Discussing midstream as the gap-filling technology between up- and downstream and alluvial filtration as the solution for efficient cell removal in just one step, this article shows how efficiency in cell removal is increased in a singleuse format which also allows for easy scale-up.
The removal of cells and cell debris takes place between fermentation (upstream) and product purification (downstream) and is referred to as midstream. The midstream process very often involves a combination of several operation units1. A highly efficient method for this is alluvial filtration (cake filtration) and can be done with FILTRODISC™ BIO SD.
Continuous process optimisation is a key factor in the bioprocessing industry. With higher and higher particle loads (>108 cells/ml), standard technologies, e.g. centrifugation, separation, membrane- and depth filtration, reach their limits. Especially mammalian cells are sensitive to breakage during clarification with high shear stress (centrifugation, separation). This results in the release of host cell proteins which can have an influence Single-Use Method Using Filter Aid Easy Removal of Midstream Cells on product stability and purity and leads to additional purification steps2. The following method describes the clarification of fermentation broths with alluvial filtration. This technology leads to a maximum product yield and highest economic efficiency.
The industry is divided in their opinion about where to put the line between upand downstream. The clarification of fermentation broths is very often treated as the stepchild of bioprocessing and assigned to either up- or downstream. Due to the importance of the clarification step within the whole process, the link between up- and downstream is called midstream (figure1).
Midstream, the clarification of fermentation broths, is the most important step in bioprocesses (figure 1). Meanwhile, cell cultures are the most important systems to produce therapeutics and diagnostics. For this purpose, the use of mammalian cells is predominant, but also bacteria, yeast and insect cells are used. Involved in the process design for the right cell removal system are questions about: process efficiency, process robustness, economic feasibility, as well as legal aspects. Challenges in process efficiency are higher and higher cell titers, amount of cell debris, scalability, robustness and flexibility in terms of process changes and future process adaptations and process optimisations. The industry asks for more efficient and more economic methods.
Alluvial filtration (cake filtration) is a well-established and economical type of depth filtration. The pharmaceutical industry has been relying on this method for decades (e.g. plasma fractionation). Instead of using just a static depth filter medium, filter aid (e.g. diatomaceous earth, DE) is added to constantly build up a filter cake during filtration. The filter cake with its resistance acts then as the actual filter medium. Alluvial filtration as a dynamic type of depth filtration, therefore, leads to a higher filter capacity3—especially with compressible particles, e.g. microbial or mammalian cells–and will extend the life cycle of subsequent sterilizing filter membranes with accomplishment of batch filtration in a much quicker and efficient manner4.
Diatomaceous earth is like the Swiss army knife—the all-purpose tool for downstream processing. (David Delvaille, MerckSerono France5)
Throughout the filtration, the filter aid particles (for e.g. diatomites) are deposited alongside the compressible solids ( for e.g. cells; figure2, b). Due to the physical properties of the filter aid particles, the permeability of the cake is sustained throughout filtration despite the compressible debris and cells. Thereby, a capacity for extraordinary particle loads will be generated5. The filtration time - and therefore the filtered volume per square meter of filter area - can be increased up to 4-fold compared to standard depth filtration (figure 3).
FILTRODSIC™ BIO SD is the first microfiltration system, which combines the advantages of standard depth filters with alluvial filtration in a singleuse system, resulting in new possibilities for midstream and downstream. Depth filters are also known for removing host cell proteins (HCP), DNA, viruses, and endotoxins3. Instead of a two-step cell removal system with centrifuges or acoustic separators as a first step and depth filters as a second step, just one step is sufficient to remove cells, microorganisms and cell debris out of a fermentation broth. The centrifugation or acoustic separation step can be completely eliminated.
FILTRODISC™ BIO SD provides solutions from process development to production scale with a simple linear scale-up (figure 4.). Therefore, a feasible filtration optimisation and a scale-up are very simple. The cake volume per litre of filtered liquid, which was determined and optimised during lab trials, is directly proportional to the cake volumes with the larger sizes of filter modules. This is also shown in the following formula:
C_P = (V_P ×C_L) / V_L
C =h ×A
C: cake volume [m3]
V: filtered volume [L]
P: production scale
h: cake height [m]
A: filter area [m2]
Scale-up calculation for alluvial filtration does not directly include the filter area, as it is dependent on the available space for the filter cake which is the important parameter in this case. Besides cells and cell debris, the FILTRODISC™ BIO SD system can remove impurities (for e.g. DNA or HCP), resulting in cost reduction for the following chromatography steps in downstream purification. A change in pH and the addition of flocculants are not necessary with this technology.
The use of alluvial filtration in midstream processing is one of the most effective, efficient, robust and easy to use methods for cell removal. Diatomite filter aids are suitable for the use in cGMP pharmaceutical processing environment6. FILTRODISC™ BIO SD provides a state of the art single-use technology for midstream processing in just one step.
1] Process Scale Bioseparations for Biopharmaceutical Industry, Chapter One: Harvest of Therapeutic Protein Product; Elisabeth Russell, Alice Wang, and Anurag S. Rathore; Taylor & Friends Group, 2007
2] Mammalian Cell Culture Clarification: A Case Study Using Chimeric Anti-Cea Monoclonal Antibodies: Mohamed Ali Abol Hassan, Abdul Wahab Mohammad, and Badarulhisam Abdul Rahman, ILUM Engineering Journal, Vol. 12, No. 4, 2011
3] Dynamic Depth-Filtration: Proof of Principle; W.E. Hurst; Technical Note AMC06; Advanced Minerals
4] Technical Bulletin, Disposable Body Feed System DBF, ManCel Associates, May 2008
5] Filtration Improvements Yield Many Benefits Down the Line, Susan Aldrige, Genetic Engineering & Biotechnology News, Vol. 30, No. 21, 2010
6] Advances in Disposable Diatomite Filter Aid Systems for cGMP Bioseparations, T. Sulpizio and J. Taniguchi, AFSS Annual Meeting, May 2008