Low Molecular Weight Cut-off Membrane Research Articles

Host cell protein platform assay development for therapeutic mAb bioprocessing using mammalian cells

Background Recombinant therapeutic proteins are usually produced by cell culture technology using genetically modified host cell lines. During the manufacturing process, a mixture of the protein of interest and host cell derived impurities, including host cell proteins (HCPs) and other process related impurities are produced. Those process related impurities will be cleared or minimized though the process by optimization of process purification. Residual HCPs in the final drug substance may affect the quality, safety and efficacy and may result in clinical adverse effects. HCPs are typically quantified using immunoassays such as enzyme-linked immunosorbent assay (ELISA). The development and the validation of those assays are really challenging mainly due to the wide variety of possible HCPs in products. Although generic ELISA kits are commercially available to quantify HCPs from different recombinant systems, a process specific assay is required before drug registration and commercialization for biologics. The reagents, polyclonal antibodies and HCP standards, need to be carefully prepared and characterized to ensure a correct quantitation of the HCPs in the final product. Here we describe the first step of the development of a production platformspecific HCP-ELISA assay for UCB’s biopharmaceuticals produced in a Chinese Hamster Ovary (CHO) cell line, i.e. the production of mock material containing the HCPs using a null cell line. Materials and methods 2L and 80L stirred tank bioreactors (Sartorius) were run for 14 days in a fed-batch mode in a chemically defined medium. Feed was added daily from day 3 onwards. If required, antifoam was added to the bioreactor by manual injections. Dissolved Oxygen (DO), pH, and temperature were controlled at set points. DO was controlled using a multi-stage aeration cascade via a ring sparger. Viable cell concentration and cell viability were measured using a ViCell cell counter (Beckman Coulter). The osmolality was measured using an osmometer (Advanced Instruments).The off-line pH was measured using a BioProfilepHOx (Nova Biomedical). The glucose, lactate, glutamine and ammonia concentrations were measured with a BioProfile Analyzer 400 (Nova Biomedical). On the day of harvest, the clarification was performed by centrifugation, depth filtration and sterile filtration. Part of the material was further clarified by tangential flow filtration (TFF) on the Uniflux 10 system (GE Life Sciences) using low molecular weight cut-off membranes and concentrated before being diafiltered into an appropriate buffer. The antigens from the mock run were assessed for their total HCP content by 2D DIGE and analyzed by the DeCyderTM 2D 7.2 software.

Purification and characterization of antioxidant peptides from enzymatically hydrolyzed chicken egg white

Egg white is considered as a rich source of high quality proteins with various bioactive peptide fractions. Enzymatic hydrolysis of proteins can be used to release bioactive fractions and different enzymes have different abilities in releasing such bioactive fractions depending on the enzyme’s site of activity on a protein. In this study, several proteases were examined for their ability to release antioxidant peptides from hen egg white and protease P was selected based on the antioxidant activity and the digestion yield of the crude protein hydrolysate. A combination of several purification steps including ultrafiltration with low molecular weight cut-off membranes, cation exchange chromatography and reversed phase high performance liquid chromatography was used to purify ‘protease P egg white hydrolysate’. Sixteen antioxidant peptides, which were derived from ovalbumin, ovotransferrin and cystatin were isolated from the most active fractions. Amino acid sequences of those peptides were determined using LC–MS/MS. Oxygen radical absorbance capacity (ORAC) values of selected short chain peptides were determined using synthetic peptides. Two peptides AEERYP and DEDTQAMP (Ala-Glu-Glu-Arg-Tyr-Pro and Asp-Glu-Asp-Thr-Gln-Ala-Met-Pro) showed the highest ORAC values. The results from this study indicate that egg white is rich in antioxidant peptides which can be used as a potential source for preparing bioactive ingredients using enzymatic hydrolysis followed by purification techniques.

Assessment of fouling-resistant membranes for additive-free treatment of high-strength wastewaters

In this contribution, we investigate the use of fouling-resistant and cleanable membranes designed for treatment of oily water in the treatment of high-strength industrial wastewaters that present challenges to classical treatment technologies. Membrane surfaces were characterized by spectroscopy and electron microscopy pre- and post-filtration to determine the extent of fouling. Low molecular weight cutoff membranes showed stable permeate fluxes without the need for intermittent cleaning, characteristic of systems with low degrees of internal fouling. For 100kDa molecular weight cutoff membranes, flux decline was more severe. While polymer-modified membranes processed ~26% more permeate than the unmodified membranes in this case, flux recovery after a membrane cleaning step was low and similar for the unmodified and modified membranes, characteristic of high degrees of internal fouling. All membranes reduced turbidity nearly 100% and chemical oxygen demand was reduced 70–84%. These results indicate that ‘tight’ ultrafiltration membranes designed for treatment of oily waters can be used for primary treatment of other highly impaired wastewaters, and the use of more ‘open’ ultrafiltration membranes will require further development and optimization of cleaning protocols.

Effect of ultrafiltration by different membranes on HPLC fingerprint of Huanglian Jiedu decorction after being

To investigate the change of HPLC fingerprint of Huanglian Jiedu decroction. Huanglian Jiedu decorction was ultrafiltrated by different membranes. Different relative molecular weight cutoff membranes were used (pes-5w, pes-3w, pes-2w, pes-1w, pes-6k) to explore its effects on content differences among all samples in HPLC. The turbidity, viscosity and other physical and chemical parameters were measured. Berberine, baicalein and jatrorrhizine became smaller while geniposide increased. Physicall and chemical parameters also showed regular changes. Taking centrifugate as reference, the similarities of the samples treated with high to low molecular weight cutoff membranes were 0.993, 0.992, 0.984, 0.982, 0.975, respectively. In this study, the ultrafiltration was used, HPLC fingerprint showed the differences of the samples, and the change of the physical and chemical parameters was explored.

A low molecular weight cut-off polymer–silicate membrane for microfluidic applications

In this article, we report a novel approach to fabricating a low molecular weight cut-off membrane that could readily be employed for several microfluidic applications. The reported structure was created by selectively retaining a precursor solution [5% (w/v) maleic anhydride, 21% (v/v) (37:1) acrylamide/bisacrylamide, and 0.2% (w/v) VA-086 photoinitiator] in a chosen location of a microfluidic network via capillary forces and then photo-polymerizing the mixture. The pores in the resulting membrane were subsequently filled with 3-aminopropyltriethoxysilane, heated, and then treated with sodium silicate solution and heated again, giving a structure having reduced porosity. The composite membrane thus created has been shown to have a molecular weight cut-off that is at least an order of magnitude smaller than other photo-polymerized microfluidic membranes reported in the literature. Moreover, this polymer–silicate structure was observed to be capable of blocking electroosmotic flow, thereby generating a pressure gradient around its interface with an open microchannel upon application of an electric field across the microchannel-membrane junction. In this study, a fraction of the resulting hydrodynamic flow was successfully guided to an electric field free analysis channel to implement a pressure-driven assay. With our current design pressure-driven velocities, up to 1.8 mm/s was generated in the electric field free analysis channel for an applied voltage of 2 kV in the pumping section. Finally, the functionality of this integrated microfluidic device was demonstrated by implementing a reverse phase chromatographic separation using the pressure-driven flow generated on-chip.

A high recovery microsampling device based on a microdialysis probe for peptide sampling

A high recovery microsampling probe based on microdialysis was devised. The new probe showed a high recovery (100%) of peptides in vitro at different perfusion flow rates (0.1–1.0 μl/min). At a high flow rate, 1.0 μl/min, a 10-fold increased in recovery of peptides compared to the conventional microdialysis probe was achieved. A probe made of a low molecular weight cutoff membrane is suitable for filtering off proteins. The new probe can be a useful tool for high recovery of peptides from living tissues.

Modes of natural organic matter fouling during ultrafiltration.

The fouling of ultrafiltration membranes by natural organic matter (NOM), isolated from a potable surface water source, was studied with an emphasis on elucidating fouling modes and the role of aggregates. NOM size was related to membrane pore sizes using parallel membrane fractionation and size exclusion chromatography, such analyses confirmed the predominance of low MW species and identified the presence of aggregates in concentrated NOM solutions. Cake formation was the dominant mode of fouling by the unfiltered feed, which contained aggregates. This was identified by a constant rate of increase in membrane resistance with permeate throughput and was independent of pore size over a 10-1000 kDa molecular weight cutoff (MWCO) range. Prefiltration (to remove aggregates) and dilution (to reduce aggregate concentration) reduced the rate of increase in membrane resistance for the low MWCO membranes but did not change the fouling mode. In contrast, such pretreatment prevented cake formation on the larger MWCO membranes and shifted the mode of fouling to pore blockage. The date lend support for the idea that an initial fouling layer of large aggregates can catalyze the fouling by lower MW species. The fouling layer could be removed from the large MWCO membranes by backwashing, but the lower MWCO membranes exhibited some irreversible fouling, suggesting that low MW species penetrated into the pore structure. A combined pore blockage-cake formation model described the data well and provided insight into how fouling modes evolve during filtration.

Apple Juice Stabilization by Combined Enzyme—Membrane Filtration Process

Stabilization against discolouration and clouding is a major problem in fruit juice production. Effects of enzymatic oxidation of polyphenols, associated with 'active filtration' and membrane filtration were evaluated, in terms of juice characteristics and stability. Laccase oxidation produces a significant decrease in the phenolic content of juices, associated with a remarkable colour increase. Active filtration by polyvinylpolypyrrolidone and activated charcoal partially decolourates the oxidized juices and stabilizes these products, removing a high quantity of phenolic compounds. Membrane filtration demonstrates to be an effective technique in terms of stabilization only if performed using low molecular weight cut-off membranes.

Sample centrifugation onto membranes for sequencing

This paper presents a new method for adsorption of proteins in solution onto a polyvinylidene diflouride (PVDF) membrane using centrifugation. The technique uses a low molecular weight cut-off membrane (LMW) placed underneath a PVDF membrane. The paired membranes are placed in a receptacle which in turn fits into a microcentrifuge tube. During sample centrifugation, the LMW acts to increase the amount of protein that is concentrated and adsorbed onto the hydrophobic surface of the PVDF membrane. By alternating between two receptacle sizes, this method can accomodate large (>10 μg) and small (<10 μg) amounts of sample. This paper demonstrates sample recovery for a variety of proteins as quantitated by radioactivity and amino acid analysis after centrifugation onto PVDF. Amino acid and sequence analysis results demonstrate the efficiency with which interfering buffers and sodium dodecyl sulfate are removed as a result of sample centrifugation and washing. Finally, we demonstrate the utility of this technique with samples in the low picomole range to obtain useful sequence information following electrophoretic isolation of cyanogen bromide fragments purified by high performance electrophoresis chromatography.

Influence of volume shifts on drug binding during equilibrium dialysis: correction and attenuation.

A time-dependent volume shift from buffer to plasma, which occurs during equilibrium dialysis, decreased the protein binding of disopyramide and its capacity constant, and had no effect on the binding association constant. The volume-dependent decrease in disopyramidine binding may be corrected for by use of a derived equation. Inclusion of dextran, 2.5% (w/v), and use of a thick, low molecular weight cutoff membrane was the most effective technique in attenuating the volume shift. The plasma (serum) protein binding of the basic drugs lidocaine, disopyramide, propranolol, and diazepam was decreased when protein was diluted to 88% or less of its undiluted concentration as a consequence of the volume shift. The protein binding of clofibrate, a highly bound acid drug, was more sensitive to volume shifts than the four basic drugs. Correction of drug binding for volume shifts was reasonably successful for most drugs. The highest binding measured for all drugs was associated with the lowest volume shift.