Bo-Lennart Johansson

Development of a cleaning in place protocol and repetitive application of Escherichia coli homogenate on STREAMLINE™ Q XL

Abstract An optimization of a cleaning in place (CIP) protocol has been performed for STREAMLINE™ Q XL separation medium using homogenised Escherichia coli as sample. The effectiveness of the different cleaning protocols was tested by analysing the content of fatty acids, phosphorus compounds and amino acids in STREAMLINE™ Q XL. Additionally, RAMAN and FT-IR measurements were used to verify the degree of contamination of separation medium. From the results of the optimization experiments, a cleaning procedure was suggested and subsequently tested with 50 repetitive applications. The study shows that of the different agents tested, 1 M NaOH is the most efficient cleaning solution and that long contact time (∼8 h) with 1 M NaOH is important. An additional cleaning step with 30% 2-propanol (v/v) is also recommended for E. coli samples.

Elution behaviour of some proteins on fresh, acid- or base-treated Sephacryl S-200 HR

The influence of sodium chloride concentration and the pH of the mobile phase on the distribution coefficient of proteins with different pI values was studied on Sephacryl S-200 HR. The non-size-related behaviour of this gel filtration packing is mainly attributed to small amounts of groups that are negatively charged within the pH range investigated (4.2-10.0). These anionic groups on the packing gave rise to ion-exchange or ion-exclusion interactions depending on the charge characteristics of the protein. Hydrophobic interactions at high ionic strength and intramolecular electrostatic repulsive interactions at low ionic strength were also observed for some proteins. The chemical stability of Sephacryl S-200 HR was studied by comparing the chromatographic results with Sephacryl S-200 HR that had been treated in acidic or basic solutions with those with fresh Sephacryl. After Sephacryl S-200 HR had been stored for 2 weeks in 0.10 M sodium hydroxide the chromatographic results at low ionic strengths clearly showed that groups that are positively charged at pH 4.2 had been formed. However, storage for 2 weeks in 0.01 M hydrochloric acid did not change the chromatographic behaviour of the proteins from that observed when injected on fresh Sephacryl S-200 HR.

Determination of the leakage from phenyl-sepharose CL-4B, phenyl-sepharose FF and phenyl-superose in bulk and column experiments

The release of ligands from Phenyl-Sepharose CL-4B, Phenyl-Sepharose FF and Phenyl-Superose has been studied in bulk and column experiments. The leakage products have been identified and quantified by liquid chromatography, fluorescence spectroscopy and proton NMR spectroscopy. It is demonstrated that the leakage occurs primarily through hydrolysis of the agarose support. However, leakage via ether cleavage of the spacer arm-ligand moiety is also observed especially for Phenyl-Superose. The release of ligands at acidic pH is in agreement with a first-order reaction and correspondingly the rate constants have been estimated for all three gels at pH 1 and 2. These show that 50% of the ligands are intact after 15 years of incubation at pH 2. Phenyl-Superose is the most stable gel at acidic pH, whereas Phenyl-Sepharose CL-4B and Phenyl-Sepharose FF are the most stable at neutral and basic pH.

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.

Characteristics of Superdex® prep grade media for gel filtration chromatography of proteins and peptides

Some chemical and chromatographic properties of Superdex® 30 prep grade, Superdex 75 prep grade and Superdex 200 prep grade, all new composite media for separation of peptides and proteins, were investigated. The selectivity of Superdex 30 prep grade was studied by constructing calibration graphs (Kav vs log Mr) of peptides and poly(ethylene glycol) (PEG) standards. The influence of the sodium chloride concentration of the mobile phase on the distribution coefficient of peptides demonstrates that Superdex 30 prep grade contains small amounts of charged groups. Titration results show that the amount of negatively charged groups (carboxyl groups) was about 1 μmol/ml gel for all three investigated Superdex media. A homologous series of n-alphatic (n=4–8) alcohols was used to study the hydrophobic interaction behaviour of Superdex media. The effects of temperature, salt concentration and methanol on Kav of the alcohols were used to characterize the elution mechanism. Hydrophobic interactions increased with the amount of dextran coupled to the agarose bead. This means that of the three Superdex media, Superdex 200 prep grade is the least hydrophobic. The functional stability of Superdex 30 prep grade was studied by testing the separation of PEG standards and peptides 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 PEG standards and the peptides was unaltered after the medium had been treated for a total contact time of 400 h in 0·1 m hydrochloric acid. However, a slight increase in retention volume was observed after treatment in basic conditions. This effect is a result of hydrolysis of covalently bonded dextran in the agarose pores. In basic conditions, the highest carbohydrate (dextran) leakage was observed for Superdex 30 prep grade and the lowest for Superdex 200 prep grade. In acidic conditions it was found that for all Superdex media, dextran and low amounts of 5-(hydroxymethyl)-2-furaldehyde (5-HMF) were released but the separation characteristics were unaltered after treatment in 0·1 m hydrochloric acid. The highest 5-HMF leakage was observed for Sepharose High Performance (the agarose bead on which Superdex media are based). The lowest amount was released from Superdex 30 prep grade.

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.