Evaluation of depth filtration to remove prion challenge from an immune globulin preparation.

BACKGROUND AND OBJECTIVES: Plasma-derived therapeutic proteins have the potential to contain transmissible spongiform encephalopathy (TSE) infectivity. This study evaluated the effectiveness and characterized the mechanism of abnormal prion protein removal during a depth-filtration step used in the manufacture of an immunoglobulin preparation. MATERIALS AND METHODS: Scrapie brain homogenate was treated with lysolecithin, sonicated and sequentially filtered through 0.45-, 0.22- and 0.1-microm membrane filters. The scrapie brain homogenate was then added (at a 1:51 dilution) to the Supernatant III fraction used in the manufacture of Rho(D) immune globulin (human). The spiked immunoglobulin preparation was then filtered through a depth filter under the same conditions used in full-scale production. After filtration, the depth filter was washed with hypertonic NaCl solutions to elute the abnormal prion protein (PrPSc) from the filter. A Western blot assay for PrPSc was used to quantify removal from the filtrate and recovery from the filter washes. A second run was performed whereby the PrPSc-spiked Supernatant III was filtered through a 0.22-microm membrane filter prior to depth filtration. A third run evaluated depth filtration of PrPSc in Tris-buffered saline (TBS). RESULTS: The depth filter removed greater than four logs of PrPSc from the Supernatant III filtrate. A significant portion of the PrPSc could be recovered from the depth filter by elution with high-molarity NaCl solutions. Prefiltration (through a 0.22-microm membrane filter) of the spiked Supernatant III prior to depth filtration removed all detectable PrPSc. Depth filtration removed less than one log of PrPSc from TBS. CONCLUSIONS: Depth filtration appears to remove PrPSc from the immunoglobulin preparation by mechanical straining rather than by adsorption to the filter matrix. The immunoglobulin preparation caused the PrPSc to aggregate from particles <0.1 microm in size to particles of >0.22 microm, probably as a result of the presence of methanol in the preparation. The depth filter failed to remove PrPSc from a purely aqueous environment.

Incorporation of an additional viral-clearance step into a human immunoglobulin manufacturing process.

BACKGROUND AND OBJECTIVES: Regulatory agencies have mandated that manufacturers of immunoglobulin products incorporate robust viral inactivation or removal steps into their purification processes. We evaluated the effectiveness of incorporating nanofiltration, a generic virus-clearance step, into an existing plasma-fractionation process for a human anti-D immunoglobulin product. MATERIALS AND METHODS: The nanofiltration process studied utilizes a 180 000-molecular weight composite membrane with well-defined pore distribution. To examine its viral-clearance capability, diluted anti-D immunoglobulin was spiked with high concentrations of human and animal model viruses and subjected to tangential-flow nanofiltration during scaled-down validation runs. Viral clearance by the membrane was determined by calculating log removal values in accordance with guidelines provided by US and European regulatory agencies. RESULTS: Nanofiltration removed viruses of varying sizes and physical characteristics. For the three non-enveloped viruses tested (porcine parvovirus, encephalomyocarditis virus and hepatitis A virus, sizes 18-30 nm), clearance was 3.3, 4.1 and > 5.1 log, respectively. For the three enveloped viruses (human immunodeficiency virus-1, bovine viral diarrhoea virus and pseudorabies virus, 50-200 nm), a substantial 5-log reduction was demonstrated. Product potency, purity and stability were unaffected. CONCLUSION: Tangential-flow nanofiltration provides substantial virus-removal capabilities for immunoglobulin preparations.

Zeaxanthin ([3R,3'R]-beta, beta-carotene-3-3'diol) as a resonance Raman and visible absorption probe of membrane structure.

When zeaxanthin ([3R,3R']-beta, beta-carotene-3,3'diol) is inserted into phospholipid dispersions and the latter heated through their gel-liquid crystal phase transitions, large changes are noted in the resonance Raman and absorption spectra of the carotenoid molecule. By analogy with the data of Carey and co-workers (J. Raman Spectrosc. 6:282) who studied the aggregation of zeaxanthin in acetone-water solutions, it is suggested that the carotenoid aggregates in the phospholipid gel state while forming a monomer in liquid crystal phases. The alterations in both the visible absorption and resonance Raman data have been used to monitor phospholipid phase behavior in dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine, (DSPC) one-component systems and binary mixtures. The phase diagram obtained for the binary system, as constructed from visible absorption and resonance Raman data, is compared with that of Shimshick and McConnell (Biochemistry. 12:2351) obtained from electron spin resonance (ESR) studies. Although the agreement between absorption and ESR data is generally satisfactory, onset temperatures for phase separation at low DSPC mole fractions deduced from resonance Raman measurements are several degrees lower than those from the other methods. Nevertheless, the use of zeaxanthin as a resonance Raman and visible absorption probe behavior will be useful in some situations where ordinary Raman spectroscopic data cannot be obtained easily. The advantage of the resonance Raman approach is illustrated in a study of the phase behavior of a phospholipid extract of a cel- mutant of Neurospora crassa. A phase separation region is observed with onset and completion temperatures of -19 and -6 degrees C, respectively.