Biomedical subjects

A Lali

Publications and source records attributed to A Lali.

5 recordsLinked to original sources

PubMed2000

Enhanced performance of expanded bed chromatography on rigid superporous adsorbent matrix.

Rigid spherical macroporous adsorbent beads with surface hydroxyl groups were prepared by cross-linking of cellulose. These beads had diameter in the range 100-200 microm and a mean pore size of about 3 microm with about 60% pore volume. The matrix (bulk density approximately 1600 kg m(-3)) could be expanded into a stable bed and used for protein chromatography. Chromatographic runs were performed on a 10 mm diameter column under non-retaining and retaining conditions on the prepared matrix (called Celbeads) and performance of the runs was measured in terms of the height equivalent to a theoretical plate (HETP). The HETP curves in both packed and expanded bed modes followed profiles typical of macroporous adsorbents, i.e. increasing and levelling with velocity. Unimpaired performance of the matrix at increasing flow-rates permitted expanded bed elution of adsorbed solutes without loss of efficiency in terms of purification factor and product concentration. As a model system, Celbeads was used to purify lactate dehydrogenase from porcine muscle homogenate by dye-affinity chromatography. The prepared matrix provided about 100 theoretical plates per meter for the enzyme system at a linear flow velocity of 1.27 cm x min(-1) in an expanded bed elution mode, and gave enzyme yields of 100% with a purification factor of 31 using an optimized procedure. The adsorbent could be cleaned in place with 5 M urea and used repeatedly without loss of performance.

Animals↗

PubMed1999

Monitoring of low concentrations of glucose in fermentation broth.

A highly sensitive glucose sensor, operating in flow-injection analysis (FIA) mode, was developed for the detection of glucose in fermentation broth. The assay system is based upon the post-column reaction of the peroxide formed in the glucose-oxidase-catalysed reaction and subsequent spectrophotometric detection of the coloured product formed. The sensor system was characterised and calibrated using standard solutions, and later used for quantification of glucose in fermentation media. Two types of enzyme column were used: one operated in packed-bed mode and the other in expanded-bed mode. Both columns were integrated into a FIA system and were found to give good analytical results. Glucose concentrations as low as 0.1 mg/l and 5 mg/l could be detected in packed- and expanded-bed modes respectively. Glucose concentrations were measured during typical fed-batch fermentation conditions in this system, and the results are presented.

Biosensing Techniques↗

PubMed1999

Expanded bed adsorption on supermacroporous cross-linked cellulose matrix.

Rigid spherical macroporous adsorbent beads (CELBEADS) prepared by cross-linking of cellulose were characterized and found eminently suitable for use as expanded bed affinity chromatography matrix. Chromatographic runs were performed on a 10 mm diameter column with three solutes tyrosine, papain and bovine serum albumin under non-retaining conditions on CELBEADS and Streamline DEAE, a commercial agarose based expanded bed matrix. Performance of the runs was measured in terms of height equivalent to theoretical plate, HETP. Variation in HETP with velocity on Streamline DEAE gave flat profiles in packed bed and increasing trend in expanded bed. On CELBEADS, the HETP curves in both packed and expanded bed modes followed profiles typical of macroporous adsorbents i.e. increasing and levelling with velocity. HETP values obtained for papain and bovine serum albumin on CELBEADS were lower than those obtained on Streamline DEAE at all velocities. Lactate dehydrogenase was purified from porcine muscle homogenate using Cibacron blue conjugated to CELBEADS using a protocol reported for supports with surface hydroxyl groups. Elution of the enzyme was investigated both in packed mode as well as in expanded mode at a flow rate of 1 ml min-1. The purification procedure took about 60 minutes and a purification fold of about 14 was achieved in both cases. The adsorbent could be cleaned in place with 5 M urea and used repeatedly without loss of performance.

Adsorption↗

PubMedDate not supplied

Effect of adsorbent porosity on performance of expanded bed chromatography of proteins.

Expanded bed or fluidized bed adsorption has emerged as an important unit operation in downstream processing of proteins. A number of specifically designed commercial adsorbents are available today for expanded bed purification of proteins. Protein purification essentially requires adsorbent matrices that have large pore size. Very large pore size or macroporous adsorbents can provide high efficiency in packed beds even at high flow rates on account of reduced pore diffusion resistance resulting from finite intraparticle flow in the macropores. This is reflected in leveling off of HETP (height equivalent to theoretical plate) versus flow curve after a threshold velocity. Expanded bed operation, on the other hand, can also show plateauing of the HETP curve, but not necessarily on account of macroporosity of adsorbent. It is shown in this article how any adsorbent intended for protein adsorption in expanded bed mode can give plateauing HETP curve, regardless of pore size. As a result, RTD measurements on an expanded bed can give equal, and at times better, performance than a corresponding packed bed. Large pore size, on the other hand, can result in lesser retention of biomass and easy flushing of the adsorbent to obtain an entirely particulate-free adsorbent prior to the product elution step. Adsorbent with larger pores is also shown to provide faster and more efficient elution both in packed and expanded bed modes.

Adsorption↗

PubMedDate not supplied

Carboxymethyl cellulose as a new heterobifunctional ligand carrier for affinity precipitation of proteins.

Affinity precipitation is a technique that imparts selectivity to the widely used primary purification step of precipitation of proteins from crude extracts. Hetero-bifunctional affinity precipitation involves use of reversibly soluble/insoluble polymers that can be used as backbones to conjugate affinity ligands for specific separations. A variety of such polymers have been reported in literature. In this work we report development of carboxymethyl cellulose (CM cellulose) as a cheap, readily available and versatile reversibly soluble polymer system. Available CM cellulose as sodium salt could be quantitatively precipitated from its aqueous solution in presence of about 50 mM calcium and 7.2% w/v polyethylene glycol-4000, and could be resolubilised in the working buffer in absence of calcium, polyethylene glycol or both. Effectiveness of the CM cellulose-calcium-polyethylene glycol system was demonstrated by purifying lactate dehydrogenase from porcine muscle extract using covalently conjugated Cibacron blue dye-ligand. By careful choice of conditions that suppressed non-specific interactions, the system was shown to be an effective affinity precipitation polymer system inspite of the polyelectrolytic nature of CM cellulose. Up to 23 fold purification of the enzyme from crude extarct was obtained in one single precipitation sequence.

Animals↗