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Your Position: Home > SEC-MALS: Accurate Determination of Protein Molecular Weight and Confirmation of Polymer State

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

Background

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.

Protein Molecular Weight Analysis Technology Platform

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:

Detection methodPrinciples and featuresResults
SEC-HPLCColumn correction methodCalculate the relative molecular weight by comparing the elution retention times of the protein to be measured and the standard protein
It is assumed that the conformation of the protein to be measured is similar in shape to the standard protein.
It is assumed that there is no non-specific interaction with the column packing, and each elution time is reproducible without drift
SEC-MALSLight scattering methodAbsolute molecular weight was determined directly by laser scattering intensity. Molecular weight determination was independent of elution volume or retention time.
The intensity of the scattered light is proportional to the square of the molar mass, concentration, and refractive index increment.
The angular dependence of scattering light is only dependent on the size of the molecule.
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.
Schematic diagram of SEC-MALS detection a. Light scattering sketch b. Molecular weight calculation of analytes

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

HSPEC-MALS determined the molecular weight of the HIV-1 coated trimer. The reliability of the results was verified by orthogonal mass spectrometry.
Researchers of 'Vaccine Production Program' in Laboratory of National Institute of Allergy and Infectious Diseases (NIAID) Vaccine Research Center [1] has determined the total molecular weight (339 KDa), the molecular weight of protein (213 KDA), and glycan component (126 KDda) of high glycosylated HIV-1 envelope trimer (Trimer 4571) vaccine by HPSEC-MALS. The assay results were further validated by orthogonal mass spectrometry, which helps to support process development and provide rapid protein characterization data. It demonstrates the broad applicability of HPSEC-MALS to many of the heterologous glycoproteins' candidate vaccines under development.
Comparison of Trimer 4571 molecular weight results obtained by SEC-MALS with MS data

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

Determination of Trimer by SEC-UV-dRI-MALS

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

Comparing the results of SEC-HPLC and SEC-MALS with the determination of the molecular weight of commercial drugs
Three known antibody drugs, OKT3, Herceptin, and Ipilimumab, were determined by SEC-HPLC and MALS, respectively. As shown in Table 1, the molecular weights of the three antibodies are within a close range of 150-160 kDa. However, the elution retention time of three kinds of antibodies was different, resulting in a molecular weight discrepancy calculated by SEC-HPLC. The OKT3 antibody was 212.5 kDa, and Ipilimumab was only 66.3 kD, which was far from the calculated molecular weight. The molecular weights of the three antibodies detected by SEC-MALS were very close to the theoretical molecular weights (Fig 5).
Therefore, MALS technology is far more accurate and reliable than the traditional SEC-HPLC method.
Tab 1. Results of molecular weight determination of three antibody drugs
Antibody NameColumnRetention time
TR (min)
Molecular weight
(SEC-HPLC)
Molecular weight
(SEC -MALS)
OKT3 G3000SWxl14.775212.5 kDa 161.6 kDa
HerceptinG3000SWxl 15.436146 kDa 151 kDa 
lpilimumabG3000SWxl 17.03966.3 kDa 172.2 kDa
Molecular weight determination of different antibodies by SEC-HPLC

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

Determination of Molecular Weight of Three Antibody Drugs by SEC-MALS

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

SEC-MALS: Ensuring the Quality of Polymeric Protein Products
Natural proteins often exist in a certain aggregation state and conformation in vivo to ensure that they play a correct biological function. The random and incorrect aggregation of recombinant proteins in the production process often causes changes in the conformation or exposure of certain key functional epitopes. It results in the inability to obtain high-quality antibody candidates targeting effective natural epitopes, which affects the results of in vitro functional verification of antibody drugs and significantly reduces the success rate of antibody-drug development.
ACROBiosystems understands the impact of recombinant protein structure on its function and the need for antibody-drug development. Through in-depth study of the structure and properties of the complexes, we designed and developed a variety of homologous/heterologous polymer products using a unique technology platform and verified the molecular weight of the products by SEC-MALS. The SEC-MALS purity results as a quality control standard to ensure more homogeneous product properties smaller inter-batch differences. At the same time, the product activity through ELISA, flow cytometry, functional and other application scenarios validation accelerate the development of antibody drugs.

Case 1: TNFSF Trimer

Tumor necrosis factor (TNF) superfamily proteins are trimeric in nature. Only when the trimeric form of TNF-α binds to the extracellular domain of TNFR1 can it induce trimerization, which effectively stimulates the downstream pathway to induce apoptosis.
Protein structure and purity verified by SEC-MALS
Cat.No. OXL-H52Q8(Human OX40 Ligand /TNFSF4 Protein, His Tag, active trimer)
The purity of Human OX40 Ligand, 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)
FACS analysis shows that human sources OX40 Ligand, 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.

Case 2: IL-2R Heterodimer / Trimer

The receptor for IL-2 (IL-2R) contains three subunits of α, β, and γ. Three subunits can form receptors with different affinities. In the absence of IL-2, α, β, and γ, a specific structure of the IL-2 receptor complex cannot directly form a high-affinity IL-2 receptor complex. ACROBiosystems has developed a range of proprietary technologies IL-2 R alpha & IL-2 R beta & IL-2 R gamma Heterotrimer protein and IL-2 R beta & IL-2 R gamma Heterodimer protein.
Protein structure and purity verified by SEC-MALS
Cat.No. ILG-H5254(Human IL-2RB&IL-2RG Heterodimer Protein, Fc Tag&Fc Tag)
The purity of Human IL-2RB&IL-2RG Heterodimer Protein

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)
The purity of Human IL-2RB&IL-2RA&IL-2RG

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.

After SPR verification, the affinity verification results of IL-2 and IL-2 receptors are consistent with the literature [2] .
SPR verified the binding affinity of IL-2 to ACRO's IL-2R construct.

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

Case 3: CTLA-4 Homodimer Proteins

With the rise of bispecific drugs, there is a wave of research and development of dual-targeted based on PD-1 / PD-L1 and CTLA-4. At present, there are many similar bispecific drugs in the clinical validation stage, and its indications also cover a variety of solid tumors. CTLA-4 (cytotoxic T lymphocyte-associated protein 4), also called CD152 (differentiation cluster 152), is a protein receptor of T cells, usually in the form of a homodimer.
Protein structure and purity verified by SEC-MALS
Cat. No. CT4-H52H9(Human CTLA-4 / CD152 Protein, His Tag, active dimer (MALS verified))
Cat. No. CT4-H52H9  (Human CTLA-4 / CD152 Protein, His Tag, active dimer (MALS verified))

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

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

The S protein is the most important target of coronavirus research. The SARS-CoV-2 virus S-protein functions as a trimer; it consists of three identical molecules encoded by the same gene. Each of these molecules has two subunits, namely S1 and S2. When the RBD in the S1 subunit binds to the host cell ACE2, it causes trimer instability. The S1 and S2 subunit dissociated, and the S2 sub-unit conformation changed into a highly stable post-fusion structure. The S protein has two conformations: prefusion and post-fusion. The researchers found that the conformational form of the S protein is unstable, even if it does not bind to the host cell receptor ACE2. There are also spontaneous conformational changes from Prefusion to Postfusion, which pose great challenges for vaccine development and neutralizing antibody screening.
Protein structure and purity verified by SEC-MALS & NS-EM

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

SDS-PAGE identified the SARS-CoV-2 S protein under reduced conditions

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.

S protein trimer can be used to detect antibody titers in serum of rehabilitated patients with a high signal-to-noise ratio
Comparative analysis of antibody titers of serum samples from rehabilitated patients detected by S-6P protein

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.

Product List
  • TNFSF

  • CD3

  • FcRn (FCGRT & B2M)

  • Interleukin

  • Spike trimer

  • Others

MoleculeCat. No.HostProduct DescriptionStructure
MoleculeCat. No.HostProduct DescriptionStructure
MoleculeCat. No.HostProduct DescriptionStructure
MoleculeCat. No.HostProduct DescriptionStructure
MoleculeCat. No.HostProduct DescriptionStructure
MoleculeCat. No.HostProduct DescriptionStructure

References

  • [1]Bender M F , Li Y , Ivleva V B , et al. Protein and glycan molecular weight determination of highly glycosylated HIV-1 envelope trimers by HPSEC-MALS[J]. Vaccine, 2021(7523).

  • [2]Sriram B , Tania C R ,  Davis A M , et al. Ligand binding kinetics of IL-2 and IL-15 to heteromers formed by extracellular domains of the three IL-2 receptor subunits[J]. International Immunology(11):1839.

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