GMP grade diatomaceous earth: the Swiss Army Knife for purification of biotechnology products
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Diatomaceous earth in biotechnology is frequently associated with dirty powdery material and old fashioned technology. |
However, diatomaceous earth is present in depth filters used for cell clarification, filtration of precipitates or prefiltration before viral filters. Even if the name Diatomaceous earth does not appear in biotechnology processes, the product is present.
Diatomaceous earth, also known as diatomite or Kieselgur is the external skeleton of unicellular microalgae. Originally this skeleton is mostly composed of silica. Diatomaceous earth included in depth filters is first calcinated at 1000°C to unify the porosity and then it is washed with an acid solution1. A clean, porous material composed of 86-93% of silica is obtained1.
The silica has 2 forms in Diatomaceous earth: silanol (SiOH) and siloxane (Si2O)2. The silanol can be charged depending on the pH (pKa = 5.4) and the siloxane allow hydropobic interactions3.
Merck Serono Biodevelopment has observed contaminant removal (Host Cell Protein - HCP) with depth filters, which contain diatomaceous earth, on 2 different processes (and molecules) at different purification steps. |
In order to understand how depth filters remove impurities, MERCK SERONO BIODEVELOPMENT, studied the adsorptive properties of GMP grade diatomaceous earth (Celpure®, ChangeXplorer – Advanced Minerals). This grade is obtained by an additional impurity removal step prior to the calcination and more frequent tests (silica >98%).
 Figure 1 : Image des pores de Celpure® |
Thus, ionic interactions of the Celpure® were studied at different pH, different ionic strengths and different hydrophobic environments. This study was conducted on 2 different molecules: an Fc-fusion molecule and an antibody with or without cells.
Results:
The elimination of contaminants without cells was evaluated on harvest solutions: |
Celpure® enabled HCP removal in a broad range of conditions with the highest impurity removal through anionic interactions. The porosity and contact time was also evaluated but no significant impact on impurity removal was observed. A contact time lower than 1 minute of the Celpure® with harvests enables over half of the HCP content in optimal pH and conductivity conditions to be removed. These optimal conditions also removed 98% of the DNA. The optimal pH for contaminant removal was applied during the clarification step: slurry of Celpure® was added in-line to crude harvests before filtration:
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The use of Celpure® in optimized conditions reduces the filter surface required to perform clarification. As the Celpure® is inexpensive, the cost of the clarification step is also reduced.
In a similar way to previous experiments, the Celpure® enables the amount of HCP to be reduced even in the presence of cells.
Conclusions:
Diatomaceous earth is a major component in depth filters. Using GMP grade Diatomaceous earth by itself enables performance in terms of filtration and impurities removal at different stages of a biotechnology product purification process to be enhanced.
References:
1 - Comparing Conventional Diatomite and Celpure Filter Aids, Technical Note AMC02 Version 3.5 ; Advanced Minerals; 2002
2 - The hydroxyl species and acid sites on diatomite surface: a combined IR and Raman study; P. Yuan, D.Q. Wu, H.P. He, Z.Y. Lin ; Applied Surface Science 227 (2004) 30–39
3 - The removal of dyes from textile wastewater: a study of the physical characteristics and adsorption mechanisms of diatomaceous earth ; M.A. Al-Ghouti, M.A.M. Khraisheh*, S.J. Allen, M.N. Ahmad ; Journal of Environmental Management 69 (2003) 229–238
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Aurore Lahille - Merck Serono Biodevelopment |
