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 process development of purification alternatives for the protein avidin

With an increased number of applications in the field of the avidin‐biotin technology, the resulting demand for highly‐purified protein avidin has drawn our attention to the purification process of avidin that naturally occurs in chicken egg white. The high‐throughput process development (HTPD) methodology was exploited, in order to evaluate purification process alternatives to commonly used ion‐exchange chromatography. In a high‐throughput format, process parameters for aqueous two‐phase extraction, selective precipitation with salts and polyethylene glycol, and hydrophobic interaction and mixed‐mode column chromatography experiments were performed. The HTPD strategy was complemented by a high‐throughput tandem high‐performance liquid chromatography assay for protein quantification. Suitable conditions for the separation of avidin from the major impurities ovalbumin, ovomucoid, ovotransferrin, and lysozyme were identified in the screening experiments. By combination of polyethylene glycol precipitation with subsequent resolubilization and separation in a polyethylene glycol/sulfate/sodium chloride two‐phase system an avidin purity of 77% was obtained with a yield >90% while at the same time achieving a significant reduction of the process volume. The two‐phase extraction and precipitation results were largely confirmed in larger scale with scale‐up factors of 230 and 133, respectively. Seamless processing of the avidin enriched bottom phase was found feasible by using mixed‐mode chromatography. By gradient elution a final avidin purity of at least 97% and yield >90% was obtained in the elution pool. The presented identification of a new and beneficial alternative for the purification of the high value protein thus represents a successful implementation of HTPD for an industrially relevant purification task. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:957–973, 2015     

High‐throughput screening techniques for rapid PEG‐based precipitation of IgG4 mAb from clarified cell culture supernatant

Locating optimal protein precipitation conditions for complex biological feed materials is problematic. This article describes the application of a series of high‐throughput platforms for the rapid identification and selection of conditions for the precipitation of an IgG₄ monoclonal antibody (mAb) from a complex feedstock using only microliter quantities of material. The approach uses 96‐microwell filter plates combined with high‐throughput analytical methods and a method for well volume determination for product quantification. The low material, time and resource requirements facilitated the use of a full factorial Design of Experiments (DoE) for the rapid investigation into how critical parameters impact the IgG₄ precipitation. To aid the DoE, a set of preliminary range‐finding studies were conducted first. Data collected through this approach describing Polyethylene Glycol (PEG) precipitation of the IgG₄ as a function of mAb concentration, precipitant concentration, and pH are presented. Response surface diagrams were used to explore interactions between parameters and to inform selection of the most favorable conditions for maximum yield and purification. PEG concentrations required for maximum yield and purity were dependant on the IgG₄ concentration; however, concentrations of 14 to 20% w/v, pH 6.5, gave optimal levels of yield and purity. Application of the high‐throughput approach enabled 1,155 conditions to be examined with less than 1 g of material. The level of insights gained over such a short time frame is indicative of the power of microwell experimentation in allowing the rapid identification of appropriate processing conditions for key bioprocess operations. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

High‐throughput miniaturized bioreactors for cell culture process development: Reproducibility,scalability, and control

Decreasing the timeframe for cell culture process development has been a key goal toward accelerating biopharmaceutical development. Advanced Microscale Bioreactors (ambr?) is an automated micro‐bioreactor system with miniature single‐use bioreactors with a 10–15 mL working volume controlled by an automated workstation. This system was compared to conventional bioreactor systems in terms of its performance for the production of a monoclonal antibody in a recombinant Chinese Hamster Ovary cell line. The miniaturized bioreactor system was found to produce cell culture profiles that matched across scales to 3 L, 15 L, and 200 L stirred tank bioreactors. The processes used in this article involve complex feed formulations, perturbations, and strict process control within the design space, which are in‐line with processes used for commercial scale manufacturing of biopharmaceuticals. Changes to important process parameters in ambr? resulted in predictable cell growth, viability and titer changes, which were in good agreement to data from the conventional larger scale bioreactors. ambr? was found to successfully reproduce variations in temperature, dissolved oxygen (DO), and pH conditions similar to the larger bioreactor systems. Additionally, the miniature bioreactors were found to react well to perturbations in pH and DO through adjustments to the Proportional and Integral control loop. The data presented here demonstrates the utility of the ambr? system as a high throughput system for cell culture process development. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:718–727, 2014     

High‐throughput ion exchange purification of positively charged recombinant protein in the presence of negatively charged dextran sulfate

Product quality analyses are critical for developing cell line and bioprocess producing therapeutic proteins with desired critical product quality attributes. To facilitate these analyses, a high‐throughput small‐scale protein purification (SSP) is required to quickly purify many samples in parallel. Here we develop an SSP using ion exchange resins to purify a positively charged recombinant growth factor P1 in the presence of negatively charged dextran sulfate supplemented to improve the cell culture performance. The major challenge in this work is that the strong ionic interaction between P1 and dextran sulfate disrupts interaction between P1 and chromatography resins. To solve this problem, we develop a two‐step SSP using Q Sepharose Fast Flow (QFF) and SP Sepharose XL (SPXL) resins to purify P1. The overall yield of this two‐step SSP is 78%. Moreover, the SSP does not affect the critical product quality attributes. The SSP was critical for developing the cell line and process producing P1. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:516–520, 2014     

Advances in clone selection using high‐throughput bioreactors

Effective clone selection is a crucial step toward developing a robust mammalian cell culture production platform. Currently, clone selection is done by culturing cells in well plates and picking the highest producers. Ideally, clone selection should be done in a stirred tank bioreactor as this would best replicate the eventual production environment. The actual number of clones selected for future evaluation in bioreactors at bench‐scale is limited by the scale‐up and operational costs involved. This study describes the application of miniaturized stirred high‐throughput bioreactors (35 mL working volume; HTBRs) with noninvasive optical sensors for clone screening and selection. We investigated a method for testing several subclones simultaneously in a stirred environment using our high throughput bioreactors (up to 12 clones per HTBR run) and compared it with a traditional well plate selection approach. Importantly, it was found that selecting clones solely based on results from stationary well plate cultures could result in the chance of missing higher producing clones. Our approach suggests that choosing a clone after analyzing its performance in a stirred bioreactor environment is an improved method for clone selection. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

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     

High‐throughput sequencing of Hop stunt viroid‐derived small RNAs from cucumber leaves and phloem

Small RNA (sRNA)‐guided processes, referred to as RNA silencing, regulate endogenous and exogenous gene expression. In plants and some animals, these processes are noncell autonomous and can operate beyond the site of initiation. Viroids, the smallest self‐replicating plant pathogens known, are inducers, targets and evaders of this regulatory mechanism and, consequently, the presence of viroid‐derived sRNAs (vd‐sRNAs) is usually associated with viroid infection. However, the pathways involved in the biogenesis of vd‐sRNAs are largely unknown. Here, we analyse, by high‐throughput pyrosequencing, the profiling of the Hop stunt viroid (HSVd) vd‐sRNAs recovered from the leaves and phloem of infected cucumber (Cucumis sativus) plants. HSVd vd‐sRNAs are mostly 21 and 22 nucleotides in length and derived equally from plus and minus HSVd RNA strands. The widespread distribution of vd‐sRNAs across the genome reveals that the totality of the HSVd RNA genome contributes to the formation of vd‐sRNAs. Our sequence data suggest that viroid‐derived double‐stranded RNA functions as one of the main precursors of vd‐sRNAs. Remarkably, phloem vd‐sRNAs accumulated preferentially as 22‐nucleotide species with a consensus sequence over‐represented. This bias in size and sequence in the HSVd vd‐sRNA population recovered from phloem exudate suggests the existence of a selective trafficking of vd‐sRNAs to the phloem tissue of infected cucumber plants.     

Development of a high‐throughput microscale cell disruption platform for Pichia pastoris in rapid bioprocess design

The time and cost benefits of miniaturized fermentation platforms can only be gained by employing complementary techniques facilitating high‐throughput at small sample volumes. Microbial cell disruption is a major bottleneck in experimental throughput and is often restricted to large processing volumes. Moreover, for rigid yeast species, such as Pichia pastoris, no effective high‐throughput disruption methods exist. The development of an automated, miniaturized, high‐throughput, noncontact, scalable platform based on adaptive focused acoustics (AFA) to disrupt P. pastoris and recover intracellular heterologous protein is described. Augmented modes of AFA were established by investigating vessel designs and a novel enzymatic pretreatment step. Three different modes of AFA were studied and compared to the performance high‐pressure homogenization. For each of these modes of cell disruption, response models were developed to account for five different performance criteria. Using multiple responses not only demonstrated that different operating parameters are required for different response optima, with highest product purity requiring suboptimal values for other criteria, but also allowed for AFA‐based methods to mimic large‐scale homogenization processes. These results demonstrate that AFA‐mediated cell disruption can be used for a wide range of applications including buffer development, strain selection, fermentation process development, and whole bioprocess integration. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:130–140, 2018     

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