In the last part of any bioprocess, the dross must be removed before generating the gold that is the biotherapeutic agent that the bioprocess has always intended. Unfortunately, the snail is bulky and varied. And biotherapeutic gold, unlike real gold, is perishable. That is, it can withstand structural damage and loss of activity. When disposing of the cartridge and collecting the gold, bio processors must be efficient and careful. They must eliminate contamination and organic waste as much as possible to ensure biotherapeutic stresses that cause stress and maintain their integrity during purification and recovery. Anything less will degrade purity and reduce yield.
To purify and restore biotherapeutics effectively and smoothly, bio processors must use the most appropriate tools and techniques. Here, we talked to some experts about what tools and techniques can help bio processors overcome difficult challenges. Some of these experts also cite new approaches that can help bio processors meet new challenges.
Hundreds of monoclonal antibodies (mAbs) are currently on the market or under development.1 with the prominence of mAbs as therapeutic agents, many companies have several antibodies in their development pipeline. These molecules are often derived from a common structure and have high homology and therefore similar physicochemical properties. This allows similar purification processes to be used when recycling different products, creating a platform process that can be used to purify similar molecules with minimal process changes.
The purification process must yield products suitable for human consumption, reliable and predictable. Impurities such as host protein, DNA, additional and endogenous viruses, endotoxins, aggregates, and other species must be removed, maintaining an acceptable yield. Furthermore, impurities introduced during the purification process must also be removed. These include protein-a-percose, resin and filter extract, process buffers, and agents such as detergents that may have been used for virus reduction.
Primary Recovery Process
The first unitary operation in a downstream process is the removal of cells and debris from the culture broth and clarification of the cell culture supernatant containing the antibody product. Given the high cell densities that can be achieved in mammalian cell cultures and microbial processes, a primary repair can be a challenge at the experimental, laboratory, and commercial production scales.
The current trend for cell culture processes is to increase productivity through the use of enriched culture media, improve cell productivity and increase cell mass. The high titer is also achieved in many cases by extending the culture time, although this can lead to a significant decrease in cell viability. These factors lead to an increase in the levels of process impurities such as host cell proteins, nucleic acids, lipids, colloids, and the generation of a wide particle size distribution in the cell culture fluid (CCF). The currently preferred process for carrying out this initial recovery is to use continuous stack disc centrifugation along with deep filtration.
Tangential flow micro filtration.
Tangential microfiltration has been successfully implemented for the collection of mammalian cells since the early days of biotechnology. Here, the CCF flows tangentially to the microporous membrane and the pressure-actuated flow of filtrate separates the soluble product from the larger insoluble cells. Membrane fouling is limited by inertial gain and shear-induced diffusion generated by laminar flow across the membrane surface.
A high-yielding crop is achieved through a series of concentration and diafiltration steps. In the first case, the volume of the CCF is reduced, concentrating the solid mass. The diafiltration step then rinses the product from the concentrated TLC mixture.
Centrifugation.
Centrifugation, along with deep filtration, has been used for the initial recovery of therapeutic proteins for many years and is suitable for experimental and commercial-scale production. Continuous disk stack centrifuges can remove cells and large cell debris; however, cells can be disrupted in the process, especially if the starting material is a culture fluid with low viability. Many submicron-sized particles cannot be removed in the centrifuge, increasing the load on subsequent depth filtration.