The affinity technology in downstream processing

Labrou, Nikolaos E.; Clonis, Yannis D.

doi:10.1016/0168-1656(94)90047-7

Cited by 116 publications

(57 citation statements)

References 122 publications

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“…Likewise, pure proteases are required in molecular biology; for example, for the cleavage of fusion proteins and protein sequencing. In such demanding applications, affinity-based purification techniques (Labrou and Clonis, 1994) play central roles.…”

Section: Introductionmentioning

confidence: 99%

Design and study of peptide-ligand affinity chromatography adsorbents: Application to the case of trypsin purification from bovine pancreas

Makriyannis

Clonis

1997

Biotechnol. Bioeng.

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The purification of trypsin from bovine pancreas was employed in a case study concerning the design and optimization of peptide‐ligand adsorbents for affinity chromatography. Four purpose‐designed tripeptide‐ligands were chemically synthesized (>95% pure), exhibiting an Arg residue as their C‐terminal (site P1) for trypsin bio‐recognition, a Pro or Ala in site P2, and a Thr or Val in site P3. Each tripeptide‐ligand was immobilized via its N‐terminal amino group on Ultrogel A6R agarose gel, which was previously activated with low concentrations of cyanuric chloride (10.5 to 42.5 μmol/g gel). Well over 90% of the peptide used was immobilized. Three different concentrations were investigated for every immobilized tripeptide‐ligand, 3.5, 7.0, and 14 μmol/g gel. The KD values of immobilized tripeptide‐trypsin complexes were determined as well as the purifying performance and the trypsin‐binding capacity of the affinity adsorbents. The KD values determined were in good agreement with the trypsin purification performance of the respective affinity adsorbents. The tripeptide sequence H‐TPR‐OH displayed the highest affinity for trypsin (KD 8.7 μM), whereas the sequence H‐TAR‐OH displayed the lowest (KD 38 μM). Dipeptide‐ligands have failed to bind trypsin. When the ligand H‐TPR‐OH was immobilized via its N‐terminal on agarose, at a concentration of 14 μmol/g gel, it produced the most effective affinity chromatography adsorbent. This adsorbent exhibited high trypsin‐binding capacity (approximately 310,000 BAEE units/mL of adsorbent); furthermore, it purified trypsin from pancreatic crude extract to a specific activity of 15,200 BAEE units/mg (tenfold purification), and 82% yield. © 1997 John Wiley & Sons, Inc.

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Section: Introductionmentioning

confidence: 99%

Design and study of peptide-ligand affinity chromatography adsorbents: Application to the case of trypsin purification from bovine pancreas

Makriyannis

Clonis

1997

Biotechnol. Bioeng.

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“…For this purpose, triazine dyes are used in columns to purify albumin and various proteins. 113,114 Immunoaffinity chromatography (IAC) We applied IAC through Cyanogen bromide (CNBr) -activated Sepharose to facilitate albumin purification and improving its purity. Initially, the pure HSA was injected to the three white New Zealand rabbits to produce of polyclonal antibody.…”

Section: Boronate Affinity Chromatographymentioning

confidence: 99%

Overview of Albumin and Its Purification Methods

et al. 2016

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“…bioaffinity ligands have proven to avoid the risks associated with toxicities in elution fractions due to ligand leakage. Additionally, these ligands typically provide for a mild elution environment, thereby retaining immunological properties of the protein, have broader immunoglobulin specificity, and are more stable in chromatographic systems in comparison to ligands such as protein A and G. [17] The use of histidyl and thiophilic ligands are limited due to their lack of specificity and selectivity. Metal chelate interaction chromatography using Cuþþ-chelate gel has been studied for the separation of Ig from blood serum and plasma.…”

Section: Introductionmentioning

confidence: 99%

A Comparative Study of Monoclonal Antibodies (Mabs) Purified from Cell Culture Supernatant on EDTPA‐Modified Zirconia Beads and Protein A‐Hyper D Support

Subramanian

Martínez

Holm

et al. 2006

Journal of Liquid Chromatography & Related Technologies

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Abstract:Colloidal zirconia was spray dried to yield zirconia particles, which were further modified with N, N, N0, N0-Ethylenediamine tetra methylenephosphonic acid (EDTPA) to yield a support for use in bioseparations. EDTPA modified zirconia particles will be further referred to as, r_PEZ. Cell culture supernatants rich in monoclonal antibody (Mab) subtypes IgG1, IgG2a, IgG2b, and IgG3 were chromatographed on a r_PEZ column, and on a protein A-hyper D column that was purchased commercially. All Mab subtypes bound to r_PEZ and process yields in the range of 88 to 99% were obtained. The purity of the Mab products were ascertained by gel lectrophoretic analysis and were estimated to be greater than 95%. The purified Mab products obtained from r_PEZ and protein A columns were compared to the reference Mab standard in biological and enzymatic assays. The value of the dissociation constant (Kd) was found to be comparable and was in the range to that obtained with reference Mab standard (0.231+0.03 M). In addition, Mabs purified with r_PEZ had the same deglycosylation profile as the reference Mab standard. Thus, it appears that the r_PEZ purified Mab is similar in activity to Mab purified with a protein A support and in addition, the zirconia surface does not adversely impact the activity of the purified Mab.

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The affinity technology in downstream processing

Cited by 116 publications

References 122 publications

Design and study of peptide-ligand affinity chromatography adsorbents: Application to the case of trypsin purification from bovine pancreas

Design and study of peptide-ligand affinity chromatography adsorbents: Application to the case of trypsin purification from bovine pancreas

Overview of Albumin and Its Purification Methods

A Comparative Study of Monoclonal Antibodies (Mabs) Purified from Cell Culture Supernatant on EDTPA‐Modified Zirconia Beads and Protein A‐Hyper D Support

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