Bioprocess: Robustness with Respect to Mycoplasma Species.

Capture bioprocessing unit operations were previously shown to clear or kill several log10 of a model mycoplasma Acholeplasma laidlawii in lab-scale spike/removal studies. Here, we confirm this observation with two additional mollicute species relevant to biotechnology products for human use: Mycoplasma orale and Mycoplasma arginini Clearance of M. orale and M. arginini from protein A column purification was similar to that seen with A. laidlawii, though some between cycle carryover was evident, especially for M. orale However, on-resin growth studies for all three species revealed that residual mycoplasma in a column slowly die off over time rather than expanding further. Solvent/detergent exposure completely inactivated M. arginini though detectable levels of M. orale remained. A small-scale model of a commercial low-pH hold step did inactivate live M. orale, but this inactivation required a lower pH set point and occurred with slower kinetics than previously seen with A. laidlawii Additionally, ultraviolet-C irradiation was shown to be effective for A. laidlawii and M. orale inactivation whereas virus-retentive filters for upstream and downstream processes, as expected, cleared A. laidlawii These data argue that M. orale and M. arginini overall would be largely cleared by early bioprocessing steps as shown previously for A. laidlawii, and that barrier technologies can effectively reduce the risk from media components. For some unit operations, M. orale and M. arginini may be hardier, and require more stringent processing or equipment cleaning conditions to assure effective mycoplasma reduction. By exploring how some of the failure modes in commercial antibody manufacturing processes can still eliminate mycoplasma burden, we demonstrate that required best practices assure biotechnology products will be safe for patients.

A consensus rating method for small virus-retentive filters. I. Method development.

Virus filters are membrane-based devices that remove large viruses (e.g., retroviruses) and/or small viruses (e.g., parvoviruses) from products by a size exclusion mechanism. In 2002, the Parenteral Drug Association (PDA) organized the PDA Virus Filter Task Force to develop a common nomenclature and a standardized test method for classifying and identifying viral-retentive filters. One goal of the task force was to develop a test method for small virus-retentive filters. Because small virus-retentive filters present unique technical challenges, the test method development process was guided by laboratory studies to determine critical variables such as choice of bacteriophage challenge, choice of model protein, filtration operating parameters, target log10 reduction value, and filtration endpoint definition. Based on filtration, DLS, electrospray differential mobility analysis, and polymerase chain reaction studies, a final rating based on retention of bacteriophage PP7 was chosen by the PDA Virus Filter Task Force. The detailed final consensus filter method was published in the 2008 update of PDA Technical Report 41. Virus Filtration.

A consensus rating method for small virus-retentive filters. II. Method evaluation.

Virus filters are membrane-based devices that remove large viruses (e.g., retroviruses) and/or small viruses (e.g., parvoviruses) from products by a size exclusion mechanism. In 2002, the Parenteral Drug Association (PDA) organized the PDA Virus Filter Task Force to develop a common nomenclature and a standardized test method for classifying and identifying viral-retentive filters. A test method based on bacteriophage PP7 retention was chosen based on developmental studies. The detailed final consensus filter method is published in the 2008 update of PDA Technical Report 41: Virus Filtration. Here, we evaluate the method and find it to be acceptable for testing scaled-down models of small virus-retentive filters from four manufacturers. Three consecutive lots of five filter types were tested (Pegasus SV4, Viresolve NFP, Planova 20N and 15N, Virosart CPV). Each passed the criteria specified in the test method (i.e., >4 log10 PP7 retention, >90% intravenous immunoglobulin passage, and passing integrity/installation testing) and was classified as PP7-LRV4.

Removal of prion challenge from an immune globulin preparation by use of a size-exclusion filter.

BACKGROUND: A size-exclusion filter (Viresolve 180, Millipore Corp.) was tested for its ability to remove transmissible spongiform encephalopathies prion protein from an immune globulin preparation during ultrafiltration. STUDY DESIGN AND METHODS: Hamster-adapted 263K scrapie brain homogenate (SBH) was spiked into Rh0(D) immune globulin (human) at 1 in 300 and 1 in 1000 dilutions. Before spiking, the SBH was treated with detergent, sonicated, and filtered through serial 0.45-, 0.22-, and 0.1-microm filters to present a rigorous filter challenge. Process variables were monitored throughout the ultrafiltration to ensure that the spiked material did not compromise the membrane flux. Removal of scrapie prion protein (PrP(Sc)) material was determined by use of a sensitive Western blot assay. RESULTS: The turbid SBH became completely translucent after sonication and passage through the 0.45-, 0.22-, and 0.1-microm filters. The filtration of the immune globulin containing PrP(Sc) material was more difficult to perform than was filtration of immune globulin spiked with the normal cellular isoform. Even during tangential flow filtration, the fibril material prevented the PrP(Sc)-spiked immune globulin from passing as readily through the filter. Western blot results indicated a removal of greater than or equal to 2.5 log PrP(Sc), while remaining within the normal filtration limits. CONCLUSIONS: The composition, physical condition, and the amount of SBH introduced have significant effects on the filtration of the immune globulin and the log removal values obtained. By use of a detergent-treated, sonicated, and filtered preparation of SBH, it was demonstrated that the Viresolve 180 effectively removes PrP(Sc) from the immune globulin.