In this second part of a series of primers with training experts from the National Institute for Bioprocessing Research and
Training (NIBRT), Ian Nelligan, Technical and Training Director, discusses downstream processing. Part 1 on upstream processing
appeared in the September issue of BioPharm International (1). NIBRT provides training, educational, and research solutions for the international bioprocessing industry in state-of-the-art
facilities. Located in South Dublin, it is based on an innovative collaboration between University College Dublin, Trinity
College Dublin, Dublin City University, and the Institute of Technology Sligo.
PREPARATION AND PURIFICATION
BioPharm: Downstream processing is largely considered the second half of a typical biopharmaceutical manufacturing process. What are
the typical steps involved in the downstream process?
Nelligan: The steps that biopharma manufacturers rely on for downstream processing are quite well established. A typical biopharmaceutial
process uses three chromatographic column steps, each preceded by ultra filtration/diafiltration (UF/DF) of product. Each
UF/DF pretreatment step is necessary to prepare the product for the following chromatography stage because all columns operate
under differing pH or molarity conditions. Basically, the product needs to be primed for chromatography use beforehand. UF/DF
concentrates and resuspends the product in the correct buffer before it is placed into the columns, making for a faster and
more efficient process
In a typical three-stage downstream purification process, the strategy is as follows:
- First, a capture phase is used to isolate, concentrate, and stabilize the target product. This capture phase must be achieved
quickly. This first step needs to be very selective because the product stream is weak (around 0.1%) and large product stream
volumes are used.
- In the next, or intermediate, phase, the objective is to remove the bulk impurities, such as other proteins and nucleic acids,
endotoxins, and viruses to increase product purity.
- In the final, or polishing, phase, the objective is to achieve high purity by removing any remaining trace impurities or closely
related substances. The selection and optimum combination of purification techniques for the capture, intermediate purification,
and polishing is crucial to ensuring a shorter time to pure product and good economy.
BioPharm: What are the major challenges associated with the chromatography?
Nelligan: Yes, there are common challenges. For instance, the gel or resin used to recover and purify product can deteriorate with
time; its activity is lost as the gel is recycled multiple times to maximize use. The gel is often made of tiny beads (a few
microns in diameter) that tend to compress when packed. If too much pressure is applied, the beads in the gel can collapse,
thereby producing small fragments or fines. Liquid flow through the columns is restricted by these fragments. As a result,
capacity and activity can be lost. A typical chromatography column can comprise many hundreds of liters of gel but the bed
height is rather flat (approximately 20 cm). Therefore, columns are shallow but wide (e.g., 2 m) to avoid over pressurization
and compression of the resin beads.
The proteins we make are very delicate. The conditions on the columns can lead to protein degradation if they are not managed
properly. Consider, for example, an everyday egg, which happens to be a protein covered in a hard shell for protection. If
we shake the egg too much, it deforms. The process is irreversible.
Biopharmaceutical products are just as friable as the egg example. If we agitate a protein too much, expose it to air for
too long, or fail to store it at the correct temperature, the protein can degrade and product loss can occur. At the end of
a typical downstream process, the protein yield is often down to 60% of the starting concentration (i.e. for every kilogram
processed, 40% is lost). Even though the industry spends a lot of time trying to optimize their processes to be efficient,
these losses are difficult to recover.