Case Study 3
Functional Analyses Following Process Changes Using a Biosensor Technology
Ion exchange chromatography is commonly used to indicate charge heterogeneity of protein products. The anion exchange chromatogram
of an Fc fusion protein has a single broad peak. The peak elution time of the drug substance produced with a new cell line
and new processes was different from that of earlier material by 1 min. The peak width and range remained unchanged. A variety
of biochemical and mass spectrometry techniques were used to determine the cause of the peak shift. Only low level variations
in the COOH terminal lysine and deamidation in the Fc domain were observed, but their relation to the peak shift was not clear.
No change in higher order structures was found by spectroscopic methods (data not shown). An SPR biosensor method (Biacore)
was then used to analyze the ligand binding of two drug substance lots, Lot 1 and Lot 2, from the early and the new processes,
respectively. Multiple concentrations of the ligand were used in triplicates. The association (ka) and dissociation (kd) rate constants were obtained by global fitting of the sensorgrams to the 1:1 kinetic model using the manufacturer-provided
software, and used to calculate the affinity constant, KD = ka/kd. Table 1 lists the results for all three sets, and for clarity, Figure 7 shows the global fitting for one set as an example.
The affinity constants of the two lots are within the experimental error. Even though the Fc effector function has not been
shown to be involved in the mode of action of this biotherapeutic, binding of the two lots with the high affinity Fcγ receptor,
FcγRI, was compared mainly to assess the structural integrity of the Fc domain. As shown in Figure 8, the two lots were comparable.
Table 1. Comparison of binding kinetics and affinity
We have demonstrated that spectroscopic methods such as circular dichroism (CD) can detect subtle differences in higher order
structure before and after changes in process and formulation. To facilitate the analysis and presentation of the comparability
of CD spectra, the RMSD calculation is proposed to semi-quantify the spectral comparison with a single numeric indicator.
This approach is not limited to CD and potentially can be applied to other types of spectra. We also presented different types
of AUC analysis for evaluating size distribution in the context of comparability assessments. The results demonstrate that
size distribution, as an important property, can be sensitive to process change. Our case studies also show that it is beneficial
to use multiple techniques for comprehensive characterization of aggregates, and it is convenient to use AUC and DLS as orthogonal
methods to enhance the analysis. Finally, we presented the use of biosensor technology to help address the question of the
likelihood that the function of a molecule will be compromised in the event that changes in some properties are detected by
other physiochemical assessments.
Figure 7. Overlaid sensorgrams for the ligand-binding of the Fc fusion protein lots from the early (Lot 1) and the new (Lot
2) processes. The Fc fusion protein was captured by preimmobilized protein A in sample flow cells. A ligand sample with a
concentration indicated in the figure was injected at 0 sec. The dissociation phase began at 280 s when the ligand injection
was switched to the flow of running buffer. The sensorgrams of one set of the triplicates for Lot 1 (pink) and Lot 2 (cyan),
as well as the fitted curves (black), are overlaid. All sensorgrams were "double corrected" for the reference flow cell signals
and the buffer injection responses.
The biophysical techniques used in these case studies are not validated and are not intended for release tests. However, these
methods enable in-depth characterization and are useful for establishing product comparability in physicochemical attributes,
which often are related to quality. As suggested in ICH Q5E, significant changes in product quality attributes must be evaluated
for their impact on product safety and efficacy.
Figure 8. Overlaid sensorgrams for the FcγRI-binding of the Fc fusion protein from the early (Lot 1) and the new (Lot 2)
processes. FcγRI with a His tag at its C-terminus was captured by an anti-His antibody preimmobilized in the sample flow cells.
Lot 1 and Lot 2 were injected at 5 and 10 nM in triplicates, and the sensorgrams are overlaid. All sensorgrams were "double
corrected" for the reference flow cell signals and the buffer injection responses.