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. 2018 May/Jun;10(4):624-635.
doi: 10.1080/19420862.2018.1445450. Epub 2018 Mar 29.

Automated high throughput microscale antibody purification workflows for accelerating antibody discovery

Affiliations

Automated high throughput microscale antibody purification workflows for accelerating antibody discovery

Peng Luan et al. MAbs. 2018 May/Jun.

Abstract

To rapidly find "best-in-class" antibody therapeutics, it has become essential to develop high throughput (HTP) processes that allow rapid assessment of antibodies for functional and molecular properties. Consequently, it is critical to have access to sufficient amounts of high quality antibody, to carry out accurate and quantitative characterization. We have developed automated workflows using liquid handling systems to conduct affinity-based purification either in batch or tip column mode. Here, we demonstrate the capability to purify >2000 antibodies per day from microscale (1 mL) cultures. Our optimized, automated process for human IgG1 purification using MabSelect SuRe resin achieves ∼70% recovery over a wide range of antibody loads, up to 500 µg. This HTP process works well for hybridoma-derived antibodies that can be purified by MabSelect SuRe resin. For rat IgG2a, which is often encountered in hybridoma cultures and is challenging to purify via an HTP process, we established automated purification with GammaBind Plus resin. Using these HTP purification processes, we can efficiently recover sufficient amounts of antibodies from mammalian transient or hybridoma cultures with quality comparable to conventional column purification.

Keywords: antibody; automation; high throughput; hybridoma; liquid handling system; purification; rat IgG2a; small scale; tip column; transient expression.

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Figures

Figure 1.
Figure 1.
(a) Tecan EVO 200 deck layout, and (b) automation process scheme for 1 mL tip column mode purification of 8 consecutive plates in a continuous run. In this layout, there are 8 boxes of regular 200 µL tips on the right side of the deck and 1 box of 500 µL Tip Columns (Tip Clmn, rectangle with dark border). Abbreviations for labware labels: P1 – P8 (plate # 1 – 8), Capt. (Capture), F.T. (Flow through), CIP (Cleaning in place buffer), Cult. (Culture Media), Clmn. (Column), Equili. (Equilibration).
Figure 2.
Figure 2.
(a) Dynamic Device Lynx 1200 deck layout, and (b) automation process scheme for 1–4 mL batch mode purification of 4 parallel plates using 4 filter plates. In this layout, there are 5 boxes of regular 1250 µL tips on the right side of the deck, 1 box for transferring PBS, 4 boxes for transferring culture media (P1 - P4). Abbreviations for labware labels: P1 – P4 (plate # 1 – 4), Reserv. (Reservoir), Vac. (Vacuum), Cult. (Culture Media), Capt. (Capture).
Figure 3.
Figure 3.
Optimizing automated HTP purification in tip columns packed with MabSelect SuRe. (a) 2 mg human IgG1 in 1 mL PBS was loaded over resin in each experiment. Concentrations before and after capture were used to calculate the amount that was bound on the resin (n = 4). Human IgG1 binding capacity was measured for batch mode capture for 16 hours at 4˚C with 20 µL resin (62±3 µg) and tip column capture by pipetting total of 2, 4, 6, 8, and 9 cycles (37±1, 46±1, 52±1, 54±1, and 55±2 µg). (b) resin-bound IgG was eluted twice from tip column with 160 µL of 50 mM phosphoric acid at pH 3.0, 3.2, 3.4, and 3.6 (n = 4 for each pH tested). Buffers at pH3.4 or above were inefficient in eluting IgG from the tip column. (c) eluted IgG concentration, and (d) IgG recovery after treating tip columns with different volumes (100 – 200 µL) of elution buffer (50 mM phosphoric acid pH3.0 buffer). Each 20 µL MabSelect SuRe tip column was loaded with 500 µg IgG in (b – d).
Figure 4.
Figure 4.
Comparing the quality of a human IgG1 purified by (i) conventional column, (ii) automated tip column and (iii) batch mode. (a) Analytical SEC profiles of purified IgG1. (b) Reduced SDS-PAGE, M: Mark 12 standard. (c) Non-reduced SDS-PAGE. (d) LC-MS intact mass analysis of light chain. (e) LC-MS intact mass analysis of heavy chain. No significant difference in purity or measured masses was observed in purified IgG from automated processes compared to a conventional column process. Glycosylation variants abbreviations: Man5 (Man5GlcNAc2), G0F (GlcNAc2Man3GlcNAc2), G1F (GalGlcNAc2Man3(Fuc)GlcNAc2), where: Man (mannose), GlcNAc (N-acetylglucosamine), Gal (Galactose), Fuc (Fucose).
Figure 5.
Figure 5.
Evaluating sensitivity of GammaBind Plus (panel a) and Protein G (panel b) to sodium chloride (NaCl) concentration and pH for binding rat IgG2a. Data from Table 2 were plotted using JMP11 software (SAS, Cary, NC). (c) Recovery efficiency with different wash condition: 150 µg rat IgG2a was loaded onto 20 µL GammaBind Plus resin tip columns, then washed with 1x PBS twice, followed by washing with low salt pH 5 (2 mM sodium phosphate, 30 mM sodium chloride) buffer or 1x PBS, finally eluted thrice with 190 µL of 10 mM citrate pH2.9 buffer, resulting in 51 ± 3% or 33 ± 1% recoveries respectively in elution 1.
Figure 6.
Figure 6.
Regeneration of MabSelect SuRe tip columns (20 µL resin). Before each use, the tip columns were regenerated using 0.2 M NaOH, followed by equilibration with 0.2 M Tris pH 7.0 and PBS. After each regeneration, known amount of human IgG1 spiked into 1 mL blank media was captured onto tip columns and purified into 160 µL final volume (single elution) in automated HTP process: (a) 500 µg IgG spiked; (b) 100 µg spiked; (c) 20 µg IgG spiked (n = 12 each panel). Less than 5% drop of recovery was observed after 22 regenerations in all tested spiking amount, giving very high re-usability of the tip columns packed with MabSelect SuRe resin. (d) a human IgG1 was expressed in replicated 96 deep-well blocks, the 1st replicate block were purified by the tip columns at 3rd use, while the 2nd replicate deep block was purified by the same set of tip columns at 7th use, with regeneration before each use. The tip columns showed consistent yield among individual columns and after different regeneration cycles (3x vs 7x).

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