What is biological processing? Upstream and downstream methods and key steps

However, the most landmark application of biological processing belongs to the field of biopharmaceuticals, and the most famous case is the industrial production of insulin - insulin is the key protein for the survival of diabetes patients.

This production method not only has low yield, poor purity, and high cost, but also often causes allergic reactions and has a short duration of efficacy.

In 1983, Eli Lilly began selling recombinant DNA human insulin expressed in E. coli. This technological breakthrough has realized the high-quality and high-yield production of insulin, significantly improved the therapeutic effect, and greatly extended the survival period of diabetes patients.

Similar to humans, microorganisms also have the ability to synthesize proteins. With the help of biological processing technology, as long as the correct "instructions" and growth conditions are provided to microorganisms, we can enable them to synthesize target proteins in a targeted manner.

In practical applications, these "instructions" usually involve introducing DNA fragments of microorganisms, while the "conditions" include key parameters such as suitable nutrients and stirring environment provided for the growth of microorganisms in the bioreactor.

Biological processing is mainly divided into two core stages: upstream processing and downstream processing.

In the upstream processing stage, suitable cell types need to be selected based on the characteristics of the target product, while determining the composition of the culture medium, the type of bioreactor, and the process scaling up plan, ultimately achieving cell harvesting and laying the foundation for downstream processing.

The downstream processing stage focuses on the separation, concentration, purification, and modification of the target product, and ultimately produces bottled formulations through the "fill sealing" process. The following text will provide a detailed analysis of the upstream and downstream processing stages.

In this container, cells achieve optimized synthesis of the target product through proliferation.

The efficient operation of bioreactors requires the establishment of precise material balance, as multiple variables such as gas flow rate, substrate concentration, liquid flow rate, and initial cell density can all affect production efficiency.

Based on determining the type, volume, and operating mode of the bioreactor, it is necessary to further optimize various process parameters for different biological sources and target products. The following text will break down the key steps of upstream biological processing.

1. The selection of cell cultures or biomaterials for bioreactor processing should be based on the characteristics of the target product (usually a metabolite or recombinant protein).

Although mammalian cells, bacteria, and yeast are the most commonly used cell types in biological processing, plant cells and insect cells can also be used in specific scenarios. Table 1 lists the advantages and disadvantages of various microbial cell culture systems.

2. Selection and optimization of culture medium provides basic nutrition and energy sources for microbial proliferation and high-value product synthesis. The selection of culture medium should be based on the characteristics of the cultured strain and cell line. 

Its core components usually include amino acids, peptides/proteins, carbohydrates, and some cell lines also require the addition of vitamins and fatty acids.

To maintain the pH stability of the suspension system, a buffer system is required; In some scenarios, gases such as carbon dioxide and oxygen need to be introduced. In addition, the culture medium usually contains salt substances to regulate osmotic pressure balance.

There are significant differences in the demand for culture medium among different cell lines, and even within the same cell line, the proliferation efficiency and product yield may vary under different culture conditions.

Therefore, it is necessary to determine the optimal nutrient concentration, pH value, osmotic pressure, and gas environment through medium optimization. 

The traditional optimization method is based on a known culture medium and uses a single factor variable method for stepwise screening, but this method is time-consuming and labor-intensive.

A more efficient optimization strategy is the "Design of Experiments" method, which uses statistical algorithms to analyze the impact of synchronized changes in multiple factors on production efficiency and the interaction between factors.

The mixing method of culture media is also a commonly used optimization method, which involves gradient mixing of different formula culture media and detection of product yield to screen for the optimal mixing ratio.

However, the drawback of this method is that the synchronous changes of multiple factors make it difficult for biological processing engineers to accurately control variables.

The new culture environment needs to provide optimal growth conditions for cells to ensure efficient synthesis of recombinant proteins, metabolites, or fermentation products. Normally, the inoculation amount is 1/20 to 1/200 of the volume of the subsequent proliferation medium.

4. Operating modes of bioreactors: The operating modes of bioreactors need to be determined based on the type of biological processing, the characteristics of biological materials, and the demand for target products. They mainly include the following four core modes:

• Batch culture mode: microorganisms proliferate with the culture time, nutrients are gradually consumed, metabolic waste is not discharged from the system, and only pH value and gas parameters are adjusted during the culture process.

• Feeding Batch Cultivation Mode: To address the issue of nutrient depletion during batch cultivation, nutrients are continuously replenished during the cultivation process to maintain a stable concentration.

• Irrigation culture mode: While supplementing nutrients, cells are retained in the reactor through a cell retention mechanism, and waste culture medium is continuously discharged to achieve continuous product harvesting.

Continuous fermentation mode: Similar to perfusion culture, it adopts a method of continuous input of nutrients and continuous discharge of waste culture medium, but without cell retention mechanism, cells are discharged together with the waste culture medium.

After the completion of cell culture and product synthesis in the harvesting process, it is necessary to remove cells and impurities through the harvesting process to create conditions for downstream processing - if there are residual cells in the system, subsequent purification and concentration steps will not be effectively carried out.

The core of harvesting technology is to remove cells, cell debris (achieved through cell lysis), sediment, and suspended particles. Common techniques include disc centrifugation, disposable centrifugation systems, deep filtration, and tangential flow filtration (TFF).

6. Process monitoring and control: The monitoring of upstream processing processes should follow the concept of "Quality by Design", which focuses on the recognition of critical quality attributes (CQAs) of products and risk management, and specifies the key process parameters (CPP) that need to be monitored.

By collecting process data in real-time, waste can be minimized, batch release speed can be accelerated, and production can be increased to the greatest extent possible. 

The achievement of this goal relies on Process Analysis Technology (PAT) - a technology system that monitors key process parameters and key quality attributes in real-time through online, in line, or near line detection methods, achieving precise control of production quality. 

Partial process analysis technology tools have been validated by regulatory agencies and widely used in the bioprocessing industry (Figure 1). 

The core tools include:Raman spectroscopy: By detecting the vibration modes of molecules and biomolecules, analysis can be achieved, and key parameters such as lactate, ammonia, glucose, glutamine concentration, and total cell density in the culture medium can be non destructively monitored online and online.

Nuclear Magnetic Resonance Spectroscopy (NMR): Traditionally used for the characterization of organic compounds, high-resolution NMR requires the use of low-temperature liquids and strong magnetic fields, and only supports offline detection. 

With the emergence of low field, desktop helium free instruments, real-time detection has become possible, enabling precise detection of various elements such as carbon, nitrogen, and phosphorus.

Mass spectrometry: a high-resolution detection tool that can monitor the purity and identity of complex biomolecules synthesized in bioreactors through high-throughput analysis, especially suitable for detecting key quality attributes such as glycosylation in the production process of monoclonal antibodies.

Dynamic Light Scattering (DLS) method: used in quality control processes to detect protein particle size distribution and aggregation, but still faces challenges as a real-time process analysis tool.

Flow cytometry: used to detect cell morphology and activity, providing key data for upstream process monitoring.

Usually, multiple concentration and purification techniques need to be used in series to enhance the effectiveness. The downstream processing of therapeutic proteins mainly includes the following key steps:

The core goal of the capture stage is to separate the target product from the mother liquor, laying the foundation for subsequent buffer replacement and purification steps. Salt induced precipitation is one of the low-cost capture technologies; 

Affinity chromatography is a highly selective mainstream technique, and protein A affinity chromatography is commonly used in the preparation of monoclonal antibodies. In addition, protein A functionalized magnetic beads can be used to achieve adsorption and capture of therapeutic proteins.

After capturing the target protein in the concentration stage, it is necessary to optimize the subsequent process efficiency and reduce the overall cost through concentration. 

The concentration process simultaneously achieves preliminary purification - salts and low molecular weight substances are removed, and proteins are enriched.

The mainstream concentration technology is based on filtration principles, including tangential flow filtration (TFF), ultrafiltration, and reverse osmosis. Suitable cut-off molecular weight parameters can be selected based on protein molecular weight and the characteristics of metabolites/salts to be removed.

3. Purification stage chromatography is the core separation technology in the purification stage, mainly including: • Volume exclusion chromatography: the separation of therapeutic protein monomers from aggregates or fragments is achieved through preparative chromatography columns. Ion exchange chromatography: Separation is achieved based on the interaction between proteins and ions in stationary and mobile phases. 

Affinity chromatography: It can also be used in the purification stage, such as nickel functionalized chromatography columns for purifying proteins containing histidine tags. Integrated column chromatography: a highly efficient separation technology developed in recent years that supports the application of 3D printed chromatography columns.

After therapeutic protein separation and purification in the biological coupling stage, biological coupling modification is usually required to achieve the following goals: adding labels for subsequent analysis and detection; 

Enhancing protein activity and therapeutic efficacy, for example, glycosylation modification can enhance the immune recognition ability of antibodies, and antibody drug conjugates (ADCs) can improve drug efficacy by targeting tumor cells; Improve protein stability, such as increasing solubility through polymer modification.

The biological coupling of proteins is mainly achieved through the side chains of lysine residues and cysteine residues, and the N-terminus and C-terminus of proteins can also serve as coupling sites due to their reactivity.

5. Formulation stage: The formulation stage is the final stage before the drug is launched, with core steps including adding excipients, adjusting buffer solutions and salt concentrations. Typically, the product needs to be made into a solid dosage form through a drying process (redissolved before use).

Due to the significant improvement in protein stability and shelf life of solid dosage forms, most therapeutic proteins on the market are sold in powder form. Common drying technologies include spray drying and freeze drying. 

Adding carbohydrates (adding polymers in some scenarios) during pretreatment can protect proteins from sudden changes in temperature and pressure.