Upstream Processing Overview

 

Upstream processing is an important stage in bioprocessing that involves preparing and manipulating biological materials before subjecting them to downstream processing. This step involves the development of viable cells, such as mammalian, microbial or insect cells, because they serve as the basis or foundation for producing therapeutic proteins, vaccines and other biologics.

Upstream processing also impacts the final products’ quality, quantity and efficiency. This article will comprehensively overview upstream processing, including its purpose, methods and applications.

What is upstream processing?

Upstream processing is the first stage in producing biopharmaceuticals, such as cell or microbial cultures, to produce the therapeutic protein. This process involves a series of steps to achieve the desired outcome.

The upstream processing aims to optimize cell or microbial culture’s growth and productivity while ensuring purity and quality. The process requires carefully controlling the growth conditions, including temperature, pH, dissolved oxygen and nutrient supply, to ensure optimal cell growth and protein expression.

Biopharmaceutical companies can increase the production process’s efficiency, reduce production costs and produce high-quality products that meet international standards.

Generally, the biopharmaceutical industry uses upstream processing to produce a wide range of products, including therapeutic proteins, enzymes, antibodies, vaccines and other biologics. It’s critical to produce these products commercially and meet the increasing demand for life-saving treatments.

What are the steps in upstream processing?

Upstream processing comprises the initial stages of manufacturing biopharmaceuticals or biological products. The initial stages or steps are discussed in detail below:

Media Preparation

The first step in upstream processing is media preparation, which involves formulating the media composition to support the growth and propagation of the desired cells or microorganisms. The media is prepared as a liquid or solid and is sterilized before use. This sterilization process is done through autoclaving, filtration or chemical treatment.

Additionally, the media’s composition is essential for the growth of cells or microorganisms. The media usually comprises carbon, nitrogen, minerals, vitamins and other important supplements for the growth process.

However, selecting these components depends on the type of microorganisms or cells being used. The media’s composition is optimized to enhance the yield of the desired product.

Inoculation

The second step is inoculation, which involves introducing the cells or microorganisms into the prepared media. It is done with a starter or seed culture containing a high concentration of the desired microorganism or cells. However, inoculation must be done under sterile conditions to prevent contamination by unwanted microorganisms.

Once inoculation is done, the culture vessel is incubated under controlled temperature, pH and aeration conditions. The incubation period promotes the growth and multiplication of the microorganisms in the media. This growth is monitored through various techniques, including optical density measurement, cell counting or metabolic activity measurement.

Cell or Microbial Culture

This step is crucial to the production of recombinant proteins. The process involves growing and multiplying cells or microorganisms in a nutrient-rich medium, and the growth conditions are carefully monitored to ensure optimal protein production. Below are the steps involved in a cell or microbial culture:

• Inoculation
• Growth
• Monitoring
• Harvesting

Harvesting

Harvesting is the process of removing cells or microorganisms from the culture medium. Harvesting aims to obtain the maximum amount of biomass or protein from the culture while minimizing the loss of product to get the desired quality of the final product.

The choice of harvesting method depends on several factors, such as the type of cells or microorganisms being used, the culture’s volume and the required purity and yield of the final product. Some common methods used for harvesting cells or microorganisms include:

Centrifugation

Centrifugation is widely used to separate cells or microorganisms from the culture medium. The culture is spun at high speeds.

Then, the sedimentation separates the cells or microorganisms from the supernatant. This method helps harvest relatively large and heavy cells or microorganisms.

Filtration

Another method to separate cells or microorganisms from the culture medium is filtration. The process involves passing the culture through a filter with a specific pore size that retains the cells or microorganisms while allowing the supernatant to pass through. This method helps harvest relatively small microorganisms.

Flocculation

A third method is flocculation, which induces the cells or microorganisms to clump together and makes them easier to harvest. Adding or involving a flocculating agent such as calcium or magnesium ions induces this method.

Sedimentation

Finally, the fourth method, i.e., sedimentation, helps separate cells or microorganisms from the culture medium depending on their density. The culture can settle, and the cells or microorganisms are separated from the supernatant based on density.

Purification

After harvesting the protein product, clarification steps must be done to reduce impurities and particulates from upstream. This gives way to the next step, purification, where the target protein is further isolated and separated from the contaminants.

This step is critical to ensure the final product’s purity and quality. Additionally, purification is done through several techniques, including chromatography, filtration and precipitation.

In chromatography, the protein binds to the stationary phase, only to be eluted using a buffer solution with specific conditions. This process can help achieve high purity levels of the desired protein.

In filtration, the filters are utilized with different pore sizes to separate the protein from the contaminants. The filter’s pore size depends on the protein’s size and can be adjusted to achieve the desired purity levels.

Precipitation is the first step in the purification process and involves adding a salt or organic solvent to the protein solution, causing the protein to become insoluble.

Upstream process development

Upstream process development involves several steps, including cell line development, media optimization, seed train and bioreactor operations. Here’s an overview of each step:

Cell Line Development

This step involves selecting a cell line to produce a desired protein or product. The cells are typically derived from mammalian, bacterial or yeast cells.

Then the cells are genetically engineered to express the desired protein or product. The cells are then screened to select the best cell line for further development.

Media Optimization

After selecting the cell line, the next step involves optimizing the growth media for protein expressions and cell growth maximization. The media must contain the necessary nutrients, vitamins and growth factors to support cell growth and protein production. It is a crucial step in the upstream process, as it directly affects the yield and quality of the final product.

Seed Train

The seed train involves cell growth in progressively larger volumes to increase the cell density and prepare the cells for the production bioreactor. This step typically consists of several stages, including inoculum expansion, seed bioreactor and production bioreactor inoculation.

Bioreactor Operations

The final step of the upstream process is bioreactor production. The bioreactor is a vessel where the cells are grown in a controlled environment.

The bioreactor conditions are carefully controlled to ensure optimal growth and protein expression. The operations include monitoring parameters such as pH, dissolved oxygen, temperature and agitation rate.

Why is upstream processing important?

Upstream processing is important in the production of biopharmaceuticals because it sets the foundation for downstream processing, which involves the purification and formulation of the final product. The quality and yield of the final product depend heavily on the quality and efficiency of the upstream process. Therefore, optimizing the upstream process is critical for producing high-quality, cost-effective and life-saving products.

Maintaining the health and productivity of cell or microbial culture is one of the important aspects of upstream processing. The growth conditions, such as temperature, pH, dissolved oxygen and nutrient supply, must be carefully controlled to ensure optimal growth and productivity. Any variation or deviation from the optimal conditions can result in reduced productivity, low yield or contamination, which can compromise the quality and purity of the final product.

Another critical aspect of upstream processing is the choice of the right cell culture or microbial culture. Different cell lines or microbial strains have different growth requirements and produce different desired protein levels. Therefore, selecting the right cell line or microbial strain is crucial for optimizing the upstream process and achieving the desired yield and quality of the final product.

Optimizing the upstream process also requires a deep understanding of the biological and chemical processes involved in the production of biopharmaceuticals. This knowledge is critical for developing and optimizing the growth media, selecting the appropriate cell line or microbial strain and controlling the growth conditions.

Examples of biopharmaceuticals produced using upstream processing include therapeutic proteins, such as insulin, erythropoietin and monoclonal antibodies, and vaccines, such as those for COVID-19. The quality and yield of these products depend heavily on the upstream process. Any optimization or improvement in this process can significantly impact the production cost and availability of these life-saving treatments.

Considerations for successful upstream bioprocessing

There are several considerations, along with some takeaways, that can help ensure successful upstream processing. These are discussed below in detail.

Cell Line Development

It is the initial step in the upstream process and significantly impacts the bioprocessing’s success. Selecting a stable cell line that has high productivity and can grow well in the selected culture media is important.

Optimization of cell line development includes genetic cell engineering, screening for high-expression clones and regulating the culture conditions.

Media Optimization

As discussed above, culture media optimization is essential for successful upstream bioprocessing. The media is optimized to provide the essential nutrients, vitamins and growth factors for optimal cell growth and protein expression. This process includes optimizing the carbon and nitrogen sources, the addition of supplements and pH optimization.

Process Control and Monitoring

Process control and monitoring are important for successful upstream bioprocessing. The bioreactor operation has to be monitored for various parameters, such as pH, dissolved oxygen, temperature and agitation rate. This information helps control the process and adjust the conditions to optimize cell growth and protein expression.

Scale-Up Strategy

Scale-up is an important consideration for upstream bioprocessing. The strategy requires a step-by-step implementation to ensure scalability without compromising product quality.

Equipment Selection

Equipment selection is also vital to carry out upstream bioprocessing. The selection is based on the specific process requirements, including the bioreactor’s volume, cell type and specific requirements for monitoring and control.

Quality by Design

The Quality by Design (QbD) approach helps ensure successful upstream bioprocessing. It is a systematic approach to developing a process that meets the required quality attributes of the final product.

The approach involves identifying the critical process parameters and understanding how they affect the final product’s quality. By using the QbD strategy in upstream bioprocessing, process optimization is possible to ensure consistent product quality.

Takeaways

• Cell line development and culture media optimization are crucial for successful upstream bioprocessing.
• Process control and monitoring are critical for maintaining optimal cell growth and protein expression conditions.
• The scale-up strategy and equipment selection has to be carefully planned to ensure the scalability and reproducibility of the process.
• Incorporating Quality by Design (QbD) into the upstream bioprocessing can help optimize the process and ensure consistent product quality.

Learn more about Avantor’s upstream processing services. Our innovative products and services are designed to meet your laboratory requirements, from chemicals and reagents to lab equipment and clean room supplies. Visit Avantor to learn how we can help you refine your discoveries and achieve your scientific objectives.