Sensitive fluorometric assay for proteins: use of fluorescamine and membrane filters.

A membrane filter assay has been developed for the determination of proteins in the submicrogram range. Proteins are: (1) treated with fluorescamine in sodium borate buffer containing MgCl₂, (2) collected on membrane filters, and (3) desorbed from the filters by an acetone-sodium borate buffer. By measuring the fluorescence intensity of the extract, as little as 0.3 μg of protein can be determined in the presence of reactive low molecular weight substances which do not interfere because they are not retained by the filter.     

MEASUREMENTS OF PHYTOPLANKTON-PROTEIN CONTENT WITH THE HEATED BIURET-FOL1N ASSAY1,2

The heated biuret-Folin method for determining protein consistently measures 90% of the total nitrogen of filtered algae samples as protein-N without the need of mechanical disruption as long as the heating period in biuret is 100 min at 100 C. Data indicate this protein assay measures total protein on all species tried and for naturally occurring mixtures of species plus detritus. Dilute algal suspensions with as little as 0.05 μg-atom particulate protein N.liter ^-1 can he concentrated by fltration on glass fiber filters to 1.0 μg-atom particulate protein-N per filter, the optimal amount of sample for a 5 ml volume of biuret. The filtered algae samples can be stored for several weeks frozen before assaying, if necessary.     

Preparation of stable and solvent-free model membranes.

A method for the preparation of solvent-free, phospholipid-impregnated filters is described. Polycarbonate filters of 13 mm diameter, 5 μm thickness and 0.1 μm pore size were employed, and 20 to 48 nmol phospholipid per filter was incorporated. Passive permeation of polar substances across the filter was determined by a flow-dialysis procedure. The presence of phospholipid led to a decrease in passive permeability by 96 – 99%. Due to their size and stability, and a partially preferential lipid orientation, these phospholipid-impregnated filters may serve as an alternative type of model membrane.     

A rapid filtration assay for cAMP

The receptor-binding assay for cAMP was improved by using polyethylenimine-treated glass filters. A polyethylenimine-treated glass filter has high protein binding capacity. This high capacity allows an increase in the amount of protein per assay tube and the use of a crude preparation, such as a beef heart extract, as specific binding protein instead of a purified protein, which has been used in the classical filtration assays involving cellulose ester filters. Since the time required for the separation of the protein-cAMP complex and the free nucleotide can be shortened by the use of polyethylenimine-treated filters, the dissociation of the bound ligand during the separation procedure, which is a serious problem with other modified assay methods involving charcoal adsorption, is minimized. Filtration through polyethylenimine-treated glass filters also gives low blanks and prevents the loss of protein or ligand due to breakage of the filters, which is often observed with fragile cellulose ester membranes. In consequence, this simple and rapid filtration assay allows more accurate and reproducible determinations.     

Two swinging-bucket ultracentrifugal filters for receptor-binding assays

Centrifugal filters for SW 25.1 and 50.1 swinging-bucket ultracentrifuge rotors have been tested up to the maximum speeds allowed, 90,000 and 300,000g, respectively. The filters are 1 and 0.5 in. in diameter and accept standard 25-mm polycarbonate filter membranes. The filter membranes are both cup-shaped to prevent loss of particulates to the support materials of the filters. The filter for the SW 25.1 rotor can take 0.75 ml and that of the 50.1 head 0.5 ml. The fluid retained after centrifuging consists of the fluid on the filter membrane and in its pores and that retained by the material filtered. The calculated volume of the pores of the 0.2-μm filter was 0.46 μl. Total liquid retentions of about 0.8 μl have been achieved with both filters using a particulate concentration of 0.5 mg/ml.     

A rapid and mild procedure for the isolation of DNA from mammalian cells

A procedure is described for the isolation of DNA from mammalian cells using polycarbonate filters. This method results in high yields of large-molecular-weight DNA, which is essentially free from protein and RNA. The procedure is rapid (approximately 2 h), does not require organic solvents, and can isolate 1.5–140 μg DNA per filter without the addition of carrier. The isolation of DNA from aflatoxin B₁-treated cells by the filter method is described in order to illustrate its advantages for the preparation of DNA containing lesions of low chemical stability.     

Tangential flow filtration of haptoglobin

Haptoglobin (Hp) is a plasma glycoprotein that scavenges cell-free hemoglobin (Hb). Hp has various potential therapeutic applications, but it has been mainly studied for treatment of acute hemolytic conditions that can arise from situations such as massive blood transfusion, infusion of stored red blood cells, severe burns, trauma, sepsis, radiation injury, and others. Therefore, Hp may also be beneficial during chronic hemolytic disease states such as hereditary spherocytosis, nocturnal hemoglobinuria, sickle-cell anemia, and malaria. Various methods have been developed to purify Hp from plasma or plasma fractions. However, none of these methods have exploited the large molecular weight (MW) range distribution of Hp polymers to easily isolate Hp from other plasma proteins. The present study used tangential flow filtration (TFF) to isolate polymeric Hp from plasma proteins using human Fraction IV (FIV) as the starting material. After removal of insoluble material from a suspension of FIV paste, the protein mixture was clarified on a 0.2 μm hollow fiber (HF) TFF filter. The clarified protein solution was then bracketed based on protein MW using HF filters with MW cut-offs (MWCOs) of 750, 500, and 100 kDa. Using untreated FIV, the Hp purity of the main bracket was ~75% with a total Hb binding capacity (HbBC) yield of 1.2 g starting from 500 g of FIV paste. However, pretreatment of FIV with fumed silica to remove lipoproteins increased Hp purity to >95% with a HbBC yield of 1.7 g per 500 g of FIV. Taken together this study provides a novel and scalable method to purify Hp from plasma or plasma fractions.     

Control of antibody high and low molecular weight species by depth filtration-based cell culture harvesting

Depth filtration-based harvesting is widely used in mAb manufacturing to remove cell and process-related impurities. However, it has not been studied on control of product-related impurities, which are very critical for product quality. In this article, we studied the interactions of depth filter with high and low molecular weight species (HMWs and LMWs) for their direct removal from cell culture. The process parameters (filter, loading, temperature, and flux) were evaluated for adsorption of HMWs and LMWs by depth filters. The adsorption is significantly dependent on filter media and loading capacity and is mainly on the basis of hydrophobic interaction during harvesting. The HMW and LMW species were characterized as HMW1, HMW2, LMW1, and LMW2. The increasing binding from LMW2 to LMW1, HMW1, and HMW2 is correlated with their increasing hydrophobicity score. Adsorption using enriched HMW sample demonstrated similar total protein binding capacity (36–40 g/m²) between depth filters D0HC and X0HC. However, X0HC has stronger HMW binding than D0HC (71% vs 43% of bound protein), indicating more hydrophobic interaction in X0HC. HMW2 DBC on X0HC reached 12 g/m², similar to protein binding on hydrophobic interaction membrane adsorbers. Further study showed LMW can induce HMW formation. This study provides a critical understanding of HMW and LMW interaction with depth filters. The strategy of HMW and LMW control by depth filtration-based harvesting was implemented successfully in mAb manufacturing.     

Glass wool filters for concentrating waterborne viruses and agricultural zoonotic pathogens

The key first step in evaluating pathogen levels in suspected contaminated water is concentration. Concentration methods tend to be specific for a particular pathogen group, for example US Environmental Protection Agency Method 1623 for Giardia and Cryptosporidium, which means multiple methods are required if the sampling program is targeting more than one pathogen group. Another drawback of current methods is the equipment can be complicated and expensive, for example the VIRADEL method with the 1MDS cartridge filter for concentrating viruses. In this article we describe how to construct glass wool filters for concentrating waterborne pathogens. After filter elution, the concentrate is amenable to a second concentration step, such as centrifugation, followed by pathogen detection and enumeration by cultural or molecular methods. The filters have several advantages. Construction is easy and the filters can be built to any size for meeting specific sampling requirements. The filter parts are inexpensive, making it possible to collect a large number of samples without severely impacting a project budget. Large sample volumes (100s to 1,000s L) can be concentrated depending on the rate of clogging from sample turbidity. The filters are highly portable and with minimal equipment, such as a pump and flow meter, they can be implemented in the field for sampling finished drinking water, surface water, groundwater, and agricultural runoff. Lastly, glass wool filtration is effective for concentrating a variety of pathogen types so only one method is necessary. Here we report on filter effectiveness in concentrating waterborne human enterovirus, Salmonella enterica, Cryptosporidium parvum, and avian influenza virus.     

Stimulation of corneal differentiation by interaction between cell surface and extracellular matrix. I. Morphometric analysis of transfilter "induction".

The present study was undertaken to determine whether or not physical contact with the substratum is essential for the stimulatory effect of extracellular matrix (ECM) on corneal epithelial collagen synthesis. Previous studies showed that collagenous substrata stimulate isolated epithelia to produce three times as much collagen as they produce on noncollagenous substrate; killed collagenous substrata (e.g., lens capsule) are just as effective as living substrata (e.g., living lens) in promoting the production of new corneal stroma in vitro. In the experiments to be reported here, corneal epithelia were placed on one side of Nucleopore filters of different pore sizes and killed lens capsule on the other, with the expectation that contact of the reacting cells with the lens ECM should be limited by the number and size of the cell processes that can tranverse the pores. Transfilter cultures were grown for 24 h in [3H]proline-containing median and incorporation of isotope into hot trichloroacetic acid-soluble protein was used to measure corneal epithelial collagen production. Epithelial collagen synthesis increases directly as the size of the pores in the interposed filter increases and decreases as the thickness of the filter layer increases. Cell processes within Nucleopore filters were identified with the transmission electron microscope with difficulty; with the scanning electron microscope, however, the processes could easily be seen emerging from the undersurface of even 0.1-mum pore size filters. Morphometric techniques were used to show that cell surface area thus exposed to the underlying ECM is linearly correlated with enhancement of collagen synthesis. Epithelial cell processes did not pass through ultrathin (25-mum thick) 0.45-mum pore size Millipore filters nor did "induction" occur across them. The results are discussed in relation to current theories of embryonic tissue interaction.     

Design and responses of Butterworth and critically damped digital filters

For many years the Butterworth lowpass filter has been used to smooth many kinds of biomechanical data, despite the fact that it is underdamped and therefore overshoots and/or undershoots data during rapid transitions. A comparison of the conventional Butterworth filter with a critically damped filter shows that the critically damped filter not only removes the undershooting and overshooting, but has a superior rise time during rapid transitions. While analog filters always create phase distortion, both the critically damped and Butterworth filters can be modified to become zero-lag filters when the data are processed in both the forward and reverse directions. In such cases little improvement is realized by applying multiple passes. The Butterworth filter has superior ‘roll-off’ (attenuation of noise above the cutoff frequency) than the critically damped filter, but by increasing the number of passes of the critically damped filter the same ‘roll-off’ can be achieved. In summary, the critically damped filter was shown to have superior performance in the time domain than the Butterworth filter, but for data that need to be double differentiated (e.g. displacement data) the Butterworth filter may still be the better choice.     

On the Pharyngeal Feeding Filter of the Salp Pegea confoederata (Tunicata: Thaliacea)

In Pegea, scanning electron microscopy of what appear to be the least damaged portions of the filter shows that it has a regular rectangular mesh consisting of thick (100 nm) fibres at right angles to thinner (50 nm) fibres. The rectangular pores of the filter are around 3.3 × 0.57 μm. These measured values from filters that have suffered shrinkage (to an uncertain degree) during preparation are considered to indicate that the actual pore size in life is some 4.0 × 0.7 μm. The mucous-net feeding filter of salps differs from that of other tunicates since flow through it results from muscular activity. Calculations based on the estimated pore size and filtering rate suggest that during part of the filtering cycle, the pressure drop across the filter is considerably greater than that across other tunicate mucous-net filters.     

Testing and evaluation of off-gas filters for bioreactors by a new bacterial aerosol challenge test method (TBAC).

A TNO bacterial aerosol challenge (TBAC) filter test rig was developed for direct assessment of the effectiveness of bioreactor off-gas filters as an alternative to routinely applied indirect wet integrity testing (IT). This TBAC test rig is based on bacterial aerosol challenging with Pseudomonas diminuta and dual monitoring by laser particle counting (LPC) and Andersen microbial sampling (AMS) of viable cells. The TBAC filter test rig is able to reproduce the various conditions encountered in fermentation processes. In experiments with several filters from one class, it was demonstrated that some filters were actually penetrated by up to 3,000 viable cells per test, despite their approval by commercially available IT test equipment. Repetitive filter use, prolonged use, and autoclaving of filters resulted in an increase in pressure drop over the filter but improved the performance of leaking/deviant filters due to building up of a filter cake (this phenomenon was identified by electron microscopy). The integrity tests used were found inadequate for accurate assessment of filter quality. Certification of filter lots by random tests of commercially available off-gas filters using sensitive direct methods such as those presented here might be advisable, as not all filters purchased were of appropriate quality.     

Impact of virus preparation quality on parvovirus filter performance

Virus‐removal filtration technology is commonly used in the manufacturing process for biologics to remove potential viral contaminants. Virus‐removal filters designed for retaining parvovirus, one of the smallest mammalian viruses, are considered an industry standard as they can effectively remove broad ranges of viruses. It has long been observed that the performance of virus filters can be influenced by virus preparations used in the laboratory scale studies (PDA, 2010 ). However, it remains unclear exactly what quality attributes of virus preparations are critical or indicative of virus filter performance as measured by effectiveness of virus removal and filter capacity consistency. In an attempt to better understand the relationship between virus preparation and virus filter performance, we have systematically prepared and analyzed different grades of parvovirus with different purity levels and compared their performance profiles on Viresolve® Pro parvovirus filters using four different molecules. Virus preparations used in the studies were characterized using various methods to measure DNA and protein content as well as the hydrodynamic diameter of virus particles. Our results indicate that the performance of Viresolve® Pro filters can be significantly impacted depending on the purity of the virus preparations used in the spike and recovery studies. More importantly, we have demonstrated that the purity of virus preparations is directly correlated to the measurable biochemical and biophysical properties of the virus preparations such as DNA and protein content and monodispersal status, thus making it possible to significantly improve the consistency and predictability of the virus filter performance during process step validations. Biotechnol. Bioeng. 2013; 110: 229–239. © 2012 Wiley Periodicals, Inc.     

Integration of Planova filters in manufacturing processes of biologicals improve the virus safety effectively: A review of publicly available data

The capacity to remove viruses by Planova filters produced by Asahi Kasei, primarily by small virus-retentive filters, were compiled from data in peer-reviewed publications and, partly, publicly available data from presentations at conferences (Planova workshops). Data from more than 100 publications and presentations at conferences covering Planova filters were assessed. The data were grouped according to the different virus filters regarding mean pore sizes and viruses of different sizes for plasma and cell culture derived products. Planova 15N and 20N filters removed parvoviruses below the limit of detection of viruses in the filtrate in approx. 50% of all studies and mean LRFs (log reduction factors) for viruses detected in the filtrate were above 4, demonstrating effective parvovirus reduction. Parvovirus removal capacity increased for Planova BioEX filters as well as for 2 Planova 20N in series. Large viruses as retroviruses (e.g., HIV and MuLV), herpesviruses, flaviviruses and togaviruses were removed effectively by Planova 15N, 20N and BioEX filters and also by Planova 35N filters. Flow interruption, transmembrane pressure, volume and protein concentration per filter area had had no substantial impact on virus removal capacity at manufacturing specification. In conclusion, the incorporation of Planova filters in manufacturing processes of biologicals remove, depending on the filter pore size, small and large viruses from the feed stream reliably. This virus reduction step with an orthogonal mechanism integrated in the manufacturing processes of biologicals, based primarily on size exclusion of viruses, improves the virus safety of these biopharmaceutical products considerably.     

Microdetermination of protein not affected by the presence of various buffers, sucrose, ATP, and eluates from polysaccharide derivatives

Paper states standard and microdetermination of protein that is not affected by the presence of various substances including buffers, sucrose, ATP, eluates from columns of polysaccharide derivatives, etc. The method was originally based on Kihara's method (4), although the basic principle is somewhat different.The protein was stained with amido black under defined condition, filtered, and washed through a small membrane filter, and the filter was extracted with SDS-methanol. The color intensity was measured. The recovery of proteins was almost 100%. From 2 to 100 μg of protein could be successfully determined on a standard scale and from 1 to 20 μg on a microscale.     

SOME PROBLEMS INHERENT IN TRANSPORT STUDIES IN SYNAPTOSOMES

A technique utilizing a 30-place manifold has been developed to study synaptosomal transport; some problems associated with such studies have been identified and clarified. The time course of L-glutamic acid uptake has been used to test variations in experimental protocol. Synaptosomes apparently become increasingly labile with increased time of incubation. This is indicated by a drop in the curve of uptake vs time after 8–12 min. Ninety seven to 98% of the glutamate taken up from a 10^?6m solution is released by osmotic shock. Synaptosomes can be stored in 0.32 m ice-cold sucrose suspension for periods up to 50 min without decline in measured uptake. Storage for 3 h or more results in a very substantial decline in measured uptake. Neither the decline in measured uptake with time, nor the decline with storage, is prevented by increasing the osmolarity of the solutions used or by use of synaptosomes from the initial 1085 g supemate rather than after sedimentation and resuspension. Although prewarming synaptosomes at 30°C for 20 min prior to their use lessened or eliminated the decline following peak uptake, the difference between stored and non-stored synaptosomes was not improved. Uptake was also much less when synaptosomes were used from the first supernate or when warmed prior to their use. Storage of tissue prior to homogenization resulted in synaptosomes that gave minimal reductions in measured uptake. Washing synaptosomes after separation from incubation medium resulted in a variable loss of substrate radioactivity, depending on such variables as brand of filter, pore size, composition of wash solution, and temperature of wash solution. The results support the hypothesis that washing causes lysis of a portion of the synaptosomes. However, with Millipore filters (0.45 μm) and a 30°C Krebs-Henseleit wash solution, the loss caused by washing is minimized (about 15%). Measured uptake is found to depend on the type of filter used. Uptake is much greater with Millipore 0.45 pm filters than with Gelman 0.45 μm filters. Use of Nuclepore (0.4 μm) filters results in measured uptakes only about 5% of that when Millipore 0.45 μm filters are used. With Millipore filters, 0.30 μm pore size filters gave uptakes only 68% of that using 0.45 pm pore size filters.     

A rapid separation of the four major deoxynucleosides and deoxyinosine by high-pressure liquid cation-exchange chromatography

A simple, rapid, and sensitive method for the measurement of DNA, RNA, and protein synthesis in cell samples is presented. The method measures the amount of isotope incorporated into these macromolecules after cell collection on filter paper and washing with a methanol:chloroform:water mixture. Samples as low as 50 μg in size can be prepared in less than 30 sec for measurement of radioactivity.     

Microassay of proteins on membrane filter in the nanogram range using cycloheptaamylase-dansyl chloride complex.

A protein assay is described in which the sample is spotted on a Sartorius membrane filter and dansylated with cycloheptaamylose-dansyl chloride complex dissolved in aqueous urea solution. The fluorescence intensity of the spot on the membrane was directly measured by a scanning fluorometer. This assay is rapid, simple, highly reproducible, and linear from 0.05 to 4 μg of protein. Very dilute solutions of protein can also be determined by filtering off the sample on the membrane filter in the presence of 0.1 m MgCl₂ and dansylating the protein collected on the membrane. There is no interference either by commonly used reagents such as Tris, citrate, guanidine, and glycine, or by naturally occurring amines and amino acids.     

Method of membrane filtration for determining the sterility and microbial contamination of antibiotics]

During membrane filtration antibiotics belonging to different chemical groups are strictly absorbed on the filters. When the filters are put into liquid thioglycol medium, the residual amounts of the antibiotics on the filters did not prevent the growth of sensitive microflora experimentally added to the drug. When the filter was put onto solid nutrient medium, only resistant forms of the microbes grew as a rule on its surface, the amount of the grown microbes being 26--43 per cent of the added one. The sensitive microbes grew only in the amount of 0.3--1.3 per cent. Subsequently the residues of the antibiotic adsorbed on the filter inhibited the growth of the sensitive and partially resistant microflora.