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Upstream and downstream processing are terms predominantly used in regards to the biopharmaceutical industry. The production of the active pharmaceutical ingredient (API) starts with its production in bacterial or mammalian cell cultures. This is followed by various purification steps, before API reaches the formulation stage at which, API becomes a part of a biopharmaceutical. After quality control, the biopharmaceutical is ready to reach the market and subsequently the consumer.

Upstream and downstream processing together lead to a safe and good quality product!


Upstream processing (USP)

Upstream processing is the first part of the biopharmaceutical production process. At this stage engineered cell lines, either microbial or mammalian, are utilized for efficient scalable production of the specific target protein or API. USP refers to all stages of cell cultivation, from early cell isolation, media development and preparation, inoculum development, cell banking and storage, all the way until harvest when the culture is terminated and the product is collected. The aim of USP is to optimize the growth of the production cell line in industrial volumes and settings and thus leading to the production of large quantities of the target product.

Initially, cells are cultivated in vials to the desired quantity and gradually scaled up using several cultivation steps to reach production bioreactor volumes of several thousand litres.

In short, upstream processing is everything related to scalable production of the cells and, consequently the product.



For the production of biopharmaceuticals microbial cultures and mammalian cells are typically used. Microbial cultures are appropriate for producing small molecules, such as peptides and enzymes. Production of big, complex proteins such as monoclonal antibodies is hindered by their lack of glycosylation mechanism, which is present in mammalian cells. This gives mammalian cell expression systems an advantage, regardless of their slow growth and complex media requirements.


After the target product is produced in the cell line expression system, the medium with the product and the cells is harvested.

Downstream processing (DSP)

The majority of biopharmaceuticals follow the three-stage purification strategy - Capture, Intermediate Purification, and Polishing (CIPP), and are purified by a similar set of methods. Each purification step leads to higher purity of the product. However, with each purification step, overall protein recovery is decreased. Besides, purification time needs to be short to maintain protein activity and stability. Thus, DSP consists of the main steps needed to ensure final product safety and quality.  Reducing the number of steps in the process also has a lowering effect on cost. Nonetheless, all the impurities should be removed to avoid an immunogenic response in the patient.

Chrom post

The main steps needed to achieve a purified end product are the following:

Primary recovery

After harvesting, the multi-step purification starts, which is a part of the downstream processing (DSP).

Harvest is processed in primary recovery as a first step in the purification process. This involves the fast separation of the target protein from the bioreactor medium, cells, and cell debris.

Proteins can be produced intracellularly or extracellularly, which influences the next steps in the DSP. In the case of mammalian cell culture, most of the protein is produced extracellularly. This means only supernatant has to be collected for further purification and product concentration. For this purpose, centrifugation is used, followed by microfiltration. The aim is to remove most of the water, medium and small molecules by product concentration. Also, by removing the bioreactor medium the product is protected from degradation by proteolytic enzymes.

In microbial production, the protein can also accumulate within the cells, therefore the cells need to be disrupted and the cell debris separated from the protein of interest. The most common cell disruption methods are high-pressure homogenization using French press of Hughes press and bead mill. By disrupting the cells, all their contents are released including proteases, therefore protease inhibitors, such as PMSF, EDTA and Pefabloc, might need to be added to prevent product degradation. However, they will need to be removed during the processing later on!

Primary recovery is focused mainly on concentrating the product as much as possible. Water is focused on primarily since it is the most abundant impurity. This is done by first performing centrifugation and some sort of filtration such as microfiltration.

Next, a series of chromatography of steps is taken for further purification, which can alternate with filtration steps.

Capture stage

After the largest impurities are removed, the product is subjected to more expensive purification steps. Primary recovery is followed by the capture stage where the target product is isolated and concentrated. This can be done by the means of chromatography. This stage usually includes 3 steps of chromatography. The three types of chromatography that are performed are affinity, ion exchange, and gel filtration chromatography.

Even though chromatography is less expensive now that the volume has been brought down, still some important financial considerations should be made here. One of the most important considerations is for example the ratio between resin affinity and cost, especially when moving from lab-scale to production scale.

Intermediate purification

Intermediate purification aims to remove most of the bulk impurities, such as other proteins, nucleic acids, and endotoxins, and to further concentrate the product. For this purpose, ion exchange chromatography can be used.


As the last step in the purification strategy, polishing is carried out. Here the focus is on the high resolution to achieve final purity. The key objective is to remove trace amounts of impurities remaining. Here size exclusion chromatography (SEC) is utilized. In this method no interaction between the column and molecules takes place, therefore the buffer with final formulation conditions can be used. In the polishing step, final purity is achieved.

Finally, after all the chromatography steps, another filtration step is performed. This is usually ultrafiltration in tangential flow filtration mode, to make sure that if the DSP was not completely sterile, all impurities will be removed, including any mammalian infecting viruses, bacterial pathogens and their endotoxins. This last step is vital for the safety of the products. After the purification process formulation follows where lyophilization of the product is used to make it more stable for storing.

All these steps together lead to the purified product.


GE Healthcare (2010) ‘Strategies for Protein Purification Handbook’, p. 167.

Subramanian G. (2004) ANTIBODIES: Novel Technologies and Therapeutic Use. Springer US.

Aldington, S. and Bonnerjea, J. (2007) ‘Scale-up of monoclonal antibody purification processes, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 848(1), pp. 64–78. doi: 10.1016/j.jchromb.2006.11.032.

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