Downstream processing is the initial step of a biomanufacturing process to harvest cell culture containing highly-expressed active pharmaceutical ingredients (APIs). This is then purified and concentrated for final product formulation and commercialization.
The entire phase requires cGMP compliance to ensure that these living organisms are maintained under safe and sterile conditions. The equipment to be used in isolation, purification, and filtration can be enclosed in a cell therapy aseptic isolator during the whole process. This technology will prevent occupational contamination and unwanted air particles to mix with the isolates.
Goals of the Downstream Process
Capture of Recovery – This involves the accelerated segregation of the product of the lead compound from the cells. The goal is/are the following:
Elimination of all micro-particulates and colloidal materials (a mixture with one substance uniformly dispersed throughout another).
Elimination of the majority of water, growth medium supplements, and small molecule solutes via product concentration.
Separation of product away from proteolytic enzymes or other degradative elements.
Intermediate Purification – This involves the elimination of bulk contaminants, including host cell proteins and adventitious viruses, as well as any potential leaching foreign bodies from other in-process materials. Often there are microscopic (but finite) specific level of leached ligand from the capture resin that can be moderately bound or otherwise co-eluted with the lead compound.
Polishing – This involves the elimination of trace contaminants and impurities, including inactive or unwanted isoform of the desired therapeutic or common impurities, including fragments or other chemical modifications thereof.
Culturing cells and allowing them to perforate in a laboratory setting to produce advantageous active pharmaceutical ingredients (APIs), is becoming one of the fastest-growing areas in the field of biotechnology. Much of this success is owed to the fast-paced modernization of technologies for cell culture such as: fermentation, harvesting, and purification.
Cell harvesting is done by fully separating the cell culture from the culture media. Main techniques utilized for this process are centrifugation and filtration, with the former being used commonly for scale ups.
Through modernized technology under Esco VacciXcell and Esco Aster, Esco can cater to various biotechnology and biopharmaceutical companies with an automated cell harvesting process, from start-up to large-scale.
Automated harvesting of cells eliminates laborious steps when manually harvesting cells from a system. Different Tide Motion bioreactors are each equipped with its own harvesting system. The harvesting process uses the same conventional method of harvesting adherent cells.
Lim, H., & Shin, H. (2013). Classification and Characteristics of Fed-Batch Cultures. In Fed-Batch Cultures: Principles and Applications of Semi-Batch Bioreactors (Cambridge Series in Chemical Engineering, pp. 62-84). Cambridge: Cambridge University Press. doi:10.1017/CBO9781139018777.006
Filtration is a crucial step in the downstream processing of all types of cell harvesting process since it is also integral to the capture of unwanted impurities, intermediate purification of the sample, and polishing stages. Moreover, filtration is utilized for the preparation of purified water and other liquids for buffering and sanitizing purposes.
Different types of filtration process:
Microfiltration can be used at the start of the downstream process to clarify the feed beyond what was accomplished in the upstream harvest and centrifugation/clarification.
Ultrafiltration is used between chromatography steps to concentrate the product and change the buffer conditions to prepare it for subsequent chromatography steps.
Sterilizing grade direct flow filtration involves the use of nanofiltration cartridges to eliminate microbial organisms and insoluble proteins, to remove adventitious and endogenous viruses, and to serve as a sterile filtration process to the product in preparation for final formulation.
Flow-through devices, assembled with the above membrane media, are formatted to affect two general flow types:
Direct Flow Filtration devices allow the process fluid to cross the membrane in essentially a perpendicular flow direction; this provides little or no prevention of particulate build-up or the concentration of other elements that do not fit through the pore structure.
A flat membrane disk is the simplest arrangement of the filter. However, as membrane surface area requirements increase, a pleated sheet either with or without a secondary supporting layer is formed. The combined filtration layer is then wrapped around a perforated collecting core. An advanced version of this type fits an even greater membrane area into the same cartridge volume by folding the pleats over, allowing a greater length of membrane along each fold.
In the typical pleated membrane DFF cartridge, flow is directed from the inlet (through the media in a perpendicular or direct path across the membrane) to the outer surface of the media. The flow the collects in the core and exits the holder to the device’s downstream port.
Tangential Flow Filtration devices orient the membrane so that process flow sweeps across the active filtration surface, which minimizes pore plugging and surface fouling by concentrated reject elements of the feed.
TFF is rapid and efficient method for separating and purifying process flow. It can be used to recover and purify solutions from small volumes (10 mL) up to thousands of liters. With TFF the feed flows tangentially over the surface of the membrane, where a portion flows through the membrane as permeate.
Lim, H., & Shin, H. (2013). Classification and Characteristics of Fed-Batch Cultures. In Fed-Batch Cultures: Principles and Applications of Semi-Batch Bioreactors (Cambridge Series in Chemical Engineering, pp. 62-84). Cambridge: Cambridge University Press. doi:10.1017/CBO9781139018777.006
Centrifugation is one of the most frequently used methods to efficiently separate molecules with different densities through the use of centrifugal force. This process utilizes a high speed rotor that will outwardly move the solid particles from the axis of rotation at a selected speed or revolution per minute (RPM).
This method uses the principle of sedimentation where the centripetal acceleration causes much denser substances to move outward while the less dense materials are displaced towards the center.
This process is used in molecular biology for the:
Collection of cells
Precipitation of DNA
Purification of virus particles
Differentiation of molecular conformation
Types of Centrifugation Methods
Differential Pelleting: This is the simplest centrifugation type employed wherein particles in a suspension will sediment at different rates based on their different densities. This method is commonly used for cellular harvesting or when producing crude subcellular fractions from tissue homogenate. The downside of this method lies with the heterogeneity of biological particles which can cause contamination and poor recoveries. But this can be further addressed through resuspension and repeating of the centrifugation steps.
Density Gradient Centrifugation: This method is mainly used for the separation of particles form living cells. But in theory, it can be applied in particle separation of materials with less than 20µm diameter.
Density Gradient Centrifugation is a procedure for separating particles in which the sample is placed on a preformed gradient like sucrose or caesium chloride.
Rate-Zonal Centrifugation: In this type of method, the cross-contamination risk seen in the former method can be avoided through layering the sample as a narrow zone on top of a density gradient. With this, the faster sedimenting particles will not be contaminated by the much slower ones. However, the disadvantage with this technique is the limitation on sample volume that can be accommodated on the density gradient.
Rate-zonal centrifugation method uses the size and mass of the particles in order to sediment, not density. With the movement of particles down through the density medium, zones will form with similar sized particles. If centrifuged long enough, the particles will form a pellet based on the fact that the particles density is greater than that of the gradient.
Isopycnic Centrifugation: This method is also known as buoyant or equilibrium separation where particles are separated based solely on their density. The sizes of particles only affect the rate at which they move until their density is the same as the surrounding gradient medium. IT must be noted that the density of the gradient medium must be greater than that of the particles to be separated.
Types of centrifuge
Low-Speed Centrifuge: This type normally runs with a maximum speed of 4000-5000 rpm and is used in most laboratories for routine sedimentation of heavy particles. This centrifuge does not have temperature control and uses two types of rotors:
- Fixed angle: this rotor does not use any moving parts, thus offering the following advantages:
Lower metal stress (longer lifetime)
Higher maximum g-force
Faster centrifugation times
However, this rotor is not flexible; the pellet position strongly depends on the angle of the tube which is located from the side of the tube when spinning. The larger angle the tubes have, the tighter the pellets are.
- Swinging bucket: This rotor is highly flexible and can be used with different tube formats as well as SBS-format plates. This rotor also has a high sample capacity.
Unlike the fixed angle, this rotor has a moving swing-bucket which can cause increased metal stress to the rotor and the buckets. Moreover, the swing-bucket rotor has a lower maximum g-force than the former one, thus causing longer centrifugation times.
High-Speed Centrifuge: This centrifuge is mostly used in more sophisticated applications where higher speeds and temperature control are both critical. High-speed centrifuge can have a maximum speed of 15,000-20,000 rpm.
There are three types of rotors for type of centrifuge:
Fixed angle
Swinging bucket
Vertical rotor: This rotor is commonly used in ultracentrifugation for isopycnic separations or separation of substances based on density differentiation. Specifically, this rotor is for the isolation of plasmid DNA and sedimentation co-efficient calculation.
This rotor has a very low K factor and have the shortest path length for the particles to travel to, which causes particle sedimentation across the tube’s diameter. With this, the advantage of vertical rotor as compared to the former two is its faster sedimentation of particles.
Ultracentrifugation: This is a specialized technique to spin samples at a remarkably high speed. Current ultracentrifuges can spin up to as much as 150,000 rpm, the downside is that it can cause overheating. To address this issue and avoid sample damage, ultracentrifuges are equipped with vacuum systems that keep a constant temperature in their rotor.
The principle of this process is the same as normal centrifugation and abides by the following:
A denser biological structure causes faster sedimentation
A bigger biological particle moves faster in a centrifugal field
A denser buffer system causes a slower particle movement
A greater frictional coefficient causes a slower particle movement
A greater centrifugal force causes a faster particle sedimentation rate
The sedimentation rate of a particle will be zero once the density of the particle and its medium are equal
Esco Pharma offers a laminar flow workstation such as the Esco Straddle Units (Single/Double) to guarantee an ISO Class 5 air quality and ensure that the samples remain free from outside contaminants. We also offer cleanroom solutions especially if sterile/aseptic facilities are required; this is also to guarantee the client’s facility is up to par with international standards and guidelines.
References:
Aryal, S. (2018). Centrifugation- Principle, Types and Applications. Microbe Notes. Retrieved from: https://microbenotes.com/centrifugation-principle-types-and-applications/
Broad Learnings. (n.d.).Types of Centrifuge Rotors. Retrieved from: broadlearnings.com/lessons/types-centrifuge-rotors/
Eppendorf. (n.d.). Basics in Centrifugation. Retrieved from: https://handling-solutions.eppendorf.com/sample-handling/centrifugation/safe-use-of-centrifuges/basics-in-centrifugation/
George, A. (2018). Density gradient centrifugation [PowerPoint Presentation]. SlideShare. Retrieved from: https://www.slideshare.net/georgeoajr/density-gradient-centrifugation
Mendes, A. (n.d.). Ultracentrifugation basics and applications. Conduct Science. Retrieved from: https://conductscience.com/ultracentrifugation-basics-and-applications/
Electrophoresis is essential in the field of molecular biology for the separation and analysis of DNA, RNA, or protein molecules based on their size and electrical charge. Samples are loaded into wells at one end of a permeable gel matrix before electrical current is applied; with one end having a positive charge and the other a negative charge. Therefore, biological molecules are separated as each migrate towards its opposite charge: a molecule with negative charge will be pulled towards the positive end, vice-versa. Moreover, separation is achieved since the smaller molecules can move faster and can cover a wider distance through the gel as compared to larger ones.
Electrophoresis has gained high importance in the era of genome sequencing with its ability to provide speed and accuracy during nucleic acid analysis. It helps to distinguish DNA fragments of different lengths.
Types of Electrophoresis
With the electrophoresis technique of separation, various gels can be used as the support medium -- either in a slab or tube form. Gel slabs are frequently used as they allow the numerous samples to be ran concurrently. But the main advantage of tube gels is that they give better resolution of the results, as such, they are usually picked for the electrophoresis of proteins.
DNA Electrophoresis: Agarose gel is most commonly used in this type of electrophoresis. Its large pore structure allows the easy movement of large molecules; however, having said that, it is not suitable for smaller molecules.
Polyacrylamide Gel Electrophoresis (PAGE): This type of gel is most suitable for quantitative analysis of samples as it allows a clearer resolution of the results. Polymerized acrylamide (polyacrylamide) forms a mesh-like matrix that allows the separation of proteins based on their size. This process is widely used in the fields of: biochemistry, forensics, genetics, molecular biology and biotechnology.
PAGE works similarly to the general principle of electrophoresis which causes the separation of molecules through a gel matrix: smaller ones migrate further due to lesser resistance. However, there are also other attributes that can influence the rate of migration of the molecules such as the structure and charge of the protein.
The most commonly used PAGE process for protein separation is the sodium dodecyl sulphate or sodium lauryl sulphate – PAGE (SDS-PAGE) which is purely based on the polypeptide chain length. Normally with general electrophoresis techniques, it is not possible to separate proteins based solely on their chain length because the molecular mobility through the gel will depend on both charge and size/structure. However, if the molecules will attain a uniform charge, then it would be possible.
In SDS-PAGE, because of the presence of SDS, along with a reducing agent, the influence of charge is eliminated. This is because the SDS chemical detergent is a strong acting protein-denaturant, and together with a reducing agent, they can cause the samples to attain uniform charge (usually negatively charged) and cleave disulphide bonds to unfold the linear chains. Therefore, when the proteins under SDS-PAGE are loaded onto the gel and placed in an electrical field, they will move towards the oppositely charged field while being separated via molecular sieving effect based solely on chain length. The protein’s size can then be calculated by comparing its migration distance to a known molecular weight ladder; the movement can be visualized via protein-specific.
Two-Dimensional (2D) Electrophoresis: This is a classical method for the separation of proteins based on two properties: charge and size. In the first dimension, proteins are separated by the isoelectric point value which refers to the pH at which a particular molecule does not have an electric charge. For the second dimension electrophoresis, its basis is the sample’s relative molecular weight.
For complex protein mixtures, this process is done in combination with two other methods: isoelectric focusing (IEF) and SDS-PAGE. In the first step, protein is separated into its charges via IEF, afterwards, they are treated with SDS and will result to negatively charged samples. Therefore, the samples focused in the pI will then be separated primarily due to their molecular weights.
References:
Aryal, S. (2018). Polyacrylamide Gel Electrophoresis (PAGE). Microbe Notes. Retrieved from: https://microbenotes.com/polyacrylamide-gel-electrophoresis-page/
Austin, C. (n.d.). Electrophoresis. National Human Genome Research Institute. Retrieved from: https://www.genome.gov/genetics-glossary/Electrophoresis
Cleaver Scientific. (2018). What is electrophoresis?. Retrieved from: https://www.cleaverscientific.com/what-is-electrophoresis/#:~:text=Electrophoresis%20is%20an%20electrokinetic%20process,and%20size%20of%20the%20molecules.
Khan Academy. (n.d.). Biotechnology: Gel Electrophoresis. Retrieved from: https://www.khanacademy.org/science/ap-biology/gene-expression-and-regulation/biotechnology/a/gel-electrophoresis
Kozlowski, L. (2017). Proteome-pI: proteome isoelectric point database. Nucleic Acids Research. doi: 10.1093/nar/gkw978
Medical & Biological Laboratories. (n.d.). The principle and method of polyacrylamide gel electrophoresis (SDS-PAGE). Retrieved from: https://ruo.mbl.co.jp/bio/e/support/method/sds-page.html
