The Polishing Step
Membrane technology is an area where major improvements in technology are anticipated in the next few years. Amelie Raveneau, purification technologies application specialist, and Suma Ray, Ph.D., virus clearance technologies product manager, both at Sartorius Stedem Biotech, discussed current chromatographic technologies in the polishing step of antibody purification.
At this stage, the goal is to reduce host-cell protein impurities, DNA, and leached protein A to acceptable levels. Flow-through membrane chromatography appears to be the superior method because of its ease of handling, high flow-through rate, and single-use character.
Virus removal is a critical component of the polishing procedure, and here Bayer Technology Services’ (BTS) UVivatec device can be integrated into the viral-clearance step. The instrument, which is jointly manufactured and marketed by BTS and Sartorius Stedim Biotech, is a helical flow, single-use reactor with hydraulic design for uniform treatment.
It employs UVC irradiation, a shortwave, germicidal wavelength that acts by causing dimerization and nicks in the DNA. The 254 nm wavelength is effective at inactivating viruses without damaging the proteins of interest. The disposable modules are robust and scalable, up to 1,000 L/hour. According to the companies, these properties make it useful in a GMP environment for polishing media, plasma and blood products, vaccines, and therapeutic proteins.
The UVivatec component inactivates either nonenveloped or enveloped virus, and due to its viral-clearance mechanism can be used in addition to virus adsorption and removal through the use of Sartobind Q and Virosart CPV membranes.
Eckhard Flechsig, Ph.D., head of virus validation at Biotest, described his company’s work optimizing the process of viral removal. Despite the risk of transmission of blood-borne diseases, there are still numerous biological products that are obtained from human blood. Therefore, it is incumbent upon producers to reduce the risk of viral transmission to an absolute minimum.
While donor selection and PCR screening for selected viruses will eliminate a large portion of the potential contamination, a viral inaction and removal step is de rigueur. These may include filtration and chromatography, but UV inactivation has proven to be one of the most robust and cost effective.
Dr. Flechsig concurred with Drs. Raveneau and Ray that UVC inactivation at a wavelength of 254 nm brings about the requisite damage to nucleic acids without causing aggregation, alteration in S-H bonds, or other adverse effects on proteins. The maximum wavelength for energy absorption by proteins is a narrow window in the higher range of 280 nm. The UVivatec system is a continuous flow system, which operates by pumping the biological fluid through a hose spiraling around the irradiation source. In model experiments it was possible to define the dose required for a greater than 4-log reduction in viability, which was required for each individual virus.