Automated high-throughput purification of antibody fragments to facilitate evaluation in functional and kinetic based assays

Journal of Immunological Methods 322 (2007) 94 – 103 www.elsevier.com/locate/jim

Research paper

Automated high-throughput purification of antibody fragments to facilitate evaluation in functional and kinetic based assays Bin Su a , Renee Hrin b , Barrett R. Harvey a , Ying-Jie Wang b , Robin E. Ernst a , Richard A. Hampton a , Michael D. Miller b , William R. Strohl a , Zhiqiang An a , Donna L. Montgomery a,⁎ a

Department of Vaccines and Biologics Research, Merck Research Laboratories, 770 Sumneytown Pike, P.O. Box 4, West Point, PA 19486, USA b Antiviral Research, Merck Research Laboratories, 770 Sumneytown Pike, P.O. Box 4, West Point, PA 19486, USA Received 14 July 2006; received in revised form 23 January 2007; accepted 6 February 2007 Available online 7 March 2007

Abstract Screening antibodies from phage displayed in vitro libraries and from affinity maturation of lead antibodies requires testing of antibody fragments (scFvs and Fabs) for function and binding affinities. Crude scFv or Fab periplasmic preparations from Escherichia coli are often not pure and/or concentrated enough for use in functional and affinity assays. We have developed an automated high-throughput approach for small and large-scale expression and purification of His-tagged scFvs and Fabs using the Qiagen BioRobot 3000 LS with optimized application software. This automated procedure enabled us to rapidly evaluate antibody fragments in functional and surface plasmon resonance (SPR) assays. We have used these procedures to make thousands of purified scFv/Fabs for several antibody maturation campaigns and significantly decreased the time needed to select the best candidates. The assay results from these purified samples were used to prioritize candidates before converting them to IgG. This protocol can process up to 300 small-scale and up to 72 large-scale scFvs or Fabs per week per full-time employee (FTE). © 2007 Elsevier B.V. All rights reserved. Keywords: High-throughput expression and purification; Automation; scFv; Fab; Functional screening; Surface plasmon resonance (SPR)

1. Introduction In the mid-1980s, recombinant DNA technology allowed the generation of chimeric mouse–human antibodies (Boulianne et al., 1984; Bruggemann et al., 1987; Morrison and Oi, 1989) and humanized antibodies (Jones et al., 1986). The demonstration that heterologously expressed peptides were displayed on ⁎ Corresponding author. Tel.: +1 215 652 4953; fax: +1 215 993 2830. E-mail address: [email protected] (D.L. Montgomery). 0022-1759/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2007.02.006

the surface of filamentous bacteriophage (Smith, 1985) and the expression of functional antibody fragments in the periplasmic space of Escherichia coli (Skerra and Plückthun, 1988; Better et al., 1988) were the essential elements for the evolution of antibody phage display (McCafferty et al., 1990). The construction of large combinatorial libraries displaying human antibody fragments on filamentous phage as single-chain Fvs (scFvs) (McCafferty et al., 1990; Breitling et al., 1991) or Fab fragments (Barbas et al., 1991; Hoogenboom et al., 1991; Kang et al., 1991) enabled the selection of antigen binding clones from such libraries. The phagemid from selected phage clones could then be

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used to produce soluble antibody fragments (Fab or scFv) in E. coli (Hoogenboom et al., 1991). Recombinant antibodies are widely used for many biomedical, diagnostic and therapeutic purposes today. With the increasing demand for antibodies for target identification/validation programs and subsequent therapeutic applications, the high-throughput generation and targeted engineering of human antibodies by phage display are especially powerful with automated high-throughput ELISA screening technology (Krebs et al., 2001). However, a set of scFvs/Fabs may need to be analyzed in functional and surface plasmon resonance (SPR) assays to select the best candidates to convert to IgG. Crude scFv or Fab periplasmic preparations are usually not very stable and often are not pure enough or concentrated enough for these kinds of assays, so purification of scFv/Fab becomes necessary. In order to analyze a large number of samples, a high-throughput expression and purification procedure is needed. In this report, we describe an automated high-throughput expression and purification approach using the Qiagen BioRobot 3000 LS with modified application software. This automated procedure enabled us to rapidly evaluate antibody fragments in functional and surface plasmon resonance (SPR) assays and to prioritize candidates before converting them to IgG. We have used these procedures to make thousands of purified scFv/Fabs for several antibody maturation campaigns and significantly decreased the time needed to select the best candidates. 2. Materials and methods 2.1. Plasmids and E. coli strains TG1 was obtained from Cambridge Antibody Technologies (CAT, Cambridge, England). TOP10F’ and DH12S were purchased from Invitrogen (Carlsbad, CA), and Max10BF’ was purchased from BIO-RAD (Hercules, CA). The scFv expression vector pCANTAB6 was obtained from CAT and the Fab expression vector pFab 3 was generated in house. The D5 and X5 scFv clones (with HIV inhibition activity) were described previously (Miller et al., 2005). The Fab clones were generated during affinity maturation of an antibody to human IL13-Rα1. 2.2. scFv/Fab expression optimization In order to obtain the optimum expression conditions in different E. coli strains, the scFv clones D5, X5 and

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8B4 were transformed into TG1, TOP10F’, DH12S and Max10BF’ strains and small-scale (5 ml) expressions were performed at the following conditions: 1) IPTG (Teknova, Hollister, CA) final concentration of 0.25, 0.5, 0.75 and 1 mM; and 2) incubation temperature after induction: room temperature and 30 °C. 2.3. SDS-PAGE and immunoblotting Samples were heat denatured and size fractionated in 4–12% Novex NuPAGE Bis–Tris polyacrylamide gels (Invitrogen, Carlsbad, CA) with 1×MES SDS buffer (Invitrogen). MultiMark (Invitrogen) and/or MagicMark XP (Invitrogen) were used as size markers. Gels were stained with Bio-Safe Coomassie Stain (BIO-RAD). For Western blots, proteins were transferred to a nitrocellulose membrane (Invitrogen) using a BIO-RAD TransBlot SD semi-dry transfer cell (BIO-RAD). Immobilized proteins were detected using mouse anti-c-myc mAb (Roche, Nutley, NJ) and WesternBreeze kit (Invitrogen). 2.4. Modification of Qiagen application software and configuration The standard application program for His-tagged protein purification for Qiagen BioRobot 3000 LS (Qiagen, Valencia, CA) was modified as follows: 1) addition of a separate container for the lysis buffer (NPI10) containing Benzonase nuclease (Novagen, San Diego, CA) and complete EDTA-free protease inhibitor cocktail (Roche); 2) a new dispensing method for the lysis buffer; and 3) multi-step vacuum pressure and duration adjustments. Modifications 1) and 2) were for the small-scale protocol and modification 3) was for both small and large-scale protocols. 2.5. Automated high-throughput expression and purification The procedure for small-scale (3 ml) expression and purification started with stock 96-well plates generated from panning outputs. Each stock plate was used to inoculate a 96-well seed plate containing 100 μl of growth medium/well: 2% glucose (Fisher Scientific, Hampton, NH), 100 μg/ml ampicillin (Teknova) in LB (L Broth: NaCl 10 g/l, Bacto-Tryptone 10 g/l and Yeast Extract 5 g/l). After overnight growth of the seed plate at 37 °C with shaking at 150 rpm, 15 μl of the culture from each well was used to inoculate 3 ml of expression medium (0.1% glucose, 100 μg/ml ampicillin in LB) in the corresponding well of four 24-well expression plates (Whatman, Florham Park, NJ). The expression medium

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was dispensed into the plates with a μFill™ Microplate Reagent Dispenser with a dispense manifold for 24-well microplate (Bio-Tek Instruments, Inc., Winooski, VT). The expression plates were incubated at 37 °C with shaking at 150 rpm until an OD600 = 0.6–0.8 (about 2–3 h). Expression was induced by addition of IPTG to a final concentration of 0.5 mM using the μFill™ Microplate Reagent Dispenser and the plates incubated overnight with shaking at 150 rpm (at room temperature for the E. coli strain TG1 and at 30 °C for strains TOP10F’, DH12S, and Max10BF’). The overnight cultures were pelleted by centrifugation at 3000 g for 10 min and the pellets frozen at − 20 °C for at least 1 h. From this point, the procedure for “Medium-Scale Purification of 6×His-tagged Proteins under Native Conditions” was followed in the Qiagen Ni–NTA Superflow BioRobot Handbook (September 2002 edition). The four 24-well expression plates were loaded on the Qiagen BioRobot 3000 LS and the following steps were automated: 1) addition of lysozyme (Pierce, Rockford, IL) and lysis buffer (NPI-10) containing Benzonase nuclease and proteinase inhibitor cocktail; 2) preparation of Ni+ columns using Ni–NTA Superflow (Qiagen) while the culture pellets were incubated with lysis buffer for 30 min; 3) transfer of lysed samples from the four 24-well expression plates to a 96-well TurboFilter plate (Qiagen); 4) collection of clarified filtrate into a 96-well QiaFilter plate (Qiagen) containing Ni+ resin for IMAC purification; 5) IMAC wash steps and elution with Imidazole; and 6) collection of the eluted scFvs/Fabs in a 96-well block (Matrix Technologies, Hudson, NH). The 96-well block containing eluted scFvs/Fabs was removed from the robot for the buffer exchange steps. An aliquot of 400 μl from each well was transferred to the corresponding well of a MultiScreen Ultracel-10 plate (10,000 MW cut off, Millipore, Billerica, MA) by using a multichannel pipette. The Ultracel-10 plate was put on top of the 96well block, the unit was centrifuged at 3000 g for 55 min at 4 °C to remove the elution buffer, 350 μl of the desired final buffer was added to each well and the plate was centrifuged again. This step was repeated 2 more times to get complete buffer exchange. The final buffer was Dulbecco's PBS without Calcium and Magnesium (Invitrogen) for samples being tested in the singlecycle HIV infectivity assay. The samples were used immediately for functional and kinetic based assays or they were stored at − 20 °C for future analysis. For large-scale purification (500 ml), up to 24 samples can be processed in each run. The 500 ml cultures were grown, induced, and expressed overnight as described for small-scale purification. Cells were

harvested by centrifugation at 4000 g for 20 min at 4 °C, and the drained cell pellets stored at either − 20 °C or − 70 °C until scFv/Fab purification. Cell pellets were lysed before the samples were put on the robot. This differs from the procedure for the small-scale samples where lysis is part of the robotic program. Another difference between the two procedures is that the 1.5 ml Ni–NTA Superflow Columns (Qiagen) are pre-packed. The robot was used for column purification only, following the protocol for “Large-Scale Purification of 6×His-tagged Proteins under native Conditions” in the Qiagen Ni–NTA Superflow BioRobot Handbook (September 2002 edition). After column elution, the 24-well plates were removed from the robot and the eluted scFvs/Fabs were transferred to Amicon Ultra-15 Centrifugal Filter Units (10,000 NMWL, Millipore) for buffer exchange. The Centrifugal Filter Units were filled with the desired final buffer and centrifuged at 3000 g for 14 min at 4 °C. This step was repeated 2 more times. The final samples were transferred to 2.0 ml Protein LoBind microcentrifuge tubes (Eppendorf, Westbury, NY) and kept at −20 °C for short term storage or − 70 °C for long term storage before analysis. 2.6. Single-cycle HIV infectivity assay This assay is based on the assay described by Joyce et al. (2002) with modifications. Briefly, P4/R5 cells (HeLa cells expressing endogenous CXCR4 and stably transfected to express CD4 and CCR5 which also contain an integrated β-galactosidase reporter gene under control of an HIV LTR promoter) maintained in DMEM, phenol red-free Dulbecco's modified Eagle's medium (Invitrogen), 10% fetal bovine serum, 1% penicillin/streptomycin (Invitrogen) were seeded in 96-well plates at 2.5 × 103 cells/well and infected the following day with the HXB2 strain of HIV-1 (Advanced Biotechnology Inc., Bethesda, MD) in the presence of titrations of scFvs and D5 IgG. Cells with and without virus addition were used to establish maximal and minimal infectivity signals, respectively. After 48 h of infection, cells were lysed and β-galactosidase was detected using Gal Screen™ chemiluminescent substrate (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. Data were obtained using a Dynex luminometer, entered into KaleidaGraph (Synergy Software, PA) and parameters such as IC50 values were obtained through data plotting and curve fitting. Although compounds that block any step of the early HIV life cycle including entry, reverse transcription, integration, and tat-mediated transcription can all inhibit production of β-galactosidase, scFvs and IgGs are considered to act specifically

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at a pre-entry step by binding to HIV and inhibiting its entry into the host cell, and therefore in this study the single-cycle HIV infectivity assay is used to evaluate the effect of scFvs in blocking HIV entry specifically (Joyce et al., 2002). 2.7. BIAcore analysis All surface plasmon resonance (SPR) data was generated using a BIAcore 3000 instrument (Piscataway, NJ). Working concentrations of 16.4 μg/ml of hIL-13Rα1 and 5 μg/ml of BSA in sodium acetate pH5 were amine coupled to lanes 2 and 1 of a CM5 (carboxymethylated dextran matrix) BIAcore chip respectively. Each lane was coupled with approximately 500RUs. Using a flow rate of 60 μl/min, each sample (diluted 2 fold in HBS-EP running buffer) was injected over the surface for 1 min and allowed to dissociate for 15 min. After each run, the surface was regenerated with 10 μl of 100 mM HCL at 60 μl/min to remove remaining bound Fab. Analysis was performed in Biaevaluation analysis software, using the off-rate to rank clones. 3. Results 3.1. Optimization of growth/induction conditions Small-scale D5 scFv cultures (5 ml) grown in test tubes were used to determine the best temperature to grow the cells after induction by different concentrations of IPTG. The results indicated that the concentration of IPTG between 0.25 and 1.00 mM made no difference in expression in the four strains tested (Fig. 1A and B and data not shown); however, the growth temperature after induction did make a difference. For the TG1 strain, the optimal growth temperature after induction was at room temperature (Fig. 1A). For the TOP10F’, Max10BF’, and DH12S strains the optimal temperature was 30 °C. Fig. 1B shows TOP10F’ as an example. The yield from any of the strains is comparable when done at the optimal temperature (Fig. 1C), but the high molecular weight bands are only generated when expression is from TG1. Similar expression was seen with 2 different scFv clones (data not shown). Since each well of a 24-deepwell plate can hold up to 5 ml of expression medium, we tested 3 and 5 ml/well of cultures of TG1 strain using the standard Qiagen protocol for 3–5 ml purification found in the Qiagen Ni–NTA Superflow BioRobot Handbook (September 2002 edition). For both volume groups, samples were lysed with and without adding Benzonase. The yields from both 3 and 5 ml cultures were improved by adding

Fig. 1. Western blot analysis of crude D5 scFv periplasmic preparation taken from 5 ml cultures. Samples were induced with the indicated concentration of IPTG and allowed to express overnight at the temperature indicated. Cells were osmotically shocked to release scFvs from the perisplasm and equal volumes loaded on the gel to show relative yields. All scFv constructs contain myc-tags, and the Western is developed with an anti-myc mAb. A) Expression in TGI strain, B) expression in TOP10 F’ strain, C) expression in TGI strain at room temperature and in TOP10 F’, MAX10BF’, and DH12S strains at 30 °C. Two concentrations of each sample have been loaded to show the yields are comparable from all strains. Lanes 1–4 were undiluted samples and lanes 5–8 are diluted 1/10.

Benzonase. The yield from 3 ml cultures with Benzonase was only slightly less than the yield from a 5 ml culture with Benzonase (on average, 8.2 μg/ml for 3 ml and 9.6 μg/ml for 5 ml, data not shown), and the columns clogged more often with the 5 ml cultures.

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Therefore, 3 ml culture/well format was chosen for expression in this system. 3.2. Automated high-throughput expression and purification When using the standard Qiagen protocol and software for operating the BioRobot 3000 LS, we observed many clogged columns at multiple steps. Therefore, multiple modifications (listed in Materials and methods) were made to the protocol and application software to make the automated steps run smoothly. Using the modified protocol/software, 3 ml/well cultures (D5 scFv in TG1 strain) in 24-well plates were purified with and without adding Benzonase. As expected, the addition of Benzonase improved the yields. The yield from 3 ml/well cultures with Benzonase was 13.3 ± 2.4 μg/well and without Benzonase was 9.7 ± 3.8 μg /well (Table 1). The yields were also improved by the software modifications. The largest yield of scFv was obtained from using the modified protocol/software with Benzonase. This combination completely eliminated the clogged columns. A diagram of the high-throughput system for small-scale samples is summarized in Fig. 2. Those steps (6–8) highlighted in blue are done by the Qiagen Robot 3000 LS and those steps (1–5, 9–12) highlighted in yellow are done manually. The steps with bold blue arrows could be done robotically if additional equipment is available. 3.3. Production of scFv preparations for functional assays In an affinity maturation campaign for D5, an antibody that inhibits HIV viral fusion to host cells by binding to the HR1 region of gp41 (Miller et al., 2005), scFvs needed to be screened in a single-cycle HIV Table 1 Comparison of the yields of D5 scFv from 3 ml/well cultures in 24well plates Sample (3 ml/well)

Modified protocol

Standard protocol

% of yield increased

Average yield (μg/well)

Average yield (μg/well)

Sample with Benzonase Sample without Benzonase

13.3 ± 2.4

8.2 ± 2.4

62.2

9.7 ± 3.8

4.2 ± 1.3

131.0

Yields of D5 scFv are shown from both the standard Qiagen protocol and from the modified protocol, with and without adding Benzonase in the purification process. The yields were calculated from 12 samples in each group.

infectivity assay (Joyce et al., 2002). It was known from earlier studies that D5 scFvs from crude periplasmic preparations were not concentrated enough to be detected in this assay. In order to utilize scFv preparations in this assay, scFv concentrations of 2 μg/ 20–30 μl were needed. The yield of D5 scFv in TG1 strain was 13.3 ± 2.4 μg/well (see Table 1) using the small-scale automated protocol. This yield is enough to be detected in the single-cycle HIV infectivity assay, as long as the final concentration is adjusted in the final buffer exchange step. In order to use the highest concentrations of the purified scFvs in the assay, samples were buffer exchanged into the culture medium used in the assay. Initially, samples of D5 and the appropriate control scFvs were tested in the assay. The samples were concentrated enough to show HIV inhibition in 3 of the 4 sample dilutions used (data not shown). We also found that samples could be stored at − 70 °C before testing in the assay with no loss of activity. Small-scale automated preparations were made of over 1200 D5 maturation scFv candidates and screened in the single-cycle HIV infectivity assay. An example of the data obtained in these initial screens is shown in Fig. 3. Duplicate samples of the parental D5 scFv were titrated along with the maturation candidates for a comparison. A group of clones which showed greater viral inhibition (lower RLU values) were chosen for a final screen. A final screen was done with large-scale automated purification. Since the concentration of the scFv was needed to determine IC50 values in the HIV inhibition assay, the purified samples were run on LabChip90 Automated Electrophoresis System (Caliper Life Sciences, Hopkinton, MA; data not shown) to determine the scFv concentrations. Fig. 4 shows the results of two final candidates compared to the parental D5 scFv and to control X5 scFv (known have greater activity than D5 as a scFv). Clone 1-20 is less active than the parent, while clone 1-25 shows activity that is better than X5. These IC50 values were used to select the candidates for conversion into IgG. 3.4. Production of Fab preparations for kinetic based assay In a maturation campaign for antibodies to human IL13R-1α, Fabs needed to be screened by SPR. Fabs expressed in the TG1 strain were purified using the same small-scale protocol described for scFv samples. Two separate batches of purified Fabs have given consistently good yields (Table 2). In the first batch, 43

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Fig. 2. Diagram of expression and purification steps using the Qiagen BioRobot 3000 LS. Those steps highlighted in blue are done by the Qiagen Robot 3000 LS and those steps highlighted in yellow are done manually. The steps with bold blue arrows could be done robotically if additional equipment is available.

maturation candidates were purified, giving a yield of 12 ± 3.7 μg/well of culture. In the second batch, 27 maturation candidates were done in duplicate wells (total 54 samples). The yield for these Fab preparations is 11.6 ± 1.5 μg/well of culture. The final concentration of the samples depends upon the final volume after buffer exchange. For these samples, the volume was either 140 μl or 90 μl, giving concentrations of about 100 ng/μl. A Western blot of randomly picked samples from both batches shows uniform band intensity of the

samples thus confirming the protein detection results (Fig. 5). There is no evidence of high molecular weight proteins that react to anti-myc in these Fab preps. A total of 70 maturation candidates were tested for their ability to bind to IL13R-α1 coupled to a BIAcore chip and the results were used to rank clones according to their off-rates within a run. Fig. 6 shows the results of several of the maturation candidates compared to the parental Fab. The concentration of these Fab samples was not known, so the on-rates could not be

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Fig. 3. Single-cycle HIV infectively assay results on scFv clones purified by the small-scale protocol. Eleven D5 maturation clones (B2–B12) were assayed with parental D5 in duplicate (D5-1, D5-2). Samples were diluted 2-fold, and titrated over 4 concentrations. The curves from the duplicate parental samples and the curves of the best maturation clones are indicated by the arrows.

determined. Note that clone 19 has a different on-rate than the other samples. The relative off-rate of clone 19 is similar to the parental kd value, but those of clone 9 (about 2-fold lower) and clone 6 (more than 10-fold lower) show affinity maturation. The amount of Fab obtained from one 3 ml preparation was enough to do two BIAcore runs. Duplicate or triplicate wells of each clone can be done to obtain enough Fab for multiple runs if needed.

4. Discussion This study was undertaken to optimize expression of scFv and Fab in E. coli and to increase the throughput of their purification. Growth temperature after induction is critical for optimal expression of both soluble scFv and Fab. The E. coli strain determines whether the optimal expression is at room temperature (TG1) or 30 °C (TOP10F’, DH12S, and MAX10BF’). The scFv yield

Fig. 4. Single-cycle HIV infectivity assay results on scFv clones purified by the large-scale protocol. Two maturation candidates (1-20 and 1-25) were tested in the same run as the parental D5 scFv and the control X5 scFv. Concentrations of purified scFv were known, so IC50 values were determined. Samples were diluted 10-fold and titrated over 10 concentrations.

B. Su et al. / Journal of Immunological Methods 322 (2007) 94–103 Table 2 Yields of Fabs from a maturation campaign after high-throughput expression and purification Sample

Fabs (43 × 3 ml/well) Fabs (54 × 3 ml/well)

Average concentration (μg/ml)

Average volume (μl)

Average yield (μg/well)

85.9 ± 26.3

140

12 ± 3.7

128.7 ± 16.5

90

11.6 ± 1.5

Two batches of clones were done. In the first batch, 43 different clones were prepared and in the second batch, 27 different clones were prepared in duplicate. All expression (3 ml/well in 24-well plates) was done at room temperature, overnight after induction. The average yield from all the preparations was 12 μg/well.

from the pCANTAB6 vector in any of the strains is comparable when expression is done at the optimal temperature (Fig. 1C), but there are additional high molecular weight proteins that react with anti-myc mAb in preparations from TG1. Although the additional high molecular weight bands are not visible in the samples from TG1 grown at 30 °C (Fig. 1A), they are produced at both temperatures. This gel was loaded to show relative yields of the scFv and the amount of scFv made at 30 °C is too low for these bands to be visible. We did not see additional high molecular weight bands when Fabs were expressed in TG1 from the vector pFAB3 (see Fig. 5) in the affinity maturation of an IL13-R1α antibody, so the additional bands appear to be a product of D5 maturation candidates expressed in the pCANTAB6/TG1 combination only. For small-scale purification, optimal yields of scFv were obtained from 3 ml cultures in 24-deepwell plates grown on a standard shaker. The yields of scFv per ml of culture was comparable to the yields obtained from 1 liter cultures grown in flasks and manually purified using a standard IMAC column (data not show), suggesting that the aeration in 3 ml cultures is very good. The standard Qiagen application protocols for both small (3 ml) and large (500 ml) scale purification did not work well for our purpose. Significant modification of the protocol and software was needed to get the system to run smoothly. The major problems with the standard small-scale purification protocol included: 1) clogged columns at multi-steps during the sample runs; 2) waste of a huge amount of Benzonase and Complete EDTAfree protease inhibitor cocktail in the initial column wash steps because they were added to a NPI-10 bottle (500 ml) and distributed through Tubing 1; and 3) pollution of Tubing 1 by Benzonase which could not be easily removed. The combination of modified condi-

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tions allowed samples to smoothly go through the whole purification process. They also significantly reduced the use of Benzonase and Complete EDTA-free protease inhibitor cocktail and kept Tubing 1 connecting the four probes free of Benzonase. This is important if the BioRobot 3000 LS is to be used for multiple applications like DNA mini preparations. Most importantly, these modifications greatly increased yields and enabled us to use the purified scFvs and Fabs in downstream assays that required a high concentration. The protocol for small-scale growth, induction and purification is only partly automated. The initial step of sample transfer from a 96-well stock plate to a 96-well seed plate is done manually. This step could be automated by using a replication robot like GeneTAC G3 from Genomic Solutions (Ann Arbor, MI) or MegaPix from Genetix Ltd (Hampshire, UK). The second step of inoculating expression plates (usually four 24-well plates) from the 96-well seed plates is also done manually. This step could also be automated by using a liquid handling robot like Mutiprobe II from PerkinElmer (Wellesley, MA) which could easily handle the complexity of transferring cultures from a 96-well plate to four 24-well plates. Automating these steps would greatly reduce the potential for human error and increase efficiency. The μFill™ Microplate Reagent Dispenser with a dispense manifold for 24-well microplate was very useful for filling 24-well plates with the desired medium and for adding IPTG when cultures were ready for induction.

Fig. 5. Western blot analysis of Fabs expressed and purified by the automated high-throughput procedure. Random samples from batch 1 were run on reducing gels to determine the quality of the product. Lanes 1–7 Fabs in TGI strain (3 ml culture/well) expressed at room temperature overnight after induction. All Fab constructs contain myctags on heavy chains so the Westerns were developed with anti-myc mAb. These reducing gels detect the heavy chain at ∼30 kDa, and show that the yields of the samples are uniform.

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Fig. 6. BIAcore data on Fab clones expressed and purified by the automated high-throughput procedures. The maturation clones are compared to the parental Fab in the same run. Concentrations of the Fab samples were not determined, so only the relative off-rates are analyzed. The first 5 min of the 15 min dissociation time for each clone is shown, which is sufficient for off-rate comparison.

After samples are processed by the robot, they are loaded onto a MultiScreen Ultracel-10 plate for buffer exchange. The advantages of using Ultracel-10 plate are the following: 1) it is a simple process and requires only a bench top centrifuge; 2) it is easy to control the final volume; and 3) it is possible to exchange purified scFv/Fab into any buffer based on the need in downstream assays. This was extremely important for screening D5 maturation samples in the single-cycle HIV infectivity assay where a high concentration of scFv was needed and was best achieved by exchanging the samples into the medium used in the cell-based assay. The large-scale purification protocol is also only partly automated. Growth and induction of the cultures are done manually in 2 l shake flasks and the cells are harvested by centrifugation. Cell pellets are kept at − 20 °C or − 70 °C overnight, or they can be stored longer without affecting yields. All that is needed is one freeze–thaw cycle to help lyse the cells. Cell pellets are lysed manually before they are put on the robot. Only the column purification is done robotically. The eluted samples are removed from the robot and manually transferred to Amicon Ultra-15 centrifugal filter units (10 k NMWL) for buffer exchange. Buffer exchange is accomplished by multiple rounds of centrifugation, usually 3 times. None of the manual steps in this protocol can be automated without custom designed instruments. Although the process is only partly automated, as many as 24 different samples can be purified in one run. Fabs and scFvs that are purified by

this procedure can be further purified on FPLC if highly purified samples are needed for downstream studies. Concentrations of the scFvs and Fabs in both smalland large-scale purification can be determined with accessory equipment such as the Caliper LabChip90 Automated Electrophoresis System or Agilent 5100 ALP (Automated Lab-on-a-chip Platform). Both instruments require only a 4–5 μl sample for analysis and can handle multiple 96-well plates. This method is more accurate than a standard protein determination since these samples do contain additional proteins other than the scFv or Fab. Use of this equipment to determine scFv/Fab concentrations will extend their use to assays that require an accurate concentration for ranking. The automated high-throughput expression and purification system has proven to be a powerful and practical way to purify antibody fragments (scFv and Fab) for a variety of purposes. One full-time employee can process up to 300 small-scale samples and up to 72 large-scale samples per week. Most importantly it enables rapid evaluation of scFv and Fab in functional assays and kinetic based assays as a way to prioritize candidates before converting them to IgG. Acknowledgments The authors would like to thank Ms. Vicki King for panning and ELISA screening the D5 maturation samples, Dr. Fubao Wang and Mr. Peter Haytko for LabChip90 (Caliper) analysis and the Qiagen technical support team for technical assistance.

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