July 29, 2020

Seed train intensification - the next big step in biopharma?

Seed train intensification is an approach in biotechnology that is gaining more and more attention. Nevertheless, with the growing demand for new drugs – not least triggered by the corona pandemic – and just in line with our generally fast-moving times, labs and manufacturers have to ensure both fast and flexible production processes. While generally applicable standards must always be adhered to within the seed train, labs and plants are expected to operate as cost-effectively as possible in all phases of process intensification, process development and production.

Novel processes such as seed train intensification by Single Use Support are being implemented increasingly, utilizing innovative lab and production systems. They can accelerate the traditional seed train process, leading to shorter production times and enhanced flexibility.

In this article, we will take a closer look at the conventional seed train and how seed train intensification can be achieved. 

Improving the seed train for cell culture – cornerstone of upstream intensification

Infrastructures and production processes are changing along with the varying volumes. Traditional compounds and blockbusters are usually produced on a large scale. However, the production of new drugs and personalized therapies such as CAR-T cell therapy or gene therapy requires only miniscule amounts of substances and/or cells. This entails a departure from established production methods, which come with their own benefits, particularly with regard to time and cost efficiency.

In line with the change requirements, any new approach in that field focuses on increased levels of agility and scalability. Processes and applications based on the lean production principle are on the advance, allowing for upstream intensification to entirely new levels. The term “lean production” refers to the time and cost-effective use of various resources, such as equipment or staff, in all business activities. As a result, it allows for a quick adaptation to new or changed customer needs thanks to a streamlined and flexible design of the production process.

Seed train intensification is a typical lean production process. It allows labs and manufacturers to grow an adequate number of cell lines for the inoculation of production bioreactors used in production. And even though it is a relatively new method, seed train intensification is gaining traction globally.

Seed train - the conventional process

The conventional seed train of cell culture an integral part in the production of several biopharmaceuticals, as it involves the production of significant amounts of biomass to produce various proteins, e.g. for antibody drug products. The different steps of the workflow have to be carried out without exposing the cells to contamination and to achieve high process throughput during scale-up.

There are certain attempts at process intensification, ranging from improving cell viability after freezing and thawing to more automated, enclosed solutions that guarantee that the product is sterile and safe to use. As the pharmaceutical landscape is changing and the demand for smaller production batches is growing, scalability also plays an important role.

Seed train – traditional procedure

The traditional seed train procedure begins with the thawing of cells that have been cultivated as the master cell bank. Fed batch size at this point is around 1 ml. As part of the upstream-process, these cells will then be fed to reach a level that is sufficient for the inoculation of a bioreactor.1

Once the number of cells for the working cell bank has been reached, they are cultivated throughout multiple steps in shake flasks, rocking motion bioreactors and stirred tank bioreactors with the required titer and viable cell density, until they have reached volumes up to 2,000 liters in a single-use bioreactor and up to 15,000 liters in a stainless steel bioreactor.2

As the traditional procedure of the production process is laid out for large-scale production in larger bioreactors, it is very time-consuming to reach a sufficient cell growth. Further, this manufacturing process requires the handling of cell cultures in the open during transfers, which leads to a higher contamination risk and can lead to genetic changes during cell expansion, causing issues with reproducibility.1 2 3 

Tools required for the seed train

In this section, we will provide a list of the tools that are needed during the conventional seed train process in biomanufacturing. The following list contains the cultivation systems in the order they are used, starting with the smallest equipment for cell culture processes and gradually moving towards larger bioprocessing devices.  

Tools required for the traditional seed train include:

  • bioprocess containers
  • Spinner
  • Wave bioreactor
  • N-3-bioreactor
  • N-2-bioreactor
  • N-1-bioreactor
  • N-bioreactor

Why seed train intensification helps save time and costs

Seed train intensification based on modern technologies applying High Cell Density Cryopreservation (HCDC) is both time-saving and cost-effective compared to conventional alternatives, like fed batch production processes.

Single use technologies are suitable to satisfy the varying needs of the pharmaceutical industry while complying with modern standards. They are not only more agile than tried-and-tested conventional stainless-steel bioreactors but also benefit from high degrees of flexibility and scalability. In other words, seed train intensification based on disposable single-use components allows small labs and global players alike to grow the exact amount of cell culture required, irrespective of whether they are needed for a clinical study, a new therapy or a personalized treatment.

While the traditional approach involves the cultivation of cell banks in stainless steel reactors, flexible systems based on the lean production approach can produce small batches in large quantities. Consequently, this increases effectiveness: Instead of constantly having to start the entire process from scratch, labs and manufacturers can simply fall back on already cultivated cells as intermediates.

How can a seed train be intensified?

Having established why seed train intensification is necessary, it is time to address the question on how to achieve this. To start protein production in productions with bacteria, e. Coli or animal cells like Chinese Hamster Ovary cells (CHO cells) in an optimized way, there are different strategies available to opt the cell lines for scale-up at faster rates.

One strategy to achieve high cell density and viability is the attachment of a cell retention device to the N-1-bioreactor.4 Through this method, it becomes possible to use a smaller bioreactor due to the higher initial cell density in the suspension cell culture and seed the production reactor. Theoretically, this step can also be performed in the earlier N-2-bioreactor stage of the work flow, saving considerable amounts of time in the production process.5

Another strategy for seed train intensification is the cryopreservation of a master cell bank with a high cell density in single-use bags. High cell density cryopreservation removes the need for traditional seed train expansion.4 


Seed train intensified with cell bank multiplication

Cell bank multiplication is an important step in seed train intensification. As speed and reproducibility are highly important factors in the production of biopharmaceuticals, the development of a working cell bank (WCB) that can be used for the production of large product quantities is essential. After a small number of cells has been extracted from the master cell bank (MCB), these cells are cultured in large aliquots with cell suspensions. Aliquoted in smaller single-use bags up to 50 ml, they are frozen to cryogenic temperatures.

What is high cell density cryopreservation?

While cell bank multiplication allows for the production of large quantities of pharmaceutical products by aliquoting smaller quantities of suspensions with high cell densities, cryopreservation plays an important role in the seed train intensification.

In high cell density cryopreservation (HCDC), intermediates with a high cell density are aliquoted in single-use bags and then frozen at -80 °C, before they are transferred into liquid nitrogen in its vapor phase. By ensuring the viability of the cells after thawing by keeping them at cryogenic temperatures ensures their reproducibility, an important aspect in the production of biopharmaceuticals. By being able to use these cells as a base, it becomes possible to skip the first culturing phase of the production process. This means process development can start earlier, making the whole endeavor more cost- and time-efficient.

It is important to note that case studies have shown good results with cryopreservation with regard to growth and recovery rates of CHO cell lines, the current cell line of choice for the production of monoclonal antibodies used in pharma production.

Seed train intensification with HCDC comes with several advantages, some of which are:

  • Time efficiency: HCDC allows for faster cell growth and higher cell densities, reducing the overall time required for the seed train process. This results in shorter manufacturing timelines and quicker production of the final product.
  • Cost-effectiveness: By achieving higher cell densities in a shorter time, HCDC can lead to reduced resource and facility usage, lowering production costs and increasing cost-effectiveness.
  • Increased productivity: HCDC enables the production of a larger number of cells in a smaller footprint, maximizing the productivity of bioreactors and improving the overall yield of the biopharmaceutical manufacturing process.
  • Consistency: The use of HCDC in seed train intensification helps maintain the consistency of the cell culture and reduces variability between batches, resulting in more reliable and predictable production outcomes.
  • Scalability: HCDC is easily scalable, allowing biopharmaceutical companies to efficiently transition from small-scale seed train operations to large-scale production without significant changes to the process.
  • Process optimization: HCDC facilitates better process optimization by providing a controlled and reproducible environment, leading to improved product quality and performance.

Tools in seed train intensification with HCDC

Seed train intensification requires innovative tools and up-to-date technologies to optimize the time and cost aspects of a project. This does not only mean that time efficiency has to be increased, but also that scalability needs to be taken into account, given that different life science projects require different product quantities.

This starts with primary packaging in single-use bags which are transferred to a homogenizing device like RoSS.PADL, ensuring an even distribution of the high cell density solution while simultaneously cooling it down.

In contrast to traditional seed trains, where the aliquoting and transferring of cells is often carried out manually, exposing the cells to contamination, fully automated aliquoting solutions like RoSS.FILL CGT erase this problem – product loss due to human errors can be reduced to a minimum.

Cryopreservation of cells for later use has to be carried out at even and controlled freezing rates, best achieved in RoSS.LNF2. This enclosed cryogenic freezer reaches temperatures down to -170 °C without exposure of cells and is the only truly controlled LN2 freezer currently on the market.

Seed train intensification with Single Use Support

As the upstream process of cells is an integral part in the production of many pharmaceutical products and applications, it does not surprise that there is a high demand for biotechnological solutions that are able to streamline and enhance the early stages of manufacturing to make the whole process more time- and cost-efficient.

This is best achieved by fully automated solutions in enclosed systems that eliminate the need to expose cells to any risk of contamination during different production steps where cells have to be transferred from one device to another.

Systems by Single-Use Support address these problems with process platforms that enable the cultivation cells as well as their preparation for the logistics process. If necessary, the cells can be filtered using single-use filtration and filled into single-use bags in an automated filling process before being frozen for sterile cell retention. Following lean production principles, the freeze-thaw process is fully automated, ensuring sterility while simplifying all process steps.

However, the solutions provided by Single Use Support are not only scalable and characterized by a high level of automation, but can also be configured to the respective needs, for instance by adding single-use filtration.

The components used can be easily disposed of, making tedious cleaning processes a thing of the past. In line with the lean production principle – and medical research – this can significantly accelerate the process of seed train intensification. In the end, this saves time and money while also contributing to increased levels of patient safety.

FAQs on seed train intensification

What is a seed train?

A seed train is a series of steps in biopharmaceutical manufacturing used to grow and expand cells for producing therapeutic proteins or biological products. It starts with a small cell culture and gradually increases in size to achieve large-scale production while maintaining quality and consistency.

What is seed train expansion?

Seed train expansion is a biopharmaceutical manufacturing process that involves sequentially increasing the volume and number of cells from an initial small culture (seed) to achieve larger cell populations suitable for large-scale production of therapeutic proteins or biological products.

What is the difference between a seed train bioreactor and a production bioreactor?

A seed train bioreactor is used for early-stage cell culture expansion, while a production bioreactor produces the final product at a larger scale later on.

  1. Seed train optimization for suspension cell culture, http://dx.doi.org/10.1186/1753-6561-7-S6-P9, Published 2013-12-04
  2. Model-based strategy for cell culture seed train layout verified at lab scale, http://dx.doi.org/10.1007/s10616-015-9858-9, Published 2015-03-20
  3. Seed Train Optimization for Cell Culture, http://dx.doi.org/10.1007/978-1-62703-733-4_22, Published 2013-12-02
  4. “Jumping Seed Train Intensification Hurdles to Maximize Yield,” BioPharm International 34, https://www.biopharminternational.com/view/jumping-seed-train-intensification-hurdles-to-maximize-yield, Published 2021
Seed train intensification - the next big step in biopharma?

Michael Eder

Senior Marketing Manager

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