Removal of viruses from human intravenous immune globulin by 35 nm nanofiltration.

Viral safety is an important prerequisite for clinical immunoglobulin preparations. A common manufacturing practice is to utilize several virus removal/inactivation process steps to ensure the safety of human intravenous immunoglobulin (IVIg). In this regard, we examined the use of Planova 35 nm filters to reduce potential loads of both non-enveloped and enveloped viruses prior to end-stage solvent detergent treatment. The nanofiltration process was validated for removal of a variety of enveloped and non-enveloped viruses ranging in size from 70 nm to 18 nm including: Sindbis virus, Simian Virus 40 (SV40), Bovine Viral Diarrhoea virus (BVDV), Feline Calicivirus, Encephalomyocarditis virus (EMC), Hepatitis A virus (HAV), Bovine Parvovirus (BPV) and Porcine Parvovirus (PPV). The filtration procedure was carried out by first spiking a 7% solution of IVIg with < 10(8) virus. The spiked IVIg solution was then filtered through a 75 nm Planova filter followed by two Planova 35 nm filters in series (75/35/35). The 75 nm prefilter is incorporated into this process to increase the capacity of the 35 nm viral removal filters. As a result of the inclusion of the 75 nm pre-filtration step it was possible to assess the removal of virus by the 35 nm filters independent of possible aggregation of the initial viral spiking material. Samples were collected at each step and immediately titred by viral plaque assay. A process control sample of the spiked load solution was held at the same conditions for the duration of the filtration process and then titred to determine the extent to which antibody neutralization may have contributed to overall viral reduction. Control assays of spiked IVIg were performed to establish the degree of toxicity of the IVIg solution to the indicator cell lines and the extent to which the IVIg interfered with plaque formation in the assay system. This combined data was used to establish assay sensitivity for the calculation of log removal by the filtration process. It was noted that toxicity/interference effects could have a significant effect upon apparent log reductions, and these effects could vary greatly, even within viruses of the same family. The results of these studies indicate that 35 nm filtration is very effective for removing substantial quantities of both non-enveloped and enveloped viruses from IVIg. Complete clearance (to the limits of detection of the assay) was obtained for all viruses larger than 35 nm. Interestingly, viruses reported to have mean diameters of less than 35 nm (EMC and HAV) were at least partially removed by the filtration (4.3 and > 4.7 logs removal, respectively). Even small viruses such as PPV were to some extent removed from the IVIg solution by the filters (2.6 logs removal). Reduction of BPV would not be assessed due to extensive neutralization and interference with plaque formation by the IVIg. Sindbis and SV40 also were subject to neutralization and assay interference due to the IVIg, though to a lesser extent. We conclude from these studies that the 35 nm mean pore size is functionally efficient in removal of smaller size viruses from spiked IVIg concentrates.

Virus removal studies using nanofiltration membranes.

Nanofiltration of immunoglobulin products was investigated as a virus removal step using Planova-controlled pore size membranes. For these studies, purified preparations of an IgG and an IgM were tested with 15 nm and 35 nm Planovace nanofiltration membranes, respectively. The results of spiking studies with four model viruses indicated that both the Planova 15 and 35 membranes removed 6-7 log10 plaque or focus forming units of murine xenotropic retrovirus, simian virus 40 and pseudorabies virus. Although two tandem Planova 35 membranes were tested for clearance of poliovirus, only the Planova 15 membrane removed this virus. Nanofiltration experiments were carried out with protein concentrations up to 12 mg/ml for the IgG and 1-2 mg/ml for the IgM, and protein recovery was excellent.

Genetic stability and recombinant product consistency.

A committee of U.S. industry scientists from the Pharmaceutical Manufacturers Association has reviewed the recent suggestions of Galibert and Center for Biologics Evaluation and Research (CBER) regarding assurances of product consistency by cloning and sequencing efforts. We disagree that their proposals will achieve this goal, and estimate that such efforts will be very costly in terms of regulatory agency time examining artifactual errors as well as industry resources. We feel that current analytical and manufacturing technology is adequate to assure recombinant product consistency without the suggested cloning and sequencing measures.

Industrial experience with the detection of retroviruses.

An important aspect of the virological assessment of cell substrates used to produce biologicals is their retroviral status. In this presentation we will attempt to document some of the practical experiences of industry when testing cell substrates for retroviruses using infectivity tests, reverse transcriptase and electron microscopy. We will also explore some important aspects of the experimental design of these retrovirus assay systems and review some results from the application of these methods in model, experimental systems.

Assessment of environmental contamination associated with a mammalian cell transformation assay.

To estimate worker exposures to, and environmental contamination from, test chemicals and organic solvents used in an in vitro assay to assess the carcinogenic potential of chemicals, sodium fluorescein, a noncarcinogenic fluorescent material, was dissolved in tissue culture medium used to maintain early passage hamster embryo cells. Personal an environmental samples were taken over a 14-d period. The assay was performed according to standard procedures in a ventilated glove box or laminar flow safety cabinet. Considerably more than 99% of the chemical contamination found was recovered from the interiors of the glove box and hood and from disposable equipment. Contamination outside the containment units (less than 1 microgram) resulted from intralaboratory transport of chemicals, treated cultures, and contaminated equipment. We conclude that the standard operating particles and procedures provided adequate safeguards for personnel and the environment.

Design and testing of a biocontainment system for chemical carcinogens.

A biocontainment facility was built for studies in which the chemical carcinogen N-methyl-N-nitrosourea (MNU) was instilled intrarectally in guinea pigs. The system operated by constant flow of uncontaminated air into carcinogen-contaminated animal isolation chambers and filtration through a high-efficiency particulate air (HEPA) filter prior to release into the environment. The facility was tested for efficiency of carcinogen containment by substituting for the MNU a similar concentration of a fluorescent tracer, sodium fluorescein, under standard operating procedures for carcinogen administration to guinea pigs. Wipe samples from the floor, isolation chambers, animal handlers and clothing, and intake and exhaust air samples were analyzed for fluorescein before and after intrarectal instillation of the tracer. The recovery of very low concentrations of total and respirable suspended fluorescein from sampling points within the facility and the absence of detectable fluorescein in the air downstream from the HEPA filter indicated that the facility provided adequate protection against contamination of personnel or the environment.

Penetration of rat skin by N-nitrosodiethanolamine and N-nitrosomorpholine.

N-Nitrosomorpholine (NMOR) and N-nitrosodiethanolamine (NDELA) were painted on the clipped upper dorsal skin of male F344 rats. NDELA was applied undiluted, dissolved in water, and dissolved in cutting oil; NMOR was applied dissolved in water and in ethyl acetate. Aqueous solutions of the nitrosamines were used for gavage. Rats were housed individually. Blood and urine samples were analyzed for nitrosamines by chromatography combined with a Thermal Energy Analyzer. Maximum penetration of NMOR was approximately equal to 34% 2 hours after application of 5 mg to the skin or by gavage; less than 1% appeared in the urine in 24 hours. Skin painting with NDELA in water (20 mg/100 microliters) and in cutting oil (25 mg/25 microliters) yielded small concentrations of NDELA (always < 25 micrograms/ml blood). When 50 mg of undiluted NDELA was painted on the skin, 130 to 220 micrograms/ml of blood was recovered after 1 hour. Administering 50 mg NDELA in water by gavage yielded similar blood concentrations. Maximum skin penetration observed with NDELA was 78% 1 hour after application of 50 mg. From 20 to 30% of the NDELA applied undiluted and by gavage appeared in the urine in 24 hours. Although animals and humans differ, skin exposure to NMOR or NDELA represents a risk due to absorption.

The penetration of rat skin by some nitrosamines of environmental importance.

N-Nitrosomorpholine (NMOR) and N-nitrosodiethanolamine (NDELA) were applied to the clipped dorsal skin (about 3.5 X 3.5 cm) of adult male Fischer 344 rats. NDELA was applied undiluted, dissolved in water and dissolved in cutting oil; NMOR was applied dissolved in water and in ethyl acetate. To compare the extent of absorption through the skin with that from the stomach, aqueous solutions of the nitrosamines were used for gavage. Blood and urine samples were analysed for nitrosamines, using GC-TEA or HPLC-TEA. Maximum skin penetration observed with NMOR was 56%, following application of 5 mg, whereas a similar proportion of the dose was regularly present after gavage of 5 mg. Less than 1% was recovered from the urine. Skin painting of NDELA in water (20 mg in 100 microliter) and in cutting oil (25 mg in 25 microliter) yielded small concentrations of NDELA in the blood; less than 25 mg/l in all cases. However, when 50 mg NDELA was painted on the skin undiluted, from 130 to 220 mg/l of blood were recovered after one hour. fifty mg NDELA in water yielded similar blood concentrations when administered by gavage. From 20 to 30% of the NDELA applied undiluted and by gavage were recovered in the urine. It is concluded that, although there are differences between animals, exposure to NMOR or NDELA represents a risk due to absorption through the skin.

Potential hazard associated with scraping preparative thin layer chromatography plates.

Scraping TLC plates in a hood leads to contamination of the hood, equipment, worker, and the environment. The magnitude of contamination is presented as a fraction of the chemical delivered, and the effects of plate thickness, dryness, and adsorbent material are examined. Frequent clean-up and decontamination are recommended.

Sources and dissemination of contamination in material handling operations.

To assess the potential risks to personnel preparing feed for carcinogen bioassay research, a tracer was mixed with a meal diet to yield 10 kg batches of 100, 3,000 and 5,890 ppm. Wipe samples were obtained from horizontal and vertical surfaces, equipment and personnel before operations began, after they were completed, and following clean-up. Total and respirable suspended particulate matter samples were obtained. All operations led to contamination of clothing and equipment; cleaning did not remove all contamination. These results, obtained in a well controlled environment in which trained and well protected personnel were working, suggest that a higher level of process control may be required for adequate protection of personnel performing material handling operations with known or suspected carcinogens.