Figure 4: Raw data obtained from near ultraviolet (UV) analysis of a mAb.
The EMA and ICH guidelines also recommend that the higher-order structure of the mAb should be characterized by appropriate
physicochemical methodologies. ICH Q6B recommends the use of "circular dichroism, nuclear magnetic resonance (NMR), or other
suitable techniques, as appropriate." Today, circular dichroism (CD) analysis is commonly used to assess the secondary structure
of mAbs. The data obtained from CD analysis of a mAb is shown in Figure 4. Assessment of the raw data using CDsstr software
allows the relative percentages of the various secondary structures to be estimated as shown in Table I.
Table I: The result of CDSSTR algorithm assessment of the near UV CD (circular dichroism) data shown in Figure 4.
In addition to the structural characterization requirements outlined above, a number of additional physicochemical properties
need to be assessed.
MOLECULAR WEIGHT OR SIZE
Figure 5: Deconvoluted mass spectrum obtained from online electrospray mass spectrometric detection (quantitative time-of-flight)
LC/ES–MS (Q-TOF) analysis of a mAb.
On-line LC/ES–MS analysis of intact and reduced mAbs using quadrupole time-of-flight (Q–TOF) instrumentation has revolutionized
the measurement of the intact molecular weight of mAbs and the released light and heavy chains. The data obtained from intact
molecular weight analysis of a mAb using this technique is shown in Figure 5.
Figure 6A: Deconvoluted mass spectrum obtained from online LC/ES-MS (Q–TOF) analysis of a mAb.
The major peaks observed are 162 mass units apart which is consistent with variation in galactosylation of the N-linked oligosaccharides present on the heavy chains of the molecule. The mass spectrometric data obtained from analysis of
the mAb following reduction are shown in Figure 6.
Figure 6B: Deconvoluted mass spectrum obtained from online LC/ES–MS (Q–TOF) analysis of a mAb following reduction.
The signal shown in Figure 6A is consistent with that expected for the light chain and the signals observed in Figure 6B are
consistent with those expected for the heavy chain with the expected N-linked oligosaccharides. Aside from confirming the molecular weight of a mAb and the component light and heavy chains, these
data also confirm the intactness of the protein backbones.