A novel, Q-PCR based approach to measuring endogenous retroviral clearance by capture protein A chromatography.

Quantification of virus removal by the purification process during production is required for clinical use of biopharmaceuticals. The current validation approach for virus removal by chromatography steps typically involves time-consuming spiking experiments with expensive model viruses at bench scale. Here we propose a novel, alternative approach that can be applied in at least one instance: evaluating retroviral clearance by protein A chromatography. Our strategy uses a quantitative PCR (Q-PCR) assay that quantifies the endogenous type C retrovirus-like particle genomes directly in production Chinese Hamster Ovary (CHO) cell culture harvests and protein A pools. This eliminates the need to perform spiking with model viruses, and measures the real virus from the process. Using this new approach, clearance values were obtained that was comparable to those from the old model-virus spike/removal approach. We tested the concept of design space for CHO retrovirus removal using samples from a protein A characterization study, where a wide range of chromatographic operating conditions were challenged, including load density, flow rate, wash, pooling, temperature, and resin life cycles. Little impact of these variables on CHO retrovirus clearance was found, arguing for implementation of the design space approach for viral clearance to support operational ranges and manufacturing excursions. The viral clearance results from Q-PCR were confirmed by an orthogonal quantitative product-enhanced reverse transcriptase (Q-PERT) assay that quantifies CHO retrovirus by their reverse transcriptase (RT) enzyme activity. Overall, our results demonstrate that protein A chromatography is a robust retrovirus removal step and CHO retrovirus removal can be directly measured at large scale using Q-PCR assays.

Robustness of virus removal by protein A chromatography is independent of media lifetime.

The robustness of virus clearance with respect to protein A media reuse was demonstrated using media with four matrix chemistries: Protein A immobilized ProSep A, Poros A50, Protein A ceramic Hyper DF and MabSelect SuRe, an alkali resistant protein A ligand. Endogenous retrovirus clearance, step yield, impurity clearance and other performance parameters were evaluated periodically in media cycled up to 300 times. Media lifetime was generally limited by either declining step yield or media fouling. However, clearance of endogenous retrovirus remained in an acceptable range, either increasing or remaining constant. Multiply cycled media were tested for clearance of three viruses (SV40, X-MuLV, and MMV); clearance was comparable to naïve media. Overall, virus clearance by protein A chromatography appears to be extremely robust with respect to media age.

Impact of multiple re-use of anion-exchange chromatography media on virus removal.

We evaluated viral clearance in multiply-cycled anion-exchange media run in flow-through mode. We found that anion-exchange columns do not lose viral clearance capacity after extensive re-use, if they are cleaned with recommended buffers that do not chemically degrade the media. In contrast, anion-exchange (AEX) columns that are not cleaned or are cleaned with buffers that chemically degrade the media lost viral clearance capacity after extended use. In these cases, other performance attributes that changed at the same time were increased band spreading, decreased DNA clearance and accumulating backpressure that prevented re-use past 80-120 cycles. Thus, our data suggests that flow through mode anion-exchange columns that are cleaned with recommended cleaning buffers, and periodically monitored for band spreading, DNA clearance and/or backpressure need not be re-evaluated for viral clearance at the end of the validated media lifetime.

Microbiological and aflatoxin evaluation of Brazil nut pods and the effects of unit processing operations.

Harvesting of Brazil nuts not only helps to preserve the Amazon rainforest but also provides income to individuals who would otherwise have little means of making a livelihood. Recently, the European Community has tightened the quality requirements for Brazil nuts, particularly with regard to aflatoxin levels and microbiological contamination. The objectives of this research were to gain a better understanding of the origin of aflatoxins on Brazil nuts and to microbiologically evaluate some of the operations involved in processing. In this regard, five Brazil nut pods were aseptically picked from trees located in each of three concessions of the Peruvian Amazon rainforest (Madre de Dios province). The exteriors of the pods and the nuts were examined for yeast and molds, including Aspergillus flavus and Aspergillus parasiticus, and for bacteria, including Salmonella and Escherichia coli. Brazil nuts obtained from various commercial process operations located in Peru were similarly evaluated. Exteriors of all Brazil nut pods did not contain A. parasiticus, and only pods from one concession yielded A. flavus isolates. All isolates tested were aflatoxigenic (630 to 915 ppb total aflatoxin). Coliforms, E. coli, and salmonellae were not recovered from any of the pods. Whole, in-shell nuts obtained after opening the pods yielded no A. flavus or A. parasiticus. Aflatoxins were not detected (detection limit 1.75 ppb) in any of the nuts. Whole, in-shell and shelled nuts from various process operations were all positive for A. flavus but negative for E. coli and salmonellae. Soaking of whole, in-shell nuts before cracking or shelling increased coliform numbers, whereas levels of A. flavus decreased. In order to gain a better understanding of the sanitary performance of the unit process operations, additional evaluations should be conducted on product lots processed on different days. Also, the microbiology of product processed from common lots should be followed through the various unit operations and compared.

Microbiological and Hydraulic Evaluation of Immersion Chilling for Poultry.

Immersion chilling has been identified as a critical control point in commercial poultry processing. A study was undertaken to investigate the impact of immersion chilling on the microbiology of carcasses within a small to medium sized commercial operation. Fifty chilled carcasses (following immersion chilling) sampled over a 10-day period using a whole bird rinse technique exhibited mean standard plate count (SPC) and coliform counts (log colony-forming units (CFU)/ml) of 3.74 and 3.03, respectively. These levels were both significantly (P < 0.005) lower (ca. 1 log unit) compared to similar numbers of prechill carcasses (birds exiting the inside-outside washer but prior to the prechiller). Water from the chiller was also shown to contain significantly (P < 0.005) lower (ca. 1 log unit) SPC and coliform levels compared to those from the prechiller. Reducing the flow of water at the inside-outside washer by 50% did not significantly (P < 0.001) affect the SPC and coliform levels of either prechilled or chilled carcasses.

Microbiological Evaluation of Vegetable Sprouts and Seeds.

Microbiological analyses of commercial sprout seeds indicated aerobic plate counts (APC) and confirmed coliforms ranging from < 30 × 102 to 40 × 103 and from 0 to > 11 × 103/g, respectively. Fecal coliforms were detected in the range from 7.3 to 11 × 102/g; Listeria or Salmonella were not detected. Seeds, pregerminated in potable water (16-18 h, 20-22°C), contained an APC ca. 1 logarithm higher than the corresponding dry seeds. Increased levels of confirmed and fecal coliforms were also detected as a result of soaking. The APC of retail sprouts ranged from 3.6 × 103 to 3.7 × 109/g. All samples contained confirmed and fecal coliforms. Coagulase positive staphylococci were detected in ca. 24% of samples analyzed.

Eugenol Induced Inhibition of Extracellular Enzyme Production by Bacillus subtilis.

Eugenol, the major essential oil of clove, in sublethal concentrations (0.02-0.03%, v/v) inhibited the production of alpha-amylase, protease, and subtilisin by Bacillus subtilis in laboratory media. Microscopic observations revealed that at these eugenol concentrations, B. subtilis cells appeared swollen and distorted and/or appeared as very long and thin filaments (> 100 µm). Of 20 amino acids investigated, only L-glutamic or L-aspartic acid (>5.0 mg/ml) prevented such morphogenic distortions when added to eugenol-containing media before inoculation. Addition of these amino acids also resulted in an increase in biomass and protease production. In contrast, the addition of serine (> 1.0 mg/ml) enhanced filamentous growth but reduced the production of protease and subtilisin.

Germination and Outgrowth of Bacillus subtilis Spores in the Presence of Selected Antioxidants.

Germination and outgrowth of Bacillus subtilis spores was investigated using laboratory media containing butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tertiary butylhydroquinone (TBHQ) and propyl gallate (PG). Although all antioxidants inhibited or retarded germination initiation and outgrowth, only BHA and TBHQ were effective at relatively low concentrations (150 and 100 ppm, respectively). Furthermore, BHA and TBHQ (150 ppm) were also shown to reduce spore growth by approximately 1 and 4 log10 within 72 and 6 h, respectively. The difference in the number of survivors between thermally (10 min at 80°C) and BHA (150 ppm)-treated germinated spores indicated that the antioxidant was effective against only a certain portion of the total heat-sensitive spore population. Sporostasis caused by BHA appeared reversible by the addition of Tween 80.

Antioxidant Activity of Selected Spices Used in Fermented Meat Sausage.

The antioxidant activity of ten spices commonly used in the formulation of a fermented meat sausage (Pastourma) were evaluated using a hemoglobin peroxidation procedure involving safflower oil in a water emulsion (10%). Clove followed by rose petals and allspice exhibited the highest antioxidant index when used in a dry form. In an aqueous-based microbiological broth, cloves again showed the highest antioxidant index followed by black pepper, ginger and rose petals. Generally anti-oxidant indices were higher in emulsions containing dry spice than in an aqueous based microbiological broth.