Application of the Protein Maker as a platform purification system for therapeutic antibody research and development.

Within the research and development environment, higher throughput, parallelized protein purification is required for numerous activities, from small scale purification of monoclonal antibodies (mAbs) and antibody fragments for in vitro and in vivo assays to process development and optimization for manufacturing. Here, we describe specific applications and associated workflows of the Protein Maker liquid handling system utilized in both of these contexts. To meet the requirements for various in vitro assays, for the identification and validation of new therapeutic targets, small quantities of large numbers of purified antibodies or antibody fragments are often required. Reducing host cell proteins (HCP) levels following capture with Protein A by evaluating various wash buffers is an example of how parallelized protein purification can be leveraged to improve a process development outcome. Stability testing under various conditions of in-process intermediates, as an example, the mAb product from a clarified harvest, requires parallelized protein purification to generate concurrent samples for downstream assays. We have found that the Protein Maker can be successfully utilized for small-to-mid scale platform purification or for process development applications to generate the necessary purified protein samples. The ability to purify and buffer exchange up to 24 samples in parallel offers a significant reduction in time and cost per sample compared to serial purification using a traditional FPLC system. By combining the Protein Maker purification system with a TECAN Freedom EVO liquid handler for automated buffer exchange we have created a new, integrated platform for a variety of protein purification and process development applications.

A microfluidic, high throughput protein crystal growth method for microgravity.

The attenuation of sedimentation and convection in microgravity can sometimes decrease irregularities formed during macromolecular crystal growth. Current terrestrial protein crystal growth (PCG) capabilities are very different than those used during the Shuttle era and that are currently on the International Space Station (ISS). The focus of this experiment was to demonstrate the use of a commercial off-the-shelf, high throughput, PCG method in microgravity. Using Protein BioSolutions' microfluidic Plug Maker™/CrystalCard™ system, we tested the ability to grow crystals of the regulator of glucose metabolism and adipogenesis: peroxisome proliferator-activated receptor gamma (apo-hPPAR-γ LBD), as well as several PCG standards. Overall, we sent 25 CrystalCards™ to the ISS, containing ~10,000 individual microgravity PCG experiments in a 3U NanoRacks NanoLab (1U = 10(3) cm.). After 70 days on the ISS, our samples were returned with 16 of 25 (64%) microgravity cards having crystals, compared to 12 of 25 (48%) of the ground controls. Encouragingly, there were more apo-hPPAR-γ LBD crystals in the microgravity PCG cards than the 1g controls. These positive results hope to introduce the use of the PCG standard of low sample volume and large experimental density to the microgravity environment and provide new opportunities for macromolecular samples that may crystallize poorly in standard laboratories.

The Protein Maker: an automated system for high-throughput parallel purification.

The Protein Maker is an automated purification system developed by Emerald BioSystems for high-throughput parallel purification of proteins and antibodies. This instrument allows multiple load, wash and elution buffers to be used in parallel along independent lines for up to 24 individual samples. To demonstrate its utility, its use in the purification of five recombinant PB2 C-terminal domains from various subtypes of the influenza A virus is described. Three of these constructs crystallized and one diffracted X-rays to sufficient resolution for structure determination and deposition in the Protein Data Bank. Methods for screening lysis buffers for a cytochrome P450 from a pathogenic fungus prior to upscaling expression and purification are also described. The Protein Maker has become a valuable asset within the Seattle Structural Genomics Center for Infectious Disease (SSGCID) and hence is a potentially valuable tool for a variety of high-throughput protein-purification applications.

Salvage and storage of infectious disease protein targets in the SSGCID high-throughput crystallization pathway using microfluidics.

The MPCS Plug Maker is a microcapillary-based protein-crystallization system for generating diffraction-ready crystals from nanovolumes of protein. Crystallization screening using the Plug Maker was used as a salvage pathway for proteins that failed to crystallize during the initial observation period using the traditional sitting-drop vapor-diffusion method. Furthermore, the CrystalCards used to store the crystallization experiments set up by the Plug Maker are shown be a viable container for long-term storage of protein crystals without a discernable loss of diffraction quality with time. Use of the Plug Maker with SSGCID proteins is demonstrated to be an effective crystal-salvage and storage method.

Nanovolume optimization of protein crystal growth using the microcapillary protein crystallization system.

The Microcapillary Protein Crystallization System (MPCS) is a microfluidic, plug-based crystallization technology that generates X-ray diffraction-ready protein crystals in nanolitre volumes. In this study, 28 out of 29 (93%) proteins crystallized by traditional vapor diffusion experiments were successfully crystallized by chemical gradient optimization experiments using the MPCS technology. In total, 90 out of 120 (75%) protein/precipitant combinations leading to initial crystal hits from vapor diffusion experiments were successfully crystallized using MPCS technology. Many of the resulting crystals produced high-quality X-ray diffraction data, and six novel protein structures that were derived from crystals harvested from MPCS CrystalCards are reported.

The plug-based nanovolume Microcapillary Protein Crystallization System (MPCS).

The Microcapillary Protein Crystallization System (MPCS) embodies a new semi-automated plug-based crystallization technology which enables nanolitre-volume screening of crystallization conditions in a plasticware format that allows crystals to be easily removed for traditional cryoprotection and X-ray diffraction data collection. Protein crystals grown in these plastic devices can be directly subjected to in situ X-ray diffraction studies. The MPCS integrates the formulation of crystallization cocktails with the preparation of the crystallization experiments. Within microfluidic Teflon tubing or the microfluidic circuitry of a plastic CrystalCard, approximately 10-20 nl volume droplets are generated, each representing a microbatch-style crystallization experiment with a different chemical composition. The entire protein sample is utilized in crystallization experiments. Sparse-matrix screening and chemical gradient screening can be combined in one comprehensive ;hybrid' crystallization trial. The technology lends itself well to optimization by high-granularity gradient screening using optimization reagents such as precipitation agents, ligands or cryoprotectants.

Using nanoliter plugs in microfluidics to facilitate and understand protein crystallization.

Protein crystallization is important for determining protein structures by X-ray diffraction. Nanoliter-sized plugs--aqueous droplets surrounded by a fluorinated carrier fluid--have been applied to the screening of protein crystallization conditions. Preformed arrays of plugs in capillary cartridges enable sparse matrix screening. Crystals grown in plugs inside a microcapillary may be analyzed by in situ X-ray diffraction. Screening using plugs, which are easily formed in PDMS microfluidic channels, is simple and economical, and minimizes consumption of the protein. This approach also has the potential to improve our understanding of the fundamentals of protein crystallization, such as the effect of mixing on the nucleation of crystals.

Using microfluidics to observe the effect of mixing on nucleation of protein crystals.

This paper analyzes the effect of mixing on nucleation of protein crystals. The mixing of protein and precipitant was controlled by changing the flow rate in a plug-based microfluidic system. The nucleation rate inversely depended on the flow rate, and flow rate could be used to control nucleation. For example, at higher supersaturations, precipitation happened at low flow rates while large crystals grew at high flow rates. Mixing at low flow velocities in a winding channel induces nucleation more effectively than mixing in straight channels. A qualitative scaling argument that relies on a number of assumptions is presented to understand the experimental results. In addition to helping fundamental understanding, this result may be used to control nucleation, using rapid chaotic mixing to eliminate formation of precipitates at high supersaturation and using slow chaotic mixing to induce nucleation at lower supersaturation.

In situ data collection and structure refinement from microcapillary protein crystallization.

In situ X-ray data collection has the potential to eliminate the challenging task of mounting and cryocooling often fragile protein crystals, reducing a major bottleneck in the structure determination process. An apparatus used to grow protein crystals in capillaries and to compare the background X-ray scattering of the components, including thin-walled glass capillaries against Teflon, and various fluorocarbon oils against each other, is described. Using thaumatin as a test case at 1.8 Å resolution, this study demonstrates that high-resolution electron density maps and refined models can be obtained from in situ diffraction of crystals grown in microcapillaries.

Microfluidic systems for chemical kinetics that rely on chaotic mixing in droplets.

This paper reviews work on a microfluidic system that relies on chaotic advection to rapidly mix multiple reagents isolated in droplets (plugs). Using a combination of turns and straight sections, winding microfluidic channels create unsteady fluid flows that rapidly mix the multiple reagents contained within plugs. The scaling of mixing for a range of channel widths, flow velocities and diffusion coefficients has been investigated. Due to rapid mixing, low sample consumption and transport of reagents with no dispersion, the system is particularly appropriate for chemical kinetics and biochemical assays. The mixing occurs by chaotic advection and is rapid (sub-millisecond), allowing for an accurate description of fast reaction kinetics. In addition, mixing has been characterized and explicitly incorporated into the kinetic model.

A synthetic reaction network: chemical amplification using nonequilibrium autocatalytic reactions coupled in time.

This article reports a functional chemical reaction network synthesized in a microfluidic device. This chemical network performs chemical 5000-fold amplification and shows a threshold response. It operates in a feedforward manner in two stages: the output of the first stage becomes the input of the second stage. Each stage of amplification is performed by a reaction autocatalytic in Co(2+). The microfluidic network is used to maintain the two chemical reactions away from equilibrium and control the interactions between them in time. Time control is achieved as described previously (Angew. Chem., Int. Ed. 2003, 42, 768) by compartmentalizing the reaction mixture inside plugs which are aqueous droplets carried through a microchannel by an immiscible fluorinated fluid. Autocatalytic reaction displayed sensitivity to mixing; more rapid mixing corresponded to slower reaction rates. Synthetic chemical reaction networks may help understand the function of biochemical reaction networks, the goal of systems biology. They may also find practical applications. For example, the system described here may be used to detect visually, in a simple format, picoliter volumes of nanomolar concentrations of Co(2+), an environmental pollutant.

Experimental test of scaling of mixing by chaotic advection in droplets moving through microfluidic channels.

This letter describes an experimental test of a simple argument that predicts the scaling of chaotic mixing in a droplet moving through a winding microfluidic channel. Previously, scaling arguments for chaotic mixing have been described for a flow that reduces striation length by stretching, folding, and reorienting the fluid in a manner similar to that of the baker's transformation. The experimentally observed flow patterns within droplets (or plugs) resembled the baker's transformation. Therefore, the ideas described in the literature could be applied to mixing in droplets to obtain the scaling argument for the dependence of the mixing time, t~(aw/U)log(Pe), where w [m] is the cross-sectional dimension of the microchannel, a is the dimensionless length of the plug measured relative to w, U [m s(-1)] is the flow velocity, Pe is the Péclet number (Pe=wU/D), and D [m(2)s(-1)] is the diffusion coefficient of the reagent being mixed. Experiments were performed to confirm the scaling argument by varying the parameters w, U, and D. Under favorable conditions, submillisecond mixing has been demonstrated in this system.