Genetic Engineering & Biotechnology News

NOV1 2018

Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D community with critical information on the tools, technologies, and trends that drive the biotech industry.

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Page 26 of 57 | Genetic Engineering & Biotechnology News | NOVEMBER 1, 2018 | 25 Application Note ADVERTORIAL Outstanding cGMP Enzymatic Technology in Purification An Efficient Method to Remove Nucleic Acids in the Production of Biopharmaceuticals Pablo Domínguez de María, Stefan Schönert N ucleic acids, like DNA and RNA, are massively re- leased to the fermentation broth when biopharmaceu- ticals—antibodies, vaccines, proteins, etc.—are produced. The viscosity of the media is then significantly en- hanced, creating membrane fouling, and leading to a cumbersome and un- predictable downstream, particularly in steps depending on fluidity, such as filtrations, centrifugation, or chroma- tography. Furthermore, nucleic acids may adhere to particles like viruses or inclusion bodies, hampering the puri- fication of such particles. Importantly, safety regulations are strict for the biopharmaceuticals in- dustry, and products to be marketed must be virtually free of nucleic acids (which are considered contaminants). Therefore, integrating nucleic acid re- moval is a challenging step, yet essen- tial for the product quality and safety criteria during the process develop- ment phase. To remove nucleic acids from biological samples, successful strategies have traditionally included chemical means, such as precipitation (using acid/base or solvent treatments), filtration (using adsorptive depth filters or tangential flow filtration), sonica- tion, or chromatographic methods, among others. These technologies often require con- ditions that can lower the yield and purity of the desired products. To tackle that challenge, regulatory authorities recommend enzymatic steps to remove nucleic acids. In this context, unselective nucleases hydrolyse nucleic acids by cleaving the phosphodiester bonds, ren- dering small oligonucleotides of several base pairs under mild conditions. To add value in this area, a genetically en- gineered form of Serratia marcescens endo- nuclease—called DENARASE ® —has been recently launched by c-LEcta (Leipzig, Ger- many). The patented production microor- ganism is a Gram-positive Bacillus sp., which is known to be an endotoxin-free strain. Moreover, the employed fermentation media is free of animal-derived feedstocks and anti- biotics, and therefore DENARASE is a BSE/ TSE-free product, with a high viral safety (as no animal-derived materials are involved in any step of its production). Remarkably, DENARASE is now avail- able at high quantities and purity, with an excellent quality-price ratio. This is due to the new production method, as Bacil- lus sp. secretes the enzyme extracellularly and is free of endotoxins. Both aspects sig- nificantly reduce costs in downstream pro- cessing. Notably, based on the production system, DENARASE manufacturing pro- cess is in full compliance with the cGMP requirements, something compulsory for red biotechnology. The biochemical features of DENARASE are outstanding and enable its use in a broad array of processing conditions. Thus, DENARASE accepts a broad substrate range, cleaving all forms of nucleic acids, RNA and DNA, single- and double-stranded, as well as linear or circular sequences. The final prod- ucts achieved are oligonucleotide fragments of 2–5 base pairs. Likewise, DENARASE is active along a wide operational frame of temperature (0–42 ºC, optimum 37ºC) and pH (5.5–10, optimum 8–9). Moreover, it re- mains active upon the addition of deleterious agents like ionic, non-ionic, or chaotropic agents, or denaturing compounds like urea, and accepts high ionic strengths of different buffers (up to 150 mM), or high concentra- tions of KCl or NaCl (typical salts of fermen- tation broths) of up to 300 mM. Overall, these excellent features for DE- NARASE broaden the options for the nu- cleic acid removal and purification steps for the development of cell- and gene-therapy treatments, vaccines (e.g., AAV, lentiviruses, etc.), as well as many other applications in the bio/pharmaceutical industry, where the digestion/removal of nucleic acid residuals appears mandatory. As stated above, a relevant application of DENARASE is the DNA/RNA removal from products obtained in fermentative processes aiming at producing biopharma- ceuticals, enzymes, vaccines, or biological compounds following biosynthetic strate- gies. For the final manufacturing of these biopharmaceuticals, and to reach their ul- timate commercialization, regulatory au- thorities restrict the nucleic acid levels to less than 100 pg per dose (applied to the end-product sample). Once DENARASE has hydrolysed the nucleic acids (Figure), its removal from the final product can be achieved by dif- ferent chromatographic steps (namely, ionic exchange or hydrophobic interac- tion chromatography). Likewise, if there are differences between the desired product and DENARASE, technologies like cross- flow filtration or depth filtration can be chosen as well. Therefore, already established purification steps of the product can be used for the removal of DENARASE, when the enzyme is used at an early stage of the whole process. The successful removal can be demonstrated by a commercially available DENARASE-ELISA kit, developed by c-LEcta, which is now available for that specific validation. In summary, DENARASE can hydrolyse all types of nucleic acids, RNA or DNA, and displays a re- markably broad application range in the production of biopharmaceu- ticals. Under many different process- ing conditions, DENARASE keeps its unselective and excellent hydro- lytic performance. DENARASE is based on the re- combinant expression in an endo- toxin-free Gram-positive Bacillus sp. strain as microbial host, rendering a high purity of >99% at competitive prices. The fermentation procedure does not use animal-derived feedstocks, conferring DE- NARASE the consideration of BSE/TSE- free product, with a high viral safety. This uniqueness, in full compliance with the cGMP requirements, is enabling DENARA- SE to generate added value to biotechnologi- cal and biopharmaceutical applications, like nucleic acid removal, biosynthesis, vaccine production, cell-therapy, oncology, CAR-T cell development, etc. Beyond biopharmaceuticals, for food and feed products, there is another new de- rivative available at c-LEcta: The so-called NuCLEANase food-grade is a nuclease which is produced according to food and feed standards, being kosher- and halal-cer- tified. NuCLEANase is yet another excellent alternative of high quality and efficiency, ac- cessible at competitive price. Interested? DENARASE can be ordered directly from c-LEcta (, or within Europe, through its new distribu- tion partner, VWR (, a part of Avantor. The supply of DENARASE is always secured, as c-LEcta keeps up to 100 MU of enzyme on permanent stock for its customers. Importantly, users of other nucle- ases in the bio/pharma industry should as- sess DENARASE in their processes as well, to have it validated as a high quality second source of nuclease, just in case of breakage of stock of their main suppliers. n c-LEcta Dr. habil. Pablo Domínguez de María CEO, Sustainable Momentum dominguez@sustainable- Dr. Stefan Schönert Head of R&D Strain & Process Development, c-LEcta. Figure. Use of DENARASE® to remove excess amounts of nucleic acids generated during the production of biopharmaceuticals

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