Predicting Column Performance in Displacement Chromatography from High Throughput Screening Batch Experiments

Rege, Kaushal; Tugçu, Nihal; Cramer, Steven M.

doi:10.1081/ss-120019089

Cited by 20 publications

(24 citation statements)

References 26 publications

(18 reference statements)

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“…Microtiter-plate techniques, based on immobilized metalaffinity chromatography, are routinely used for the high-throughput purification of histidine tagged proteins (Draveling et al, 2001;Paborsky et al, 1996). Recently, parallel screening techniques have resulted in the rapid discovery of selective and high affinity displacer candidates for protein purification by displacement chromatography (Mazza et al, 2002;Rege et al 2003Rege et al , 2004.…”

Section: Introductionmentioning

confidence: 99%

High-throughput process development for recombinant protein purification

Rege

Pepsin²,

Falcon³

et al. 2006

Biotechnol. Bioeng.

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Methods development in chromatographic purification processes is a complex operation and has traditionally relied on trial and error approaches. The availability of a large number of commercial media, choice of different modes of chromatography, and diverse operating conditions contribute to the challenging task of accelerating methods development. In this paper, we describe a novel microtiter-plate based screening method to identify the appropriate sequence of chromatographic steps that result in high purities of bioproducts from their respective culture broths. Protein mixtures containing the bioproduct were loaded on aliquots of different chromatographic media in microtiter plates. Serial step elution of the proteins, in concert with bioproduct-specific assays, resulted in the identification of "active fractions" containing the bioproduct. The identification of a successful chromatographic step was based on the purity of the active fractions, which were then pooled and used as starting material for screening the next chromatographic dimension. This procedure was repeated across subsequent dimensions until single band purities of the protein were obtained. The sequence of chromatographic steps and the corresponding operating conditions identified from the screen were validated under scaled-up conditions. Various modes of chromatography including hydrophobic interaction, ion exchange (cation and anion exchange) and hydrophobic charge-induction chromatography (HCIC), and different operating conditions (pH, salt concentration and type, etc.) were employed in the screen. This approach was employed to determine the sequence of chromatographic steps for the purification of recombinant alpha-amylase from its cell-free culture broth. Recommendations from the screen resulted in single-band purity of the protein under scaled-up conditions. Similar results were observed for an scFv-beta-lactamase fusion protein. The use of a miniaturized screen enables the parallel screening of a wide variety of actual bioprocess media and conditions and represents a novel paradigm approach for the high-throughput process development of recombinant proteins.

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Section: Introductionmentioning

confidence: 99%

High-throughput process development for recombinant protein purification

Rege

Pepsin²,

Falcon³

et al. 2006

Biotechnol. Bioeng.

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“…In the past decade, fast approaches have been developed for sorbent performance evaluation and screening [1][2][3][4][5][6][7][8]. Recently, high-throughput (HT) screening platforms were investigated using ProteinChip arrays carrying functionalities equivalent to those of chromatographic sorbents [9][10][11] or using micro-volumes of chromatographic sorbents deposited in 96-well plates [12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27]. This micro-volume technique was improved by using 96-well filter plates.…”

Section: Introductionmentioning

confidence: 99%

Rapid screening of purification strategies for the capture of a human recombinant F(ab′)2 expressed in baculovirus-infected cells using a micro-plate approach and SELDI-MS

Pezzini

Brochier²,

Barrouillet

et al. 2009

Journal of Chromatography B

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“…Protein A purification is one of the most widely used chromatographic techniques for the production of monoclonal antibodies 1,2 but it is under-represented in the literature with regards to development of scaledown purification methods. Advances in high throughput purification methods focus on purification optimization for cation exchange chromatography screening in 96 well-plate [3][4][5][6][7][8][9][10][11] or miniaturized column formats. 12 These techniques offer rapid methods for purification optimization with minimal protein requirements.…”

Section: Introductionmentioning

confidence: 99%

Development of a high throughput protein a well‐plate purification method for monoclonal antibodies

Hopp¹,

Pritchett²,

Darlucio³

et al. 2009

Biotechnology Progress

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We have developed a new high throughput method for the purification of monoclonal antibodies from harvested cell culture fluid for analytical characterization. This method uses Protein A resin in a 96 well-plate format with protein loading sufficient to perform multiple analyses per well. Resin and buffer conditions were optimized to obtain aggregate and charge variant comparability with three preparative Protein A purified monoclonal antibodies. We are able to successfully demonstrate comparability for aggregate within 0.25% based upon size-exclusion chromatography. Acidic species were found to be within 2% from the preparative purified control based upon cation-exchange chromatography, 5% based upon capillary zone electrophoresis, and 3% based upon imaged capillary isoelectric focusing. Glycan distribution was analyzed and was within 1% of the preparative purified controls. A tryptic digest was performed and all peaks in the preparative purified control were found in the first elution from the well-plate format.

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Predicting Column Performance in Displacement Chromatography from High Throughput Screening Batch Experiments

Cited by 20 publications

References 26 publications

High-throughput process development for recombinant protein purification

High-throughput process development for recombinant protein purification

Rapid screening of purification strategies for the capture of a human recombinant F(ab′)2 expressed in baculovirus-infected cells using a micro-plate approach and SELDI-MS

Development of a high throughput protein a well‐plate purification method for monoclonal antibodies

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