High-performance liquid chromatographic methods for antibodies, glycosidases and membrane proteins

Josić, Djuro; Schütt, Wolfgang; Renswoude, Jos van; Reutter, Werner

doi:10.1016/s0021-9673(01)87071-0

Cited by 22 publications

(11 citation statements)

References 6 publications

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“…Anion-exchange as well as cation-exchange HPLC was used for the isolation of both monoclonal and polyclonal antibodies (14,15). A problem that arises with the use of ion-exchange HPLC is the dissimilar behaviour of different antibodies, including monoclonal ones, in Chromatographie runs, even under identical conditions.…”

Section: Resultsmentioning

confidence: 99%

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Isolation of Immunoglobulins and Their Use in Immunoaffmity HPLC

Josić¹,

Hofmann²,

Habermann³

et al. 1988

Clinical Chemistry and Laboratory Medicine

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Summary:For the isolation of monoclonal and polyclonal antibodies different high performance liquid chromatography (HPLC) and high performance affinity chromatography (HPAC) methods were investigated. Specially designed "mixed-bed" ion-exchange and hydroxylapatite columns as well as hydrophobic interaction columns were efficiently applied to the isolation of monoclonal antibodies. When these methods are used for the isolation of polyclonal antibodies from antiserum, the sample has to be pre-treated, e. g. by removal of serum albumin.Protein A HPAC is an easy method and quick to handle, especially for the preparative isolation of antibodies. The antibodies that do not bind to protein A, can be purified by protein G HPAC. If this method cannot be used because of the rather extreme elution conditions, hydroxylapatite, ion-exchange or hydrophobic interaction HPLC have to be considered as alternatives.We further concentrate on immunoaffinity HPLC with immobilized antibodies. This method has proved to be very effective for one-^step isolation of antigens, even from very complex samples such as plasma membrane extracts. The problem with immunoaffinity HPLC is the quick deterioration of the columns, caused by increasing denaturing of the immobilized antibodies during elution. In order to solve this problem, an indirect method is recommended for analytical immunoaffinity HPLC. For this purpose, the antibodies are bound to a protein A HPAC column. The solution containing the antigens is then applied. After washing, the antigenantibody complex is eluted from the column.

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Section: Resultsmentioning

confidence: 99%

“…The albumin is removed from the samples by chromatography on an afil-blue-gcl column (Bio Rad) (14).…”

Section: Sample Preparationmentioning

confidence: 99%

Isolation of Immunoglobulins and Their Use in Immunoaffmity HPLC

Josić¹,

Hofmann²,

Habermann³

et al. 1988

Clinical Chemistry and Laboratory Medicine

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“…Protein conformational changes are known to occur when exposed to the strong hydrophobic surfaces of reversed phase chromatography (RPC) (Benedek et al, 1984;Cohen et al, 1985;Lu et al, 1986;Gooding and Regnier, 1990;Oroszlan et al, 1992;Fernandez, 1999, 2001;). Losses of enzyme activity (Kato et al, , 1986Josic et al, 1986) and low chromatographic recovery (Ichinose et al, 1985;Fausnaugh et al, 1984a,b;Kato et al, 2002) have also been reported for some HIC media, even though they are the much less hydrophobic than RPC resins. In particular, unstable proteins can exhibit complex chromatographic peak shapes during HIC, ranging from broad peak widths and shoulders to split peaks.…”

Section: Introductionmentioning

confidence: 92%

Hydrophobic interaction chromatography selectivity changes among three stable proteins: conformation does not play a major role

Jones¹,

Fernandez²

2004

Biotech & Bioengineering

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Interesting retention and selectivity changes have been noted for a number of proteins in hydrophobic interaction chromatography (HIC). In this study, we investigated the degree to which conformational changes may be responsible for selectivity changes of stable proteins. Hydrogen-deuterium isotope exchange detected by mass spectrometry was used to investigate changes in solvent accessibility during adsorption on HIC media. Lysozyme was determined to exhibit EX2 hydrogen exchange kinetics both in solution and adsorbed to Butyl Sepharose 4 Fast Flow and Phenyl Sepharose 6 Fast Flow high sub surfaces. A small, but significant, increase in solvent accessibility was observed upon adsorption. Similar approaches were used to analyze solvent accessibility of three stable proteins with melting temperatures above 50jC exhibiting significant selectivity changes on Butyl Sepharose and Toyopearl Butyl 650M. While all three proteins (lysozyme, chymotrypsinogen A, and ovalbumin) exhibited enhanced exchange while adsorbed, no differences in solvent accessibility on the different adsorbents were observed. More detailed studies of lysozyme showed no significant changes in labeling prior or during elution. These results demonstrate that HIC surfaces examined here do not dramatically alter the structure of these stable proteins and that differences in conformation are not responsible for the selectivity changes observed. Thus, other factors such as different preferred binding orientations or variations between the media pore structure, size, and/or surface chemistry must be responsible. B 2004 Wiley Periodicals, Inc.

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“…Therefore, favourable interactions can happen if these surface sites are complementary to the groups on the stationary phase surface. On the other hand, several authors have reported that these hydrophobic interactions between proteins and some HIC media could produce loss of enzyme activity [32,33], low chromatographic recovery [34][35][36], and in the case of unstable proteins (␣-lactoalbumin, lysozyme), partial or total unfolding may occur [37][38][39]. For instance, there are reports about two peaks in HIC of ␣-lactoalbumin; the less retained was identified as native, and the more retained as an "unfolded mixture of species."…”

Section: Hydrophobic Interactions and Retention Mechanisms In Hydrophmentioning

confidence: 99%