Amicon filter

A sample of poly (2,2-diraethyl-4,7-dihydro-l,3-dioxepin-alt-maleic anhydride) polymers was fractionated and briefly examined for antiviral properties (31). The copolymer was fractionated through Amicon filters into a 1,000-10,000 and 10,000-30,000 molecular weight cuts. Both molecular weight fractions were examined for effect against mice inoculated (ip) with 106 Ehrlich Ascites tumor cells. Five days after inoculation the animals were sacrificed and total peritoneal exudate cells were counted with a hemo-cytometer. Under these conditions, Ottenbrite (31) showed that the 1,000-10,000 molecular weight fraction of the ID-MA copolymer was as effective as Pyran (control in experiment) for control of Ehrlich Ascites tumor cells. Pyran, the copolymer of divinyl ether-MA is a well known antitumor agent (32) and interferon inducer (33). 

Buffer Proteins to be microinjected were dialyzed overnight into 2 mM Tris-acetate, pH 7.0, or a buffer compatible with both the cell (e.g., low calcium, low buffering capacity, near-neutral pH, limited salt and magnesium, nontoxic) and the protein. Collodion bags were used for the dialysis of small volumes and for the concentration of protein solution by vacuum dialysis. Amicon filters were used for concentration of some proteins. The solution was clarified by centrifugation at 25,000 rpm for 20 min in a Beckman type 42.2 Ti rotor. 

For total serum AGE fluorescence or pentosidine fluorescence, serum was diluted 1/100 in PBS, for low molecular weight AGEs, serum was diluted 1/2 with PBS, ultra-filtered through 10 kDa cut-off Amicon filters in a refrigerated centrifuge at 4 C and 4,000 g for 8 h and read without further dilution as previously described... 

Table 1 Clinical characteristics and serum AGEs in neonates and adult controls. Data are presented as mean SD and range. AGE fluorescence was measured as described in Methods. Low molecular weight serum fractions were obtained by ultrafiltration using 10 kDa Amicon filters as described in Methods. Significant differences between cord blood and adult control values are shown in the right column.

Dehydrase activity was monitored and the fractions exhibiting a high activity were pooled and concentrated on an Amicon filter (YM 30) under low pressure at 4°C. 

Figure 5. Anlytical HPLC separation of 300 pg venom peptides (A) and 10 ml conditioned water (B) from Conus textile. Details on preparation of the substances are given in the legend to Fig. 4. Separations were performed on an analytical CIS reverse phase column (Vydac wide pore, 4.6 x 250 mm, 5 pm particle size) at a flow rate of 0.5 ml/min. Substances were loaded on the column in aqueous 0.1% tri-fluoroacetic acid (TFA) and eluted with a linear gradient of 0-60% acetonitrile in 0.1% aqueous TFA in 0-60 minutes. On-line detection and spectral analysis was performed with a Hewlett-Packard diode array detector. The spectrum of the main peak obtained from the CW (B) is not identical to those of any of the venom derived peptides (A) that are eluted at similar times from the column (not shown). Attempts to isolate the active component(s) of Conus textile CW on reverse phase cartridge columns and Amicon filters were not successful, due to loss of the biological activity.

The sampling of solution for activity measurement is carried out by filtration with 0.22 pm Millex filter (Millipore Co.) which is encapsuled and attached to a syringe for handy operation. The randomly selected filtrates are further passed through Amicon Centriflo membrane filter (CF-25) of 2 nm pore size. The activities measured for the filtrates from the two different pore sizes are observed to be identical within experimental error. Activities are measured by a liquid scintillation counter. For each sample solution, triplicate samplings and activity measurements are undertaken and the average of three values is used for calculation. Absorption spectra of experimental solutions are measured using a Beckman UV 5260 spectrophotometer for the analysis of oxidation states of dissolved Pu ions. 

Fractionation of the Sediment Extract. Ultrafiltration methods (17) have produced some useful data about the nominal molecular size of the active component. Crude sediment extract was filtered (Amicon, 50 psi) and separated into three fractions having the following nominal molecular fractions <10,000, <2,000, <500. The fractions were tested for hydrilla-inhibition, and the two lower M.W. fractions proved to be inactive against the plant. The fraction having a M.W. greater than 2,000 but less than 10,000 showed inhibitory action somewhat enhanced over that of the crude extract (12). 

Pool fractions containing PPO and concentrate to 5 mL using YM 10 Amicon ultrafiltration filters. 

The material eluted was loaded onto the column once more subsequently, the column was washed with 350 mL of 20 mM KH2PO4 (pH 7.4) and then with 350 mL of 20 him potassium phosphate buffer (pH 7.4) supplemented with 1 mM imidazole. PAMO-P3 was eluted with 100 mL of 50 mM tris-HCl (pH 7.4) containing 200 mM imidazole. In total, 25 mL of yellow eluate was collected and concentrated to 12.5 mL by centrifuge filters (Amicon, MWCO 10000). 

Prq[iaration of clarified digests sludge. Samples were removed under anaerobic conditions and moved immediately into the anaerobic chamber. Before analysis, the samples were clarified by centrifugation and filtration with 0.22-jLtm Acrodisc filters. Concentrated digester supernatant was also examined, and it was prepared by ultrafiltration in the anaerobic chamber with an Amicon stirred cell ultraconcentrator and Amicon PMIO membranes. 

Step II Diafiltration. Extracts to be fractionated by HPLC were diafiltered into buffer A using Centricon 10 microconcentrators (Amicon Div., Grace Co., Danvers, MA) and then passed through 0.22 jim filters. 

Purification of Cationized Lignin. The purification was carried out either by ultrafiltration of the neutralized product mixture through a Diaflo UM2 membrane (Amicon Corp., exclusion limit 1000 MW) or by slurrying a finely ground 1 g sample of dry crude cationic ether at room temperature in 160 mL 94% ethanol. The filtered powder was washed with 50 mL 94% ethanol and then with 10 mL diethyl ether and brought to a constant weight in a vacuum desiccator. The ultrafiltration was preferred when the nitrogen content of the purified cationic ether exceeded 3%. 

Amicon stirred concentrating cell (Series 8000, 50 or 200 mL) with a 10,000 Mr cutoff filter for concentration F(ab)2 and products... 

Centrifugation alone is not sufficient to separate Np solid material from the solution (j) ). Thus, the Np solution concentrations and oxidation state analyses were determined from aliquots filtered through approximately 1.8 nm pore-size Centriflo membrane cones (Amicon Corp., Lexington, MA). Each filter cone was pretreated with an initial aliquot of the suspension to be filtered. Tests using successive filtration confirmed that no significant amounts of Np were being sorbed by the filter cones. The solutions were alpha counted by liquid scintillation techniques. Major cations in the solutions were determined by inductively coupled plasma spectrometry (ICP) analyses. 

After 24 h and 48 h of hydrolysis, the hydrolysate was separated from the residue solids by centrifuging at 11,950y for 10 min. The supernatant was then filtered through a 10-kDa membrane at 4°C in a 160-mL Amicon ultrafiltration unit (Beverly, MA). Fresh buffer was added during the filtration to remove the soluble end products. The retained enzymes in the Amicon unit were added back to the solid residues, and fresh buffer was added to the initial volume without any additional enzymes. 

The postmicrosomal supernatant was filtered through Amicon membrane filters of varying pore size, and assayed for its effectiveness in the presence of retinol. These results are summarized in Table IV which shows that the enhancing factor has a molecular weight between 50,000 and 100,000 daltons. Finally, this factor is not precipitated from the soluble fraction by (NH )aSO in the range from 1.0 to 2.0 M. 

Different types of syringe filters were then evaluated to try to remove the problematic excipients. Figure 10.4c shows that the use of an Amicon Ultra-4 centrifugal filter (Millipore, Billerica, MA) removed the residual HPMC from the sample solution but did not remove the starch. The Amicon Ultra-4 filter, however, does introduce later eluting filter-related peaks that fortunately do not interfere with any components of interest. Starch was not removed by any of the filters tested. As a result, excipient placebo samples were used as part of the method. These placebo samples were extracted and injected onto the HPLC system to confirm which peaks in the fixed combination tablet samples were excipient-related and these peaks were not quantitated or reported as drug-related degradants. 

Figure 10.4 Representative HPLC chromatograms (a) excipient placebo for the high dose active IR portion of the bilayer tablet (b) excipient placebo for the low-dose active sustained release portion of the bilayer tablet (c) Amicon Ultra-4 filtered fixed combination tablet sample. Annotation 1 = low-dose active ingredient 2 = high-dose active ingredient = pregelatinized starch-related peak = HPMC-related peak = filter-related peaks.

Ultrafiltration YM2 filters, cutoff of 2 kDa (Amicon, Danvers, MA) are useful (see Note 8). 

Protein Production, Isolation, and Purification. The expression and purification of chicken lysozyme mutant proteins in yeast are performed as described by Malcolm et al. with the following modifications. The 50-ml minimal medium second seed yeast culture is used to inoculate a 2.8-liter Fembach flask containing 500 ml of 1% yeast extract/2% Bacto-peptone/ 8% glucose (w/v) medium and is then incubated for 7 - 9 days at 30°. Cells are harvested, washed twice with 60 ml of 0.5 M NaCl, and collected by centrifugation. The supernatants are pooled, diluted 5-fold with deionized water, and loaded onto a 20-ml column of CM Sepharose Fast Flow (Pharmacia, Piscataway, NJ) equilibrated with 0.1 M potassium phosphate, pH 6.24. The column is washed with the same buffer, and lysozyme is eluted with 0.5 M NaCl/0.1 M potassium phosphate, pH 6.24. Fractions are assayed by activity (decrease in A450 of Micrococcus lysodeikticus cell wall suspensions per minute). Fractions containing lysozyme are concentrated in Centricon-10 (Amicon, Danvers, MA) filter units, washed with 0.1 M potassium phosphate buffer, pH 6.24, and stored at 4°. The protein concentration is determined from e 1 = 26.4.15... 

The protein solution is concentrated and desalted against KP 6.2 in Centricon-10 filter units (Amicon, Danvers, MA) as directed by the manufacturer. 

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