Team:TAS Taipei/Hardware

HARDWARE

Summary

We developed specific hardware to mediate the enzymatic conversion process in our blood type conversion kit in response to feedback from hematologists and blood banks. Our hardware addresses concerns of mechanizability and biocompatibility, is airtight, and utilizes components that conform to existing industry norms.

Our hardware components include nickel sepharose beads with immobilized enzymes, leukodepletion filters, biocompatible mesh grids, columns, and a narrow open collection tube with a unique narrow open cap in our device. The entire device was constructed with Computer Aided Design (CAD) using SOLIDWORKS.

Ni Sepharose Beads:

During the protein purification process, enzymes are immobilized on Nickel sepharose beads, something we plan to use to our advantage (Cube Biotech, 2020).

In contrast to normal protein purification procedures, we will not be detaching the enzymes from those beads. Instead, we can directly harvest beads along with their immobilized enzymes. Since beads will be highly concentrated with enzymes, they will be diluted with beads without immobilized enzymes. Specifically, beads immobilized with α-Gal will be diluted by 400x and 22 mL of bead solution will be used. Beads with NAGA immobilized will be diluted by 60x and 22 mL will be used. Beads with endo-β-Gal immobilized will be diluted by 400x and 18 mL of it will be used (refer to the Enzyme Kinetics Model page for details).

We will then directly incorporate these beads into our hardware device, saving us the additional steps of eluting enzymes from the beads and immobilizing them on another surface. This will both increase our yield of enzymes and streamline the time spent on purification.

However, studies have raised some concerns of using Nickel sepharose beads in our prototype, primarily regarding how Nickel may negatively affect RBC function when the solution is passed through the column (De Luca, et al., 2007), (Tkeshelashvili, et al., 1989). Yet, as shown in Fig 1, the Nickel ion is bound only to the polyhistidine tag of the protein and thus should not have much of an effect on RBCs. Another concern regarding the nickel involves the protein/enzyme detaching from and exposing the nickel ion to the RBCs. However, without imidazole or a low pH solution, the polyhistidine tag and the enzyme will not easily detach. The low nickel concentration is also unlikely to pose any significant negative effects on the RBCs. To test this theory, we performed experiments using porcine RBCs and Nickel Sepharose beads. We found that no significant hemolysis resulted from passing the red blood cells through the bead columns.

Figure 1 - Structure of enzyme immobilization on Nickel Sepharose beads with polyhistidine tags (Magdeldin & Moser, 2012)

Hardware Device:

Our hardware device takes in RBC in saline solution from a blood bag. The solution is passed through the columns that contain enzymes to cleave the antigens off the RBCs. It is then collected in a tube for later purification of cleaved RBCs (removing fallen antigens, antibodies, agglutinated RBCs and enzymes) (Fig 2).

Figure 2 - Overview CAD diagram for our hardware labelled with dimensions. Assembled blood type conversion system (left), individual conversion column (middle), and narrow open collection tube (right).

Conversion Columns:

Each column in the device contains one type of enzyme. We designed our device to be modular such that different combinations of our columns can convert different blood types (Fig 3).

Figure 3 - Modularity of enzymatic conversion kit. Columns can be assembled in 3 configurations according to blood type.

Each column is detachable and made of biocompatible plastic connected by PVC tubes for the enzyme cleavage process of our prototype (Fig 4).

Figure 4 - CAD diagram for one conversion column with dimensions

All connections will be joined by luer locks to prevent any leakage of liquids and make sure the system is airtight and thus safe. Each of the columns will have a total height of 16 cm with an internal diameter of 3 cm for the main body (Fig 4). The columns will be filled with Nickel sepharose beads immobilized with the relevant enzyme. These beads have an average size of 90 μm (Fig 4).

To prevent these beads from flowing to the next part of the prototype, the beads will be placed on leukodepletion filters with pore sizes of 50 μm (Fig 4). These leukodepletion filters are already implemented in blood processing centers for leukocyte filtering and are likely safe for our purposes (Dzik, 1993). A 50 μm pore size will allow RBCs (7-8 μm) to pass through but prevent the 90 μm beads from flowing through (Kinnunen et al, 2011). To support the filter and prevent it from tearing as a result of bead weight, we added a hard, curved mesh made with biocompatible material with approximately 2 mm holes. It will be placed 4 cm above the bottom of the column (Fig 4). This mesh will have a height of 1 cm and will allow everything that passes through the leukodepletion filter to flow through the 2 mm holes. Since this part of the device will be able to operate hands-off, it can easily be mechanized if desired.

We used SOLIDWORKS to simulate fluid flow through an individual conversion column (Fig 5). The pipes represent the flow trajectory of washed RBCs. We closed off both ends of the column, then ran 220 iterations to perform the simulation model.

Figure 5 - SOLIDWORKS simulation of fluid flow (washed RBCs) through an individual conversion column. Pipes represent flow trajectory.

Narrow Open Collection Tube:

After the solution has flowed through a series of columns, it will enter a collection tube with a narrow open cap (Fig 6). The narrow open cap allows for the entire process to remain airtight as blood exits the column and into the collection tube.

Figure 6 - CAD diagram for the narrow open collection tube with dimensions

After collection, this part of the device will then serve as the container used to purify cleaved RBCs and remove unwanted components from the solution via centrifugation and filtration (Fig 6) .

Since the solution will contain free antigens and potentially minor amounts of detached enzymes in addition to processed RBCs, we will centrifuge the solution in the narrow open collection tube and resuspend the processed RBCs in saline to remove any unwanted substances (Fig 7). 100 μL of the stock solution of antibodies provided by the kit will be used to agglutinate the RBCs that have not had their target antigens fully cleaved (for more information on the specifics, please visit the Antibody-Antigen model). Then we will proceed to pass this solution through a 35 μm leukodepletion filter with pore size to separate the agglutinated from the non-agglutinated. Finally, we centrifuge the processed blood again and resuspend it in saline to remove free antibodies in the remaining solution. The final product will thus only be cleaved RBCs, and will be ready for transfusion.

Figure 7 - Illustration of post-conversion purification process. Through centrifugation and filtration, cleaved RBCs can be purified from a solution of free/removed antigens, enzymes, and uncleaved RBCs.

References:

“Affinity Chromatography: Principles and Applications | IntechOpen.” n.d. Accessed October 21, 2021. https://www.intechopen.com/chapters/33046.

“Cube Biotech.” n.d. Cube Biotech. Accessed October 21, 2021. https://cube-biotech.com/guide-to-magnetic-beads/magbeads-for-protein-purification.

De Luca, Grazia, Tiziana Gugliotta, Giulia Parisi, Pietro Romano, Antonella Geraci, Orazio Romano, Adriana Scuteri, and Leonardo Romano. 2007. “Effects of Nickel on Human and Fish Red Blood Cells.” Bioscience Reports 27 (4–5): 265–73. https://doi.org/10.1007/s10540-007-9053-0.

Dzik, S. 1993. “Leukodepletion Blood Filters: Filter Design and Mechanisms of Leukocyte Removal.” Transfusion Medicine Reviews 7 (2): 65–77. https://doi.org/10.1016/s0887-7963(93)70125-x.

Kinnunen, Matti, Antti Kauppila, Artashes Karmenyan, and Risto Myllylä. 2011. “Effect of the Size and Shape of a Red Blood Cell on Elastic Light Scattering Properties at the Single-Cell Level.” Biomedical Optics Express 2 (7): 1803–14. https://doi.org/10.1364/BOE.2.001803.

Tkeshelashvili, L. K., K. J. Tsakadze, and O. V. Khulusauri. 1989. “Effect of Some Nickel Compounds on Red Blood Cell Characteristics.” Biological Trace Element Research 21 (1): 337–42. https://doi.org/10.1007/BF02917273.