Investigating SARS-CoV-2 Therapeutic Structure with SEC-MALS

The surface glycoprotein responsible for viral entry in SARS-CoV-2 is trimeric in form, as is the case in many other viruses.

A provider of full-length SARS-CoV-2 spike proteins for research and development purposes, ACROBiosystems, uses SEC-MALS to make sure that the material it supplies is made up of the native trimeric form for optimal utilization in developing inhibitors, vaccines, and diagnostics. Mike Chen, CEO of ACROBiosystems, was asked to provide more details on this.

Front and top view of the trimeric coronavirus spike protein ectodomain obtained by cryo-electron microscopy analysis. Three S1 protomers (surface presentation) are colored in red, blue, and green. The S2 trimer (cartoon presentation) is colored in light orange (RCSB PDB structure 6VYB) .

Figure 1. Front and top view of the trimeric coronavirus spike protein ectodomain obtained by cryo-electron microscopy analysis. Three S1 protomers (surface presentation) are colored in red, blue, and green. The S2 trimer (cartoon presentation) is colored in light orange (RCSB PDB structure 6VYB) . Image Credit: ACROBiosystems

Q: Please describe the products and services provided by your company.

A: ACROBiosystems is a leading manufacturer of recombinant proteins and other critical reagents that are used to aid the development of target therapeutics. They utilize an application-oriented development strategy, with a particular emphasis on quality control, product design, and solution-based support.

Their products and services allow anyone in the field of drug development to possess a more streamlined, intuitive process. ACROBiosystems' catalog has a comprehensive list of drug targets and disease-associated biomarkers from humans to other common species.

In order to adhere to the rigorous standards of pharmaceutical research and development, all of their products are created using high quality and batch-to-batch consistency.

By promoting training and communication opportunities, supplying additional technical resources and custom services, initiating collaborations in the bio-industry community, and facilitating transactions in the dynamic global and niche markets, ACROBiosystems continues to grow and adapt to bring more value to their clients.

Q: Recently you have offered both full-length spike protein trimers of the 2019 novel coronavirus, and an inhibitor screening kit. Please tell us about those. How do you foresee customers using them, and what is unique about them?

A: An article which was published in Science [Wrapp et al, Science, 2020 Mar 13;367(6483):1260-1263. http://doi.org/10.1126/science.abb2507] established a 3.5-angstrom-resolution cryo-electron microscopy structure of the SARS-CoV-2 S trimer in the prefusion conformation.

In order to facilitate medical countermeasure development, the full-length S [spike – WTC] protein with a trimer structure was developed by ACROBiosystems. The trimer proteins are more suitable for antibody screening and immunization.

The main state of the trimer has one of the three receptor-binding domains (RBDs) rotated up in a receptor-accessible conformation. structural and biophysical evidence that the SARS-CoV-2 S binds ACE2 with higher affinity than SARS-CoV S is also shared by the authors.

The principle of SARS-CoV-2 inhibitor screening kit.

Figure 2. The principle of SARS-CoV-2 inhibitor screening kit. Image Credit: ACROBiosystems

In addition, it is crucial to develop SARS-CoV-2 inhibitors quickly, for instance, therapeutic antibodies and small molecular compounds, plus vaccines against COVID-19, as there is no effective treatment for COVID-19 in the market to date.

ACROBiosystems developed the SARS-CoV-2 inhibitor screening kit for that reason. The time-consuming labeling process is eliminated and the experimental procedures are greatly simplified by supplying proteins pre-labeled with biotin.

This inhibitor screening ELISA kit is designed to facilitate the identification and characterization of SARS-CoV-2 inhibitors. The S protein receptor binding domain (RBD) verified by MALS is also provided.

Q: You emphasize that the trimer spike protein structure is verified by SEC-MALS. Can you tell us a little about sec-mals, and why that is so important to your product validation?

A: Multi-angle light scattering (MALS) is a method which is used to establish the molecular weight of proteins based on the relationship between the concentration, MW, and the intensity of scattered light. This technique does not depend on the elution volume, and protein standards are not required for its calibration.

a) Schematic diagram of light scattering; b1) The intensity of the scattered light is related to the product of molar mass, concentration, and square of the refractive index increment; b2) calculation of the sample’s MW.

Figure 3. a) Schematic diagram of light scattering; b1) The intensity of the scattered light is related to the product of molar mass, concentration, and square of the refractive index increment; b2) calculation of the sample’s MW. Image Credit: ACROBiosystems

The correct folding and conformation of recombinant proteins is crucial for antibody screening and identification, immunization, antibody drug quality control, functional verification of antibody drug candidates, and clinical sample analysis.

The oligomeric state of proteins in solution will influence the structure and exposed active sites. Only when the quaternary structure of the protein is correct will the protein activate signal pathways and physiological functions effectively.

Yet, particularly in the case of glycoproteins, the protein’s native conformation may not correspond to globular protein column calibration standards. Trying to establish the molecular weight of such proteins against a column calibration curve invariably produces results which are incorrect.

The result is more reliable and accurate than the traditional HPLC-SEC analysis in which the MW is calculated according to the elution time, since MALS does not rely on retention time to calculate the MW.

SEC-MALS was used to establish the MW of three commercially available antibody drugs including Herceptin, OKT3, and Ipilimumab. The molecular weights of these three antibodies are actually very similar, but the retention times of the three antibodies are quite different, as shown in Figure 3.

The OKT3 antibody was estimated at 212.5 kDa, and Ipilimumab was only 66.3 kDa by column calibration, both of which are nowhere near the true molecular weight. On the other hand, as shown in Figure 4, the MW of the three antibodies established by SEC-MALS is very near to their theoretical (sequence) MW.

The molecular weights of three different antibodies determined by SEC-MALS are very close to the sequence weights. A) OKT3, b) Herceptin, c) Ipilimumab.

Figure 4. The molecular weights of three different antibodies determined by SEC-MALS are very close to the sequence weights. A) OKT3, b) Herceptin, c) Ipilimumab. Image Credit: ACROBiosystems

Q: Do customers recognize the value in the sec-mals validation of the oligomeric state of the sars-cov-2 spike protein?

A: Despite the inherent value of SEC-MALS for establishing the oligomeric state of proteins in solution, it is thought that the majority of customers do not understand the advantage of this technique fully. ACROBiosystems intend to share more data to show that accurate determination of MW by SEC-MALS is crucial for biopolymer analysis.

About ACROBiosystems

ACROBiosystems is a leading manufacturer of recombinant proteins and other critical reagents to support the development of target therapeutics. The company employs an application oriented development strategy, with a particular focus on product design, quality control and solution based support. Our products and services enable anyone in the field of drug development to have a more intuitive and streamlined process.

To respond to coronavirus pandemic, ACROBiosystems has developed SARS-CoV-2 antigens specifically designed and optimized for serological test kits, including Spike-derived antigen S1, RBD and Nucleocapsid protein. Proteins have been supplied to diagnostic companies in large quantity.


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Last updated: Jun 18, 2020 at 6:31 AM

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