Lysozyme separation by hollow-fibre ultrafiltration

This paper discusses the purification of lysozyme from chicken egg white using hollow-fibre ultrafiltration (30kDa MWCO, polysulphone membrane). Lysozyme is preferentially transmitted through the membrane while the membrane largely retains other egg white proteins. Improvement in system hydrodynamics resulted in an increase in permeate flux while lysozyme transmission remained unaffected, leading to higher productivity. The percentage purity of lysozyme obtained was generally insensitive to system hydrodynamics. The permeate flux and productivity increased with increase in transmembrane pressure (TMP) before levelling off around 0.7bar. However, the TMP did not have any pronounced effect on the transmission and the purity of lysozyme. Experiments carried out in the diafiltration mode showed that moderately pure lysozyme (80-90%) could be obtained in an extended operation.     

Fractionation of bovine serum albumin and monoclonal antibody alemtuzumab using carrier phase ultrafiltration

Protein transmission and hence selectivity of separation can be significantly affected by solution pH and ionic strength in protein fractionation using ultrafiltration. Using parameter scanning ultrafiltration, the transmission of bovine serum albumin (BSA) and monoclonal antibody alemtuzumab (Campath-1H) through 300 kDa polyethersulfone (PES) ultrafiltration membranes were studied over a range of pH and salt concentrations, with focus on the likely conditions for achieving "reverse selectivity," i.e., obtaining purified alemtuzumab (approximately 155 kDa) in the permeate. Experimental results demonstrate that the reverse selectivity could be obtained by manipulating the operating conditions such as the solution pH, ionic strength, permeate flux, and system hydrodynamics. With a two-stage batch ultrafiltration process under suitable conditions, the monoclonal antibody alemtuzumab with a purity of > 98% was obtained in the permeate from a feed solution initially containing 0.50 g/l each of BSA and alemtuzumab. Further purity can be expected by selecting more suitable membranes and optimizing operating conditions.     

Separation of proteases from yellowfin tuna spleen by ultrafiltration

Separation of protease, trypsin and chymotrypsin from yellowfin tuna spleen extract by ultrafiltration (UF) using regenerated cellulose membranes with molecular weight cut off (MWCO) 30 and 100 kDa was studied. The 100 kDa membrane had a higher transmission of enzymes than that of the 30 kDa membrane. The enzyme transmission varied from 0.01 to 0.18 and from 0.6 to 0.8 for the 30 kDa membrane and 100 kDa membrane, respectively. The protein transmission was about 0.8 for both membranes. Increasing cross-flow rate and transmembrane pressure (TMP) increased permeate flux. The limiting fluxes at cross-flow rate 120, 240 and 360 L/h for the 30 kDa membrane were 17.3, 43.9 and 54.7 L/m2h, respectively and the limiting fluxes at the same flow rate for 100 kDa membrane were 34.1, 51.1 and 68.4 L/m2h, respectively. The separation of these proteases was achieved using the 30 kDa membrane. The purities of proteases were increased more than ten times at TMP 1.5 bar and cross-flow rate 360 L/h by diafiltration using 30 kDa membrane.     

A novel membrane based process to isolate photosystem-I membrane complex from spinach

The isolation of photosystem-I (PS-I) from spinach has been conducted using ultrafiltration with 300 kDa molecular weight cut-off polyethersulfone membranes. The effects of ultrafiltration operating conditions on PS-I activity were optimized using parameter scanning ultrafiltration. These conditions included solution pH, ionic strength, stirring speed, and permeate flux. The effects of detergent (Triton X-100 and n-dodecyl-beta-D-maltoside) concentration on time dependent activity of PS-I were also studied using an O₂ electrode. Under optimized conditions, the PS-I purity obtained in the retentate was about 84% and the activity recovery was greater than 94% after ultrafiltration. To our knowledge, this is the first report of the isolation of a membrane protein using ultrafiltration alone.     

Purification of densonucleosis virus by tangential flow ultrafiltration

Purification at commercial scale of viruses and virus vectors for gene therapy applications and viral vaccines is a major separations challenge. Tangential flow ultrafiltration has been developed for protein purification. Here tangential flow ultrafiltration of parvoviruses has been investigated. Because these virus particles are small (18-26 nm), removal of host cell proteins will be challenging. The results obtained here indicate that 30, 50, and 100 kDa membranes reject the virus particles, whereas 300 kDa membranes allow some virus particles to pass into the permeate. The decrease in permeate flux for the 300 kDa ultrafiltration membrane is much greater than for the 30, 50, and 100 kDa membranes, indicating possible entrapment of virus particle in the membrane pores. The permeate flux and level of protein rejection is strongly affected by the cell culture growth medium. The results indicate that when developing a new process, it is essential that the cell culture and purification operations be developed in parallel.     

Purification of monoclonal antibodies derived from transgenic goat milk by ultrafiltration

With the goal of recovering heterologous immunoglobulin (IgG), which comprises 10-15% of the total proteins, from transgenic goat milk at 80% yield and 80% purity, we have developed and tested a two-step membrane isolation and purification process. In the first step, reported earlier by Baruah and Belfort, microfiltration was used to fractionate the milk proteins and recover > 90% of the original IgG at a purity of about 15-20% in the permeate stream. Here, we focus on ultrafiltration (UF) to increase the purity of the target protein to 80%, while maintaining a relatively high IgG yield (80%). Tangential flow UF experiments in diafiltration mode were conducted with 100 kDa cellulosic membranes to evaluate the optimal pH, ionic strength, and uniform transmembrane pressure (TMP). The TMP was kept uniform by permeate circulation in co-flow mode. The traditional approach of conducting the UF process close to the pI of the predominant whey proteins (15-40 kDa, pI 5.2), to transmit these proteins while retaining heterologous IgG (155 kDa), could not be applied here because of precipitation of residual casein at pH values lower than 8.5. Instead, the packing characteristics of the cake layer on the membrane wall, as elucidated in the Aggregate Transport Model presented by Baruah et al. was utilized to achieve a selectivity of > 15, which was sufficient to meet the stated goals of purity and yield for this difficult separation. This combined process is expected to reduce the load on subsequent purification and polishing steps for eventual therapeutic use.     

Separation and purification of phycocyanin from Spirulina sp. using a membrane process

The highest purity ratio of phycocyanin extract was obtained when fresh biomass was used as raw material. The crude extract was purified by membrane process using microfiltration and ultrafiltration. Membrane of pore sizes 5 μm, at feed flow rate of 150 mL min⁻¹, permeate flux of 58.5 L h⁻¹ m⁻² was selected for coarse filtration and membrane with pore size 0.8/0.2 μm at the flow rate of 100 mL min⁻¹, permeate flux of 336 L h⁻¹ m⁻² was selected for fine filtration, giving phycocyanin recovery of 88.6% and 82.9%, respectively. For ultrafiltration, membrane with MWCO at 50 kDa, 69 kPa and 75 mL min⁻¹ of flow rate with a mean permeate flux 26.8 L h⁻¹ m⁻² and a retention rate of 99% was found to be optimal. Under these filtration conditions, food grade phycocyanin with the purity around 1.0 containing c-phycocyanin as the major component was obtained.     

Separation of monoclonal antibody alemtuzumab monomer and dimers using ultrafiltration

This article examines the feasibility of using ultrafiltration to separate the monomer of the monoclonal antibody alemtuzumab (Campath or Campath-1H) from a mixture of dimer and higher-order oligomers (collectively called "dimers" here). Using parameter scanning ultrafiltration, we initially assessed the suitability of the following membranes: 100 kDa and 300 kDa polyethersulfone (PES) membranes, and a 100 kDa polyvinylidene fluoride (PVDF) membrane. A detailed study was then carried out to examine the effects of operating conditions (such as solution pH, ionic strength, stirring speed, and permeate flux) on the separation of the monomer from the dimers using 300 kDa PES and 100 kDa PVDF membranes. Results of the experiments carried out in the carrier phase ultrafiltration (CPUF) mode indicate that the size-based protein-protein separation critically depends on the membrane used as well as the system hydrodynamics. The separation of the monoclonal antibody monomer and dimers using 100 kDa PVDF membranes in the diafiltration mode was also examined. Experimental results demonstrate that under suitable conditions, it is feasible to obtain the alemtuzumab monomer with a purity of more than 93% and a yield of more than 85% (from a mixture of 75% monomer and 25% dimers, which is the typical composition obtained after affinity chromatography). Simulation study indicates that this could be further improved to a purity of more than 96% and a monomer yield of more than 96% by increasing the selectivity of separation or by employing a two-stage diafiltration process.     

Enzyme recovery during gas/liquid two-phase flow microfiltration of enzyme/yeast mixtures

The effect of a gas/liquid two-phase flow on the recovery of an enzyme was evaluated and compared with standard crossflow operation when confronted with the microfiltration of a high-fouling yeast suspension. Ceramic tubular and flat sheet membranes were used. At constant feed concentration (permeate recycling) and transmembrane pressure, the results obtained with the tubular membrane were dependent on the two-phase flow pattern. In comparison with single-phase flow performances at the same liquid velocity, the enzyme transmission was maintained at a high level with a bubble flow pattern but it decreased by 70% with a slug flow, whatever the flow rate ratio. Identical results were obtained with flat sheet membranes: for the highest flow rate ratio, the enzyme transmission was reduced by 70% even though the permeate flux was improved by 240%. During diafiltration experiments with the tubular membrane, it was found that a bubble flow pattern led to a 13% higher enzyme recovery compared to single-phase flow conditions, whereas with a slug flow the enzyme recovery was strongly reduced. With bubble flow conditions, energy consumption was minimal, confirming that this flow pattern was the most suitable for enzyme recovery.     

Selective separation of cationic peptides from a tryptic hydrolysate of beta-lactoglobulin by electrofiltration

Electrofiltration (EF) was used to selectively separate cationic (basic) peptides contained in a tryptic beta-lactoglobulin (beta-LG) hydrolysate, with particular emphasis on the isolation of basic sequence beta-LG 142-148, which is a potential antihypertensive peptide. Both the influence of feed solution pH and operating parameters (transmembrane pressure, feed velocity) were assessed to find optimum conditions enabling the fractionation between peptides during EF. The cathode (-) was inserted in the permeate side to increase the separation of basic peptides contained in the tryptic beta-LG hydrolysate as compared to conventional NF. The highest separation factor between basic and neutral peptides was obtained at pH 9 using G-10 membrane with a molecular weight cut-off (MWCO) of 2,500 g/mol, at 5 V with the lowest transmembrane pressure (0.344 MPa) and feed velocity (0.047 m/s). The transmission behavior of the peptides during EF was better explained when taking into account the positive/negative charge ratio. Because of its 3+/1- charge ratio, beta-LG 142-148 had the highest transmission during EF. Consequently, its relative concentration was raised from 3.5% in the initial tryptic beta-LG hydrolysate up to 38% in the permeate. The electric field seemed more effective when the convective/shearing forces were minimized.