Ingrid Drevin

Chemical properties of and solute-support interactions with the filtration medium Superdex 75 prep grade

Abstract The influence of the ionic strength and pH of the mobile phase on the distribution coefficient of proteins with different pI values was studied on Superdex 75 prep grade. Superdex contains small amounts of negatively charged groups which are responsible for electrostatic interactions between ionic solutes and the gel. Ion-exchange or ion-exclusion interactions were observed at low ionic strengths when the pH of the mobile phase was lower or higher than the pI values of the proteins. For some proteins, hydrophobic interactions were also observed at high ionic strengths. The chemical stability of Superdex 75 prep grade was studied by comparing the chromatographic results from Superdex treated in acidic or basic solutions with those from untreated Superdex. The separation characteristics of Superdex 75 prep grade were unaffected after 25 washes with 1.0 M sodium hydroxide solution or 0.1 M hydrochloric acid with a contact time of 4 h for each wash. However, storage for 2 weeks in 0.01 M hydrochloric acid or 0.1 M sodium hydroxide solution partly hydrolysed the covalently bounded dextran in the agarose pores. This hydrolysation resulted in leakage of dextran and an increase in the Kav values of the test proteins.

Determination of polyethylene glycol bonded to epoxy-activated Sepharose 6B

Determination de PEG couplee a du Sepharose 6B par du bis (epoxy-2,3 propoxy)-1,4 butane par coupure quantitative des liaisons ethers dans le ligand et determination subsequente par chromatographie gazeuse des produits de coupure

Chemical and chromatographic characterization of a new bioprocess™ medium for hydrophobic interaction chromatogrpahy: Butyl Sepharose® 4 FastFlow

Abstract The authors have studied the influence of ligand density, temperature, eluent pH and Triton X-100 on the separation performance of Butyl Sepharose 4 Fast Flow. The proteins used during the study were α-chymotrypsinogen, cytochrome C, ribonuclease A and lysozyme. Small adjustments to the chromatographic conditions can affect the retention times of the proteins. The effects from changes in temperature prove that the separation process is entropy-driven. The functional stability of Buty Sepharose 4 Fast Flow was studied by testing the separation of a protein in mixture during repeated cleaning-in-place (CIP) treatments with 1·0 m sodium hydroxide solution and 0·1 m hydrochloric acid. The separation behaviour of the protein mixture was unaltered after the medium had been treated for a total contact time of 4 weeks with 1·0 m sodium hydroxide solution. However, a slight decrease in retention times was observed in acidic conditions. The study shows that butyl ligands are released due to hydrolysis of the agarose support in acidic conditions. This explains the effect on retention time observed after CIP at pH 1. For this study, a method for determining the ligand content was developed. The amount of butyl groups coupled via ether linkage to the agarose matrix was determined by gas chromatography after cleavage of ether bonds by boron tribromide. The authors have also investigated the clearance of ethanol, 2-propanol and Triton X-100 from an HR 10/10 column packed with Butyl Sepharose 4 Fast Flow. For a column treated with Triton X-100, a regeneration procedure with 2-propanol was necessary in order to retain the original retention behaviour of the medium.

Column performance of Q-Sepharose HP in analytical- and preparative-scale chromatography

Abstract The chromatographic behaviour of two Q-Sepharose® HP HR 16/10 columns was tested under analytical- and preparative-scale conditions. Protein clogging of the top filter and the packing was the main reason for the observed decrease in retention volume, gel bed height and peak height during 300 analytical separations of a protein mixture (5 mg of protein per injection). Different washing procedures proved that adsorbed protein molecules decreased the availability of the sample molecules to the ion-exchange groups. By using a column where the top filter was fixed to the adaptor (XK 16/20 column), the stability of the column bed height was improved. Purification of ovalbumin from egg white with large sample loads showed that washing with 0.1% pepsin solution maintained the optimum recovery. The column performance was evaluated in 50 purification cycles, corresponding to ca. 25 g of purified ovalbumin.

Characterization of the chemical and functional stability of DEAE Sepharose® fast flow

The release of amines from the ion-exchange groups in DEAE Sepharose® Fast Flow has been studied under static and column conditions. The leakage compounds have been identified and quantified by gas ...

Three independent methods for quantitative determination of octyl covalently coupled to sepharose CL-4B

Quantification of the octyl content in Octyl-Sepharose CL-4B was accomplished by three independent methods. Firstly, the 1H NMR spectrum was registered on a deuterium chloride hydrolysed gel. Secondly, gas chromatography was applied to the ether-linked ligands cleaved by boron tribromide. Finally, the gel was combusted to carbon dioxide and elemental carbon analysis was performed. The results from the three methods indicate only random errors at a confidence level of 95%. All developed methods are therefore usable for the determination of the ligand content.

Stability of Superdex 75 prep grade and Superdex 200 prep grade under different chromatographic conditions

Abstract The chemical stability of two gel filtration media, Superdex 75 prep grade and Superdex 200 prep grade, was studied in bulk and column experiments. The release of agarose and dextran from these two composite media was measured by three different methods: a specific nephelometric method based on the use of antidextran antiserum, an anthrone method and a gel filtration chromatographic method with a light-scattering detector. Dextran fragments were released from Superdex 75 and 200 prep grade under extreme basic and acidic conditions. However, Superdex withstands many short-term incubations (contact time ca . 4 h each time) at pH 14 and 1 without any influence on the chromatographic behaviour. Equilibration of a Superdex column with a neutral buffer after these short-term treatments lowered the concentration of dextran in the eluate to an undetectable level after about three bed volumes. The ability of Superdex columns to withstand practical mistakes such as pumping air into the column was also investigated.

Quantitative determination of the ligand content and the negative and positive charges on butylamine-substituted sepharose 4B

Abstract Analytical methods based on ion-exchange reactions have been developed for the quantitative determination of the amount of positive and negative charges on butyl-Sepharose 4B at different pH values. A quantitative 1 H NMR procedure is also described for measuring the degree of substitution. These methods constitute the basis for an evaluation of the relative amounts of different coupling structures where butylamine is linked to cyanogen bromide-activated Sepharose 4B.

Characterisation of process separation media by pyrolysis gas chromatography–mass spectrometry

Abstract Pyrolysis capillary gas chromatography–mass spectrometry has been used for characterization of a large number of different process separation media. Media aimed for ion-exchange, affinity and gel filtration chromatography were included in this study and the media matrices were based on Sepharose, Sephacryl or Sephadex. The pyrograms showed many common pyrolysis products but also several unique derivatives from the different ligands and matrices. It has also been shown that the pyrogams from ion-exchange media are affected by the type of counter-ion used at the sample preparation. All investigated media could simply be differentiated by their pyrograms. Specific organic structures of low content were also easily identified. It is therefore concluded that pyrograms of separation media based on polysaccharides can be useful in the study of chemical variations between different production batches.