Clarification Primer

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Clarification of mammalian cell culture involves multiple steps and, typically, a systematic combination of unit operations which can include centrifugation, tangential flow filtration, depth filtration, and sterile filtration. The ultimate goal of clarification is to prepare the cell culture for downstream chromatography and purification.

Not all components of the cell culture fluid are completely removed by a single unit operation. Understanding process inter-dependencies, since more than one unit operation may be required to clarify the feed stream, is a key factor in establishing an optimized and robust clarification scheme.

Three main challenges faced when developing a clarification scheme are:

1. Fluid complexity
2. Selection of optimal separation technologies
3. Technology integration

1. Fluid Complexity

Cell culture harvest is an extremely complex process stream with many constituents influencing the efficiency of clarification. These constituents include the following:

• Solids – The level of solids present in the harvest can directly impact the capacity of clarification technologies. The type of cells and organisms used to produce the protein of interest range from delicate mammalian and insect cells to more robust bacteria and yeast cells.
• Colloids - Negatively charged particles, which are deformable and sticky, often have a negative impact on downstream process steps.
• Soluble Entities - Proteins, buffers, anti-foams, and cell culture media can prove troublesome to clarification technologies.

2. Selecting Optimal Separation Technologies

When selecting the optimal set of separation technologies to prepare the process fluid for purification, there are four factors that should be considered during the selection process (in addition to capacity):

• Fluid Variability – All cell cultures will be subject to some level of variability including variation in cell density and viability. It’s important to understand the level of variability to be expected and whether those variations are controllable. The selected technologies must ultimately be robust enough to handle the anticipated process variation.
• Batch Size and Frequency – The frequency of processing, and the production batch volume are the most important factors in selection of separation technologies. Will the batch size be 1L or 10,000L? Is the harvest frequency <10x per year or more?
• Downstream Process Steps – The technologies which will follow the clarification step will be impacted by the choice of clarification technology. Does TFF UF immediately follow your clarification step and do you need to worry about volume processed?
• Process Constraints – Are there limiting factors such as process time or cleaning requirements that would drive the selection of technology?

3. Integrating Selected Technologies

There are a wide variety of technology options available for harvest and clarification, and many inputs to consider when selecting the best technologies. Three of these technologies include: Tangential Flow Filtration (TFF), depth filtration, and centrifuge.

When all evaluations are completed and technologies selected, the next major challenge is how to successfully integrate the chosen process train. Factors to consider during process integration are:

• Goals or application objectives
• Where is the molecule in the development pipeline
• Media composition
• Cell viability and density

Merck  offers a full line of clarification products and services to help speed your MAb and plasma-derived products to market faster and more cost effectively.