Purification of Antibodies by Chromatographic Methods

Vandevyver, Caroline; Freitag, Ruth

doi:10.1007/978-1-4419-8875-1_5

Cited by 6 publications

(5 citation statements)

References 118 publications

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“…Protein A chromatography, in general, delivers a product‐related purity of greater than 98% and removes most of the process impurities including proteases. The ensuing process unit operations are considered as polishing steps, responsible for separation of product‐related isomers and removal of trace amounts of host cell proteins, DNA, endotoxin, and potential viruses (7–13).…”

Section: Introductionmentioning

confidence: 99%

Basic Concepts in Q Membrane Chromatography for Large-Scale Antibody Production

2006

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The large-scale production of recombinant human monoclonal antibodies demands economical purification processes with high throughputs. In this article we briefly describe a common antibody process and evaluate the Q membrane adsorber for process-scale antibody production as an alternative to a Q-packed-bed column in a flow-through mode. The scientific concepts underlining Q membrane technology and its application are reviewed. The disadvantages and advantages of using Q membrane chromatography as a purification unit in large-scale production are discussed, including problems initially seen with the Q membrane scale-down model but solved with the invention of a new scale-down model. The new Q-membrane unit operation has a process capacity greater than 3,000 g/m(2) or 10.7 kg/L with a LRV over 5 for four model viruses. In this Review, a cost analysis illustrates that Q membrane chromatography is a viable alternative to Q column chromatography as a polishing step in a flow-through mode for process-scale antibody production.

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Section: Introductionmentioning

confidence: 99%

Basic Concepts in Q Membrane Chromatography for Large-Scale Antibody Production

2006

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“…The covalent immobilization of proteins to supports and surfaces has been used for many years in applications such as immunoassays, affinity chromatography, enzyme reactors, and protein microarrays. − It is generally desirable in these applications to have an immobilized protein that closely retains the structure and activity of the same protein in its native form. However, there is often little information available on the structure and orientation of proteins after they have been immobilized.…”

mentioning

confidence: 99%

Identification and Quantitative Studies of Protein Immobilization Sites by Stable Isotope Labeling and Mass Spectrometry

Cerny²,

Hage

2006

Anal. Chem.

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A method was developed for characterizing immobilization sites on a protein based on stable isotope labeling and MALDI-TOF mass spectrometry. The model for this work was human serum albumin (HSA) immobilized onto silica by the Schiff base method. The immobilized HSA was digested by various proteolytic enzymes in the presence of normal water, while soluble HSA was digested in 18 O-enriched water for use as an internal standard. These two digests were mixed and analyzed, with the 18 O/ 16 O ratio for each detected peptide then being measured. Several peptides in the tryptic, Lys-C, and Glu-C digests gave significantly higher 18 O/ 16 O ratios than other peptides in the same digests, implying their involvement in immobilization. Analysis of these results led to identification of the N-terminus and several lysines as likely immobilization sites for HSA (e.g., K4, K41, K190, K225, K313 and K317). It was also possible from these results to quantitatively rank these sites in terms of the relative degree to which each might take part in immobilization. This method is not limited to HSA and silica but can be used with other proteins and supports.

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“…Moreover, the range of K D values of 2.2–5.5 μM observed for these new adsorbents is clustered around the values of dissociation constants typical of other adsorbents currently in use for antibody purification, for example, the K D values for the interaction of serum IgGs with Protein A has been reported to be 10 −7 M (Vandevyver and Freitag, ), with a biomimetic A2C7I1 peptoidal ligand 53.4 (El Khoury and Lowe, ) or 9 μM with mercaptoethylpyridine immobilized onto cellulose (Lin et al ., ). Further, the derived static binding capacities (21–75 mg/ml resin) of the new adsorbents developed in the current study compare favourably with other commercially available Protein A adsorbents (e.g.…”

Section: Resultsmentioning

confidence: 97%

N-Heterocyclic-based adsorbents for antibody purification-effect of ligand structure

et al. 2014

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A new set of ligands based on substituted pyridine and other N-heterocyclic structures, possessing an aliphatic primary amino group tether and an exocyclic sulphur atom, has been prepared and immobilized onto epoxy-activated matrices such as Sepharose 6 Fast Flow®. The derived adsorbents have been evaluated for their utility to capture and purify humanized monoclonal antibodies. Favourable binding properties were assessed from screening assays to determine optimal conditions for the capture and elution of the monoclonal antibodies. Static and dynamic binding experiments were employed to derive the equilibrium dissociation constants KD 's and binding capacities Qmax 's. Typically, the KD values were in the range of 2-5 μM and the Qmax values between 20 and 75 mg mAb/ml resin, depending on the stereo-electronic properties of the substituent in the N-heterocyclic ring structure. The effect of ligand structure on the selectivity of these adsorbents was also investigated, and criteria for their use in the purification of monoclonal antibodies from cell culture supernatants established.

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Purification of Antibodies by Chromatographic Methods

Cited by 6 publications

References 118 publications

Basic Concepts in Q Membrane Chromatography for Large-Scale Antibody Production

Basic Concepts in Q Membrane Chromatography for Large-Scale Antibody Production

Identification and Quantitative Studies of Protein Immobilization Sites by Stable Isotope Labeling and Mass Spectrometry

N-Heterocyclic-based adsorbents for antibody purification-effect of ligand structure

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