Intact mass analysis is the assessment of a protein’s total molecular weight by mass spectrometry (MS) without prior digestion or fragmentation of the molecule of interest.
Monoclonal antibodies (mAbs) are a very important class of biopharmaceutical molecules. As a protein drug, thorough characterization of the mAb is required in each of the manufacturing steps. Intact mAb analysis offers rapid assessment on determining the accurate molecular weight of an mAb product and its degree of heterogeneity, such as post-translational modifications (PTMs), antibody-drug conjugate (ADC), mAb sequence variations, or degradation products. Quadrupole Time-of-flight (Q-TOF) LC/MS systems are often used to analyze intact proteins or antibodies due to excellent resolution at the high mass range.
In contrast to an enzyme-linked immunosorbent assay (ELISA) for quantifying MAbs, mass spectrometry-based assays do not rely on MAb-specific reagents such as recombinant antigens and/or anti-idiotypic antibodies, and time for development is usually shorter. Furthermore, using molecular mass as a measurement tool provides increased specificity since it is a first-order principle unique to each MAb.
Intact protein characterization is performed on samples in solution using electrospray ionization mass spectrometry (ESI-MS). Because the mass spectrometer measures the component’s mass-to-charge ratio, and electrospray ionization of proteins can generate ions with multiple charges, proteins of molecular weight up to 250kDa have been analyzed successfully.
Advantages of ESI-MS Intact Protein Analysis Service:
- High sensitivity, high accuracy, easy to operate and fast detection.
- ESI-MS can be effectively used in combination with chromatography, and is suitable for the identification or structure determination of trace substances in complex systems.
- Suitable for the analysis of strong polarity, difficult volatilization or thermal instability compounds.
- The limited detection conditions are few and the signal is easy to be analyzed.
- The information of transition state can be obtained, and the composition and conformation of polypeptide and protein molecules can be studied effectively.
- Avoid detergents and keep buffer concentration at a minimum.
- Quantity 10-40ug.
- Acceptable buffers: ammonium acetate, ammonium bicarbonate, ammonium formate.
- Avoiding buffer: HEPES, PBS, MES, MOPS, and Tris.
- Avoiding detergents: SDS, PEG, PPG, Tween, CHAPS, Triton, and urea.
- Avoiding salts: Alkylammonium salts, guanidinium salt, metal cations and inorganic anions such as phosphate, sulfate, and halides.
Concentration & Volume
- For routine intact mass analysis, the minimum amount of protein required depends on the MW of the peptide or protein.
- Good results have been obtained with 25 pmol at 5 kD, 100 pmol at 20 kD, 200 pmol at 40 kD, and 500 pmol at 60 kD.
- Sample concentration should be such that the appropriate amount of protein is contained in 20-25 uL
AxisPharm provides intact protein analysis using Maldi-TOF, ESI-TOF, and LC-HRMS to determine proteins with mass upto 300 KDa. We also provide SEC and FPLC protein purification services. Please contact us for details and tech support.
Protein Separation And Purification
Protein Precipitation Technical Guide