Characterization and design of chemically selective cationic displacers using a robotic high‐throughput screen

A robotic high‐throughput displacer screen was developed and employed to identify chemically selective displacers for several protein pairs in cation exchange chromatography. This automated screen enabled the evaluation of a wide range of experimental conditions in a relatively short period of time. Displacers were evaluated at multiple concentrations for these protein pairs, and DC‐50 plots were constructed. Selectivity pathway plots were also constructed and different regimes were established for selective and exclusive separations. Importantly, selective displacement was found to be conserved for multiple protein pairs, demonstrating the technique to be applicable for a range of protein systems. Although chemically selective displacers were able to separate protein pairs that had similar retention in ion exchange but different surface hydrophobicities, they were not able to distinguish protein pairs with similar surface hydrophobicities. This corroborates that displacer‐protein hydrophobic interactions play an important role for this class of selective displacers. Important functional group moieties were established and efficient displacers were identified. These results demonstrate that the design of chemically selective displacers requires a delicate balance between the abilities to displace proteins from the resin and to bind to a selected protein. The use of robotic screening of displacers will enable the extension of chemically selective displacement chromatography beyond hydrophobic displacer‐protein interactions to other secondary interactions and more selective displacement systems. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

A practical strategy for using miniature chromatography columns in a standardized high‐throughput workflow for purification development of monoclonal antibodies

The emergence of monoclonal antibody (mAb) therapies has created a need for faster and more efficient bioprocess development strategies in order to meet timeline and material demands. In this work, a high‐throughput process development (HTPD) strategy implementing several high‐throughput chromatography purification techniques is described. Namely, batch incubations are used to scout feasible operating conditions, miniature columns are then used to determine separation of impurities, and, finally, a limited number of lab scale columns are tested to confirm the conditions identified using high‐throughput techniques and to provide a path toward large scale processing. This multistep approach builds upon previous HTPD work by combining, in a unique sequential fashion, the flexibility and throughput of batch incubations with the increased separation characteristics for the packed bed format of miniature columns. Additionally, in order to assess the applicability of using miniature columns in this workflow, transport considerations were compared with traditional lab scale columns, and performances were mapped for the two techniques. The high‐throughput strategy was utilized to determine optimal operating conditions with two different types of resins for a difficult separation of a mAb monomer from aggregates. Other more detailed prediction models are cited, but the intent of this work was to use high‐throughput strategies as a general guide for scaling and assessing operating space rather than as a precise model to exactly predict performance. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:626–635, 2014     

His tag effect on solubility of human proteins produced in Escherichia coli: a comparison between four expression vectors

We have compared four different vectors for expression of proteins with N- or C-terminal hexahistidine (His6) tags in Escherichia coli by testing these on 20 human proteins. We looked at a total recombinant protein production levels per gram dry cell weight, solubility of the target proteins, and yield of soluble and total protein when purified by immobilized metal ion affinity purification. It was found that, in general, both N- and C-terminal His6 tags have a noticeable negative affect on protein solubility, but the effect is target protein specific. A solubilizing fusion tag was able to partly counteract this negative effect. Most target proteins could be purified under denaturing conditions and about half of the proteins could be purified under physiological conditions. The highest protein production levels and yield of purified protein were obtained from a construct with C-terminal His tag. We also observe a large variation in cell growth rate, which we determined to be partly caused by the expression vectors and partly by the targets. This variation was found to be independent of the production level, solubility and tertiary structure content of the target proteins.     

High‐throughput mAb expression and purification platform based on transient CHO

A high‐cell‐density transient transfection system was recently developed in our laboratory based on a CHO‐GS‐KO cell line. This method yields monoclonal antibody titers up to 350 mg/L from a simple 7‐day process, in volumes ranging from 2 mL to 2 L. By performing transfections in 24‐deep‐well plates, a large number of mAbs can be expressed simultaneously. We coupled this new high‐throughput transfection process to a semiautomated protein A purification process. Using a Biomek FX^p liquid handling robot, up to 72 unique mAbs can be simultaneously purified. Our primary goal was to obtain >0.25 mg of purified mAb at a concentration of >0.5 mg/mL, without any concentration or buffer‐exchange steps. We optimized both the batch‐binding and the batch elution steps. The length of the batch‐binding step was important to minimize mAb losses in the flowthrough fraction. The elution step proved to be challenging to simultaneously maximize protein recovery and protein concentration. We designed a variable volume elution strategy based on the average supernatant titer. Finally, we present two case studies. In the first study, we produced 56 affinity maturation mAb variants at an average yield of 0.33 ± 0.05 mg (average concentration of 0.65 ± 0.10 mg/mL). In a second study, we produced 42 unique mAbs, from an early‐stage discovery effort, at an average yield of 0.79 ± 0.31 mg (average concentration of 1.59 ± 0.63 mg/mL). The combination of parallel high‐yielding transient transfection and semiautomated high‐throughput protein A purification represents a valuable mAb drug discovery tool. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:239–247, 2015     

High-throughput purification of recombinant proteins using self-cleaving intein tags

High throughput methods for recombinant protein production using E. coli typically involve the use of affinity tags for simple purification of the protein of interest. One drawback of these techniques is the occasional need for tag removal before study, which can be hard to predict. In this work, we demonstrate two high throughput purification methods for untagged protein targets based on simple and cost-effective self-cleaving intein tags. Two model proteins, E. coli beta-galactosidase (βGal) and superfolder green fluorescent protein (sfGFP), were purified using self-cleaving versions of the conventional chitin-binding domain (CBD) affinity tag and the nonchromatographic elastin-like-polypeptide (ELP) precipitation tag in a 96-well filter plate format. Initial tests with shake flask cultures confirmed that the intein purification scheme could be scaled down, with >90% pure product generated in a single step using both methods. The scheme was then validated in a high throughput expression platform using 24-well plate cultures followed by purification in 96-well plates. For both tags and with both target proteins, the purified product was consistently obtained in a single-step, with low well-to-well and plate-to-plate variability. This simple method thus allows the reproducible production of highly pure untagged recombinant proteins in a convenient microtiter plate format.     

InsectDirectTM System: Rapid, High-level Protein Expression and Purification from Insect Cells

A fundamental challenge in high-throughput (HT) expression screening is to rapidly identify the appropriate expression system for many targets in parallel. Known or unknown open reading frames (ORFs) are typically amplified by PCR and then cloned into a variety of vectors, producing recombinants used to direct target protein expression in Escherichia coli, insect cells, mammalian cells, or yeast. To facilitate rapid expression and purification in Spodoptera insect cells (Sf9), we developed transient expression vectors that include an enterokinase cleavage site immediately upstream of a ligation-independent cloning site (Ek/LIC). We also developed a high-efficiency insect cell transfection reagent, and automation-compatible fusion protein purification system for insect cells to facilitate expression screening and protein production. Positive clones identified from the small-scale screening were subjected to a larger scale production. Using this InsectDirect^TM approach, we successfully expressed milligram quantities of different human proteins including heat shock proteins, phospholipases, and protein kinases.     

Bispecific antibody process development: Assembly and purification of knob and hole bispecific antibodies

Production of knob and hole dual light chain bispecific antibodies poses several unique challenges for development of a feasible industrial scale manufacturing process. We developed an efficient process for the assembly and purification of knob and hole dual light chain bispecific antibodies. Two distinct half‐antibodies targeting two different antigens were expressed separately in Escherichia coli cells and captured independently using Protein A chromatography. When combined, the knob and hole mutations in the CH3 domains promoted heterodimer formation. The hinge region disulfides were reduced and reoxidized to form the disulfide bridge between the two complementary half antibodies. Unreacted half antibodies, noncovalently linked homodimers, covalently linked homodimers, and noncovalently linked heterodimers are impurities closely related to the product of interest and are challenging to remove by standard processes. Characterization of the molecular properties of the half antibodies and high‐throughput screening predicted column chromatography performance and allowed for rapid development of downstream purification steps for removal of unique product‐related and process‐related impurities. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:397–404, 2018