Harvest window

Getting high cell titer is crucial for bioprocess industries such as probiotics, vaccines, agricultural biotechnology, and live biotherapeutic products. Choosing the right harvest time is one of the most important parameters for getting a good cell titer. But it is far from trivial.

In this explainer we will discuss some of the aspects of perfecting the harvest timing

Use a harvest window instead of a harvest point

When discussing the optimal harvest point, it may seem that a specific incubation time is always used. In practice, it is better to define a harvest window rather than a fixed time point. Bioprocesses cannot be perfectly standardized, and unavoidable process variability will lead to fluctuations in the timing of optimal harvest.

A harvest window, for example 18–24 hours post-inoculation, is used to accommodate normal process variability and mitigate the impact of fluctuations. Within this window, the process is monitored using one or more Process Analytical Technology (PAT) tools, such as online pH, dissolved oxygen measurements, off-gas analysis, and/or BactoBox® measurements. These PAT signals provide real-time process insight and support informed decisions regarding the optimal harvest time.

The overall strategy is to define the harvest window and associated acceptance criteria based on PAT data. Harvest is initiated when predefined criteria are met, such as achieving a target cell titer while maintaining impurity levels below specified limits. This approach ensures robust and consistent process performance despite inherent batch-to-batch variability.

Use a harvest window instead of a harvest point.

BactoBox® provides insights on harvest window and harvest point

Frequent sampling for off-line analyses is tedious. With BactoBox® you can keep this at a bare minimum.

• First do BactoBox® measurements on a few spread out sampling points in exponential stage to forecast/project when the culture will be within the harvest window.

• Once inside the narrow harvest window, use more frequent BactoBox® measurements to perfect the harvest time.

This requires an accurate mathematical understanding of your bioprocess. Get this by the Track growth curve workflow. The forecasting features are work-in-progress. Stay tuned for updates.

Downstream survival may depend on harvest time

Onset of stationary stage at carrying capacity may serve as a tentative indicator of harvest time. This is the point in time where cell concentrations are no longer changing.

In practice, real-life conditions are often more complex than this. In some cases, your bacterial cultures may benefit from being harvested either earlier or later than the onset of the stationary phase (see the info box below).

Empirical downstream processing tests may be needed to find the harvesting time that results in maximal CFU after processing. In principle this may be done in the following manner.

1. Get bioprocess samples during deceleration, stationary and decline Growth phases.

2. Do a pilots-scale downstream processing simulation of the processing steps, e.g. harvest, cross-flow filtration, and drying.

3. Determine CFU for the dry product.

4. Create an overlay of the data, e.g. as in the below hypothetical example.

5. Decide which harvest time gives the highest CFU/g after processing. Identify which CIZE and cell concentration is associated with this harvest time.

The cells/mL, CIZE, and harvest window parameters will guide your future production runs.

In the (hypothetical) example below the time point with max CFU/mL was also the time point that results in the highest CFU/g after downstream processing.

Best harvest time for max cell titer can be inferred from the CFU values after downstream processing (CFU/g). Cell concentrations are represented on the primary axis, while CIZE is shown on the secondary axis. The transparent blue box illustrates the harvest window. The CFU/g is a hypothetical example, while the other data are from the MDP-1 workflow Identify harvest time.

Best harvest time may not always be at onset of stationary phase

In this workflow we use onset of stationary phase as determinant for when to harvest. This is not always the case.

• Earlier harvest: If the bioreactor is huge it may take several hours to cool and harvest. Harvest prior to stationary phase accounts for processing time

• Deferred harvest: Bacteria may demonstrate better tolerance to downstream processing when they remain in stationary phase for a while. This is partially attributable to accumulation of protective compounds like trehalose and glycogen and alteration of membranes for higher resilience

Summary

Harvest timing is one of the most important factors for maximizing cell titer and final product quality. Rather than relying on a fixed harvest point, bioprocesses should use a harvest window guided by PAT tools such as BactoBox®. By combining real-time process monitoring with downstream performance data, you can identify robust harvest criteria that account for process variability and maximize viable cell yield.