Understanding Upstream and Downstream Bioprocessing

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Understanding Upstream and Downstream Bioprocessing

Reading Time: 8 minutes
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In biopharmaceutical manufacturing, active pharmaceutical ingredients (APIs) are synthesized using a biological source. Biomanufacturing of these APIs, also called biologics or biopharmaceuticals, involves living organisms such as bacteria or fungi.

In this blog, we’ll look at the biomanufacturing process, and how upstream and downstream bioprocessing contribute to the creation of a final biologic that is safe and effective.

A History of Biopharmaceutical Manufacturing

The history of biomanufacturing is long and storied, dating back to at least 9,000 years in the Neolithic era. These earliest examples of biomanufacturing include the fermentation of sugars into alcohols (for drinks) and acids (for pickling).

An essential step in bringing biomanufacturing to today’s state was the discovery and manufacture of insulin for treating diabetes mellitus. Despite a long history of treatments with varied efficacy, a major milestone occurred in 1889 when Oskar Minkowski and Joseph von Mering found that the removal of the pancreas in dogs led to the development of diabetes. Over the next one hundred years, advances in biotechnology included optimized isolation techniques. By the 1970s, the pancreas glands sourced from approximately 31 cows and/or pigs were required to procure enough insulin to keep one patient alive for a year. In 1978, City of Hope and Genentech scientists developed a method for manufacturing a biosynthetic version of human insulin using recombinant DNA technology. By 1982, the Food and Drug Administration (FDA) approved Humulin, the biosynthesized insulin and the first biosynthetic therapeutic.

These early steps in the development of biotechnology paved the way for the development of modern biopharmaceutical practices.

How Are Biologics Made?

Biomanufacturing is a broad term that encompasses many different processes.

For example, citric acid is a food additive that acts as a preservative and is found in virtually all processed foods. Today, the fermentation of sugars and minerals using Aspergillus niger, a fungus, is used to manufacture over 100 million tons of citric acid annually. This process occurs in large tanks called bioreactors, where scientists carefully optimize conditions for the fungus to thrive. But biomanufacturing includes more than just fermentation. For example, producing an mRNA virus involves incubating the genetic sequence of interest with elements that can make copies of it in a bioreactor. When conditions are correct, the reaction can be triggered to begin synthesizing mRNA. Similarly, biomanufacturing can describe the process of using cell lines that are genetically engineered to produce host cell proteins of interest.

It can be challenging to define biomanufacturing due to the variety of approaches that fall under its scope. In general, bioprocessing can be broken down into two steps, which are generally referred to as upstream and downstream bioprocessing:

  1. An approach is selected based on the desired end product, and the components are put together in a bioreactor. This can include the use of an organism to convert sugar to another chemical (fermentation), using an organism to secrete a protein (recombinant peptides or antibodies), or even creating a large population of cells for use in cell therapy. The container, components, and conditions are optimized to produce the final product. For example, the bioreactor may be disposable to ensure sterility. Or, the media in the bioreactor may be stirred by a physical paddle, by rocking motions of the entire reactor, or not stirred at all if the end product is fragile.
  1. The end product needs to be harvested. If the product is inside cells, the cell membranes must be disrupted. If the end product is a cell, the cells must be delicately separated from the other products in the media. Separation and further purification through technologies — like filtration, chromatography, and centrifugation — play a critical role during this phase.

What is the Difference Between Upstream and Downstream Bioprocessing?

Due to the division of expertise, processes, and techniques in these two approaches, the biotechnology field has coined a term for each phase. Upstream processing includes everything from engineering the microbial or mammalian cell lines up to and including optimizing conditions inside the bioreactor to maximize the production of the end product. Subsequently, downstream processing includes harvesting (the process of collecting the desired product from a bioreactor) and the separation and purification processes needed to produce the final biologic.

Despite the usefulness of discussing upstream and downstream bioprocessing, the terms do not have a unified and agreed-upon definition in the pharmaceutical industry. Some biopharmaceutical companies regard harvesting as an upstream step and only begin considering purification as downstream processing. Other companies narrowly define upstream and downstream processes such that secondary tasks are considered a third “support” process. For convenience, we’ll consider upstream processing as the initial steps described here (with harvesting as a downstream process).

What is Upstream Processing?

Upstream processing is a critical step in biomanufacturing, as it is essential to ensure that the cells are healthy and produce the desired product. This step is complex and challenging, as it requires a high level of control over cell cultures and the environment in which the cells are grown. Here, we’ll consider several of the most common upstream processing steps.

Cell Banking

As biomanufacturing depends on a living organism to create a final product, the quality of the cells used for manufacturing can significantly affect the quality of the final product. One strategy often used by biopharmaceutical companies is the establishment of cell banks. A master cell bank (MCB) is a collection of cells grown from a selected single cell line. These cells are ideally identical and will be used to produce the biologic and so must be carefully selected and characterized. The MCB cells are then cryopreserved and stored at multiple sites. When production requires new cells, a small amount is removed from the MCB and is used to create a working cell bank (WCB). Cells from the WCB are directly used in the biomanufacturing process.

The MCB and WCB are important quality control tools. These cell banks guarantee that the final product is made from cells with the same genetic makeup and properties, ensuring that the end product is produced consistently.

There are several challenges to creating and using MCBs and WCBs, including contamination and quality control. MCBs and WCBs are susceptible to contamination, which can lead to the production of unsafe and ineffective products. Consequently, MCBs and WCBs must be rigorously tested to ensure they are free of contamination and meet the required quality standards.

Media Preparation

The upstream process of media preparation consists of preparing a nutrient-rich solution to grow cells in a bioreactor. The media must provide the cells with the nutrients they need to grow and produce the desired product.

The challenges associated with media preparation include contamination, composition, and timelines. Contamination can produce unsafe, inconsistent, and ineffective products. Once assured of sterility, the media must also be of the correct composition and must be prepared in a timely manner. Any deviations from an optimal process can lead to cell growth problems and the production of low-quality products.

Bioreactor Calibration

Bioreactor calibration is the process of ensuring that the bioreactor is operating according to designed parameters meant to optimize the biomanufacturing process.

There are several challenges associated with bioreactor calibration, including complexity and accuracy. Bioreactors are complex machines with different components, and each component must be calibrated correctly. For example, ensuring that a large liquid vat is at a consistent temperature throughout requires significant design and engineering of temperature controls and surfaces.

What is Downstream Processing?

After upstream processing is complete, downstream processing includes the operations needed to separate and purify the biologic of interest. In general, the end product of the downstream process will be in one of three places:

  1. If secreted by the cells, the end product will exist in the media, mixed in with waste products, cells, and unused media.
  1. For end products that are inside the cells, the cell membranes will have to be disrupted. The techniques for breaking open cells can roughly be divided into mechanical (e.g., sonication) and non-mechanical (e.g., application of a detergent) approaches.
  1. The end product may be a population of cells that must be isolated from their surrounding media.

Steps in Downstream Processing

The current biotechnology standard for isolating the product of interest is a three-step process known as capture, intermediate purification, and polishing (CIPP) to ensure the purity of the end product. In the capture (C) step, the end product is isolated and stabilized. Intermediate purification (IP) removes the bulk of the impurities, leaving a relatively pure end product. In the polishing (P) step, any remaining impurities are removed, leaving the end product at a high purity level, ready for formulation and fill/finish. Here, we’ll consider several of the most common downstream processing steps.

Cell Membrane Disruption

Disrupting cell membranes is critical in producing biopharmaceuticals when the desired product is inside the cells. There are several methods for disrupting cell membranes, each with advantages and disadvantages.

One common method is to use mechanical force, such as sonication or homogenization. These methods are effective at disrupting cell membranes, but they can also damage the desired end product. Another approach is to use chemical agents, such as detergents or enzymes. These methods can be less damaging to the desired end product but can be less effective at disrupting cell membranes. The choice of method depends on several factors, including the type of cells being used, the desired end product, and the desired yield.


Affinity chromatography has been the biomanufacturing workhorse for purifying proteins for the last few decades. Affinity chromatography uses beads or a resin attached to a ligand with an affinity for the target molecule. The media from a bioreactor (including cells, waste products, and the target molecule) are passed through the column, allowing the ligands to bind to the target molecules preferentially. The target molecules are then eluted from the column.

If a highly selective ligand exists for a product, affinity chromatography can perform all three steps of the CIPP process. In other cases, other types of chromatography can be used. For example, ion exchange chromatography can be used if the end product is a charged molecule. Alternatively, ion exchange chromatography can remove charged impurities such as proteins and nucleic acids. If the end product has a significantly different physical size than other elements in the media, then size exclusion chromatography can be employed.


Despite advances in biotechnology and biomanufacturing techniques, the final product can be contaminated with bacteria or viruses introduced intentionally for production purposes or through environmental exposure. Inactivation is a process used to destroy or inactivate these harmful microorganisms in biopharmaceutical products. It is an essential step in the manufacturing process, as it helps to ensure the final product’s safety.

Several methods can be used to inactivate microorganisms, including heat, chemical treatment, and radiation. Heat treatment, one of the most common inactivation methods, involves heating the product to a high. Chemical treatment consists of using chemicals to destroy microorganisms. Common chemicals used for inactivation include formaldehyde, glutaraldehyde, and ethanol. Techniques that use radiation involve exposing the product to high-energy radiation, such as gamma rays or ultraviolet light. Ultimately, the choice of inactivation method depends on the specifics of the setup, including the type of contaminant that needs to be inactivated, the sensitivity of the biopharmaceutical product, and the cost of the inactivation process.

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Optimization of Upstream and Downstream Bioprocessing

Process intensification is a set of methods and techniques used to improve the efficiency and productivity of biomanufacturing processes.

In the upstream steps, process intensification is used to improve the yield of the desired product. This can be done by using more efficient cell culture systems, improving the efficiency of cell disruption, or selecting different combinations of methods for product recovery. In downstream processing, process intensification is used to enhance the purity of the desired product. This can be accomplished by using more efficient isolation and separation techniques or by altering the product formulation or fill-finish.

That’s a Wrap

In summary, biomanufacturing is a complex and challenging process essential for producing life-saving biotherapeutics. New technologies are being developed to improve the efficiency and productivity of upstream and downstream bioprocessing, reduce the cost of production, and improve the quality of the end products. An important lesson learned from the biomanufacturing of vaccines during the global pandemic is the importance of distributed production. There is a growing trend in the pharmaceutical and biopharmaceutical industry to use smaller, more distributed facilities to produce not just the final biologics but also raw components. Advances in biotechnology and manufacturing approaches have the potential to revolutionize the way companies in the biopharmaceutical industry synthesize life-saving therapeutics.

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