g., XCell ATF Technology, Repligen) have enabled N-1 cell culture intensification processes up to 3000 L to seed 15,000-L to 18,000-L reactors globally, adds Gebski. Along with bioreactor design and control systems, these devices have enabled the higher cell densities attained with intensified seed train culture, she says.
Are there particular synergies to using seed train intensification together with single-use bioreactors and/or single-use technologies, or is the end result (optimized yield) the same whether using single-use bioreactors or stainless-steel? Verlenden says that the single-use vs. stainless-steel factor depends on the manufacturing context. “Some companies have made the choice to implement N-1 perfusion in stainless-steel bioreactors with volumes greater than 3000-L to seed 10-L to 15,000-L stainless-steel bioreactors operating in typical fed-batch. This allows maximum productivity in fed-batch at large scale for commercial molecules while taking advantage of seed train intensification,” he says.
Gebski also says that the seed train intensification process and outcome is the same whether stainless-steel reactors or single-use reactors are used. “Seed train intensification, and the benefits provided, continue to be demonstrated and clinically or commercially implemented in many mid-scale and large-scale cell culture processes,” she says.
In addition, she makes the distinction that single-use components and technologies are implemented for their shorter implementation time, reduced equipment cleaning requirements, and ease-of-use. “Single-use has not paved the way for seed train intensification approaches, but rather single-use and intensified seed train approaches drive efficiency gains into the manufacturing process, regardless of bioreactor type,” she states. The addition of closed, single use alternating tangential flow devices has simplified and expedited this adoption even further aligning with the trend towards single use facilities.
Meanwhile, as perfusion in the production stage with continuous harvest of proteins becomes more adopted in the industry, the concept of scaling processes out—as opposed to scaling processes up—will unleash its full power, Verlenden states. “This will be only possible with extensive use of single-use bioreactors and components in order to maximize process flexibility,” he adds.
The use of single-use technologies is a key enabler of process intensification overall, Verlenden also says, citing the Biophorum Operations Group’s technology roadmap issued in 2017 (1). He notes that the enabling benefit of single-use technologies is even more true in the seed train, where typical early stages are manual and open. The use of single-use technologies, such as HCDC bags, make the entire upstream process functionally closed, which ultimately reduces the burden and costs associated with environmental testing required for room classification, he explains.
“The adoption of intensified seed train at larger scale—late-stage clinical and commercial manufacturing—remains slower than initially anticipated by the industry, and this is not necessarily because of a lack or inefficiency of single-use technologies,” Verlenden goes on to clarify. He explains that the long-standing perception within the industry is that media cost for implementation of perfusion in the seed train outweighs the benefits of increased throughput and process flexibility. “While a greater quantity of media is needed to run a N-1 bioreactor in perfusion, recent cost model data clearly show that the media price and cost have a [negligible] impact on the upstream manufacturing costs per grams of protein produced. A better understanding and use of process economics modelling would likely alleviate this industry perception in the future,” he concludes.
1. Biophorum Operations Group, Biomanufacturing Technology Roadmap, www.biophorum.com, 2017.
Feliza Mirasol is the science editor for BioPharm International.
Vol. 34, No. 3
Pages: 20–22, 30
When referring to this article, please cite it as F. Mirasol, “Jumping Seed Train Intensification Hurdles to Maximize Yield,” BioPharm International 34 (3) 2021.