SEC-MALS: Accurate Determination of Protein Molecular Weight and Confirmation of Polymer State

Recombinant protein antigens are involved in various R&D processes, including antibody-drug development, pre-immunization, antibody screening, identification, antibody-drug candidate function verification in vivo and in vitro, quality control, and clinical sample analysis. The quality and activity of the recombinant protein can have a tremendous impact on the success and even cycle of antibody-drug development.

Recombinant protein antigens must maintain correct folding and consistent conformation with the native proteins. Therefore, ensuring the natural activity of the protein antigens and minimal batch-to-batch inconsistency, will ensure a high success rate of drug development. As an industry leader in recombinant proteins for drug development, ACROBiosystems is committed to "Where Proteins and Innovation Advance Biomedicine." Our company's commitment extends beyond recombinant protein design, expression, and production process of strict control into an established, accurate, and reliable molecular weight determination analysis method. Our methods can accurately characterize the aggregation state of recombinant proteins under physiological conditions in solution and guide the design, production process optimization, and quality control of recombinant protein antigen products.

Several methods are used to analyze molecular protein weight, including:

(1) An interpolation method based on SEC-HPLC (High-Performance Molecular Sieve Liquid Chromatography) to estimate the retention time;

(2) Molecular weight absolute quantification based on SEC-MALS (Multi-angle Laser Scattering).

Both techniques can determine the molecular weight of proteins in solution, but there are some differences in principle. The similarities and differences between the two techniques are as follows:

Fundamentals of SEC-MALS technology

The MALS technique is based on the direct correlation between the molecular weight and the light scattering intensity to determine the absolute molecular weight. There is no need to use a standard protein independent of the elution volume. The components separated by SEC enter the MALS detector. Light scattering occurs when the laser irradiates the analyte. The intensity of the scattered light is measured simultaneously by MALS from multiple angles (Fig. 1.a). Formula b1 shows that the scattering light intensity is proportional to the square of molar mass, concentration, and refractive index increment. Further, the absolute molecular weight of the analyte can be calculated directly by formula b2.

Therefore, in biopharmaceutics, SEC-MALS is widely used in R & D and analytical experiments, characterization of proteins and polymers, evaluation of solution properties, process development, and quality control.

Fig 1. Schematic diagram of SEC-MALS detection a. Light scattering sketch b. Molecular weight calculation of analytes

Fig. 2. Comparison of Trimer 4571 molecular weight results obtained by SEC-MALS with MS data

Fig 3. Determination of Trimer by SEC-UV-dRI-MALS

Tab 1. Results of molecular weight determination of three antibody drugs

Antibody Name	Column	Retention time TR (min)	Molecular weight (SEC-HPLC)	Molecular weight (SEC -MALS)
OKT3	G3000SWxl	14.775	212.5 kDa	161.6 kDa
Herceptin	G3000SWxl	15.436	146 kDa	151 kDa
lpilimumab	G3000SWxl	17.039	66.3 kDa	172.2 kDa

Fig 4. Molecular weight determination of different antibodies by SEC-HPLC

Fig 5. Determination of Molecular Weight of Three Antibody Drugs by SEC-MALS

Case 1: TNFSF Trimer
          Case 2: IL-2R Polymer
          Case 3: CTLA-4 Homodimer Proteins

          Case 4: Spike Trimer

         

Case 1: TNFSF Trimer

Protein structure and purity verified by SEC-MALS

Cat. No. OXL-H52Q8(Human OX40 Ligand /TNFSF4 Protein, His Tag, active trimer)

Figure 6. The purity of Human OX40 Ligand, His Tag (active trimer) (Cat. No. o. OXL-H52Q8 ) is more than 95% in HP-SEC, and the molecular weight of this protein is around 60-80 kDa verified by SEC-MALS.

Cell functional activity verified by FACS

Cat. No. OXL-H52Q8(Human OX40 Ligand /TNFSF4 Protein, His Tag, active trimer)

Figure 7. FACS analysis shows that human sources OX40 Ligand, His Tag (active trimer) (Cat. No. OXL-H52Q8 ) can bind to 293T cells overexpressing human OX40. The concentration of OX40 Ligand was 0.1 μg / mL.

Protocol

Case 2: IL-2R Heterodimer / Trimer

Cat. No. ILG-H5254(Human IL-2RB&IL-2RG Heterodimer Protein, Fc Tag&Fc Tag)

Fig. 8. The purity of Human IL-2RB&IL-2RG Heterodimer Protein, Fc Tag&Fc Tag (Cat. No. ILG-H5254 )is more than 90% and the molecular weight of this protein is around 1 145 ~ 165 kDa verified by SEC-MALS.

Cat. No. ILG-H5257(Human IL-2RB&IL-2RA&IL-2RG, Fc Tag&Fc Tag)

Fig 9. The purity of Human IL-2RB&IL-2RA&IL-2RG, Fc Tag&Fc Tag (Cat. No. ILG-H5257 ) is more than 90% and the molecular weight of this protein is around 175-190 kDa verified by SEC-MALS.

Fig 10. SPR verified the binding affinity of IL-2 to ACRO's IL-2R construct.

Case 3: CTLA-4 Homodimer Proteins

Fig 11. The purity of Human CTLA-4, His Tag (Cat. No. CT4-H52H9 ) is more than 90% and the molecular weight of this protein is around 45-60 kDa verified by SEC-MALS.

CTLA-4 dimer affinity verified by SPR

Fig 12. SPR verified that the affinity of CTLA-4 dimer is more than ten times higher than that of monomer

Case 4: Spike Trimer

Cat. No. SPN-C52H9(SARS-CoV-2 S protein, His Tag, Super stable trimer (MALS & NS-EM verified))

Fig 13. SDS-PAGE identified the SARS-CoV-2 S protein under reduced conditions. His Tag, Super stable trimer (MALS & NS-EM verified) (Cat. No. SPN-C52H9 ) The purity is over 90%, and the molecular weight is about 550-660 kDa by SEC-MALS. The size and appearance of the SARS-CoV-2 trimer verified with NS-EM were like those reported in the published literature.

Fig 14. Comparative analysis of antibody titers of serum samples from rehabilitated patients detected by S-6P protein, S-2P protein, and S protein. The results showed that the signal-to-noise ratio of S-6P was significantly higher than that of S protein, S-2P, and other S proteins. It suggested that S-6P was more suitable for antibody titer detection in serum samples and could detect antibody titers in serum early after injection of vaccine, which was helpful for evaluation of vaccine immunogenicity.