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How to Scale-Up Lentiviral Vector Production Part 1: Considerations for Upstream Processing

Lentiviral vectors (LVV) are a key component in the production of cell and gene therapies. They are most often used to deliver genetic material that will modify cells and confer therapeutic properties. Today, even with the proliferation of cell and gene therapies in development, LVV is still produced using legacy methods employed in basic research. Overcoming technical challenges in the scale-up of LVV production is a major focus for the industry. Scalable LVV production platforms are critical for manufacturing affordable cell and gene therapies and making them more widely available. For an overview of LVV manufacturing and process optimization considerations see our previous post.

We will start our series on LVV scale-up by exploring the principles that govern the scaling of upstream processing (USP).

First, what do we mean by upstream processing? This includes the culture of producer cell lines (e.g. HEK293 cells), transfection with plasmids encoding viral constructs for transient processes, and harvesting viruses from cell culture media.

USP scale-up for commercial production of LVV will almost always require a switch from culturing adherent cells in flasks (e.g. laboratory method) to culturing cells in suspension in a bioreactor. If a process has already been adapted to a bioreactor, the question may be how to go from a smaller bioreactor to a bigger one.

To accomplish this there are a few key principles to remember:

Scaling Up is NOT Just About Numbering Up

Process scale-up is not simply about increasing to the next order of magnitude. Instead, scale-up is a technique that is applied differently for every process. The first step in this technique is clearly defining the scale change criterion. For instance, if you want to scale-up to a bioreactor that is 4X bigger, what do you mean by that? Will you use a bioreactor that has four times the surface area, or four times the volume? To be successful in scaling-up, you need to ensure geometric similarity when moving from one bioreactor to another. This is not always easy, as the dimensions of a 1L bioreactor may not be the same as a 5L bioreactor even when they are made by the same manufacturer!

Process = Chemistry + Physics

Parameters related to the biology and chemistry of a process are often scale-independent (e.g. media composition, cell density), while physical parameters (e.g. mechanical stress on cells and mixing time) are scale-dependent. Your scaled-up process must consider all of these parameters to produce the desired final product.Q3-free-download.jpg

When producing a virus in a transient transfection process, transfection efficiency is another critical parameter that needs to be tested as part of scale-up. Transfection efficiency is measured most reliably towards the end of USP, when viral titre is assessed following harvest from cell culture media. At this stage, a significant amount of time and money will have gone into completing a process. Therefore, to avoid waste, it is desirable to have a sense of transfection efficiency first at small-scale in a stirred tank reactor platform.

Start Small to Go Big

To save time and money, it is recommended to begin testing parameters at a small scale before moving to a larger scale. For example, the agitation rate of cells producing a virus in suspension is a critical parameter in LVV production because viruses are very susceptible to shear stress. It is important to consider that an agitation rate that is fine for the cells themselves may not be good for the virus. One key step in de-risking a large-scale process would be to test agitation rates first at smaller scales. However, this is not always possible. For instance, stirred-tank, single-use bioreactors, like the XDR reactor that are quickly becoming the industry standard, may not be available in scaled-down versions. Developing an understanding of how to scale effectively from one vessel to another is a critical piece in going from cost effective, small scale reactors for process development to larger scale reactors for feasibility runs and eventually to production size scale reactors.

Scalable and Consistent vs. Optimized and Non-Scalable

CCRM’s experienced process development team has learned that when it comes to scaling-up LVV production, clients are most interested in processes that are efficient and reliable at scale as opposed to processes that are highly optimized. We have also learned that different clients have different priorities in this regard, and we are building a flexible development platform that can be customized depending on the needs of the client.

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