Biophysical Characterization for Product Comparability - Spectroscopic methods such as circular dichroism can detect subtle differences in higher order structure before and after changes in process


Biophysical Characterization for Product Comparability
Spectroscopic methods such as circular dichroism can detect subtle differences in higher order structure before and after changes in process and formulation.

BioPharm International Supplements

Case Study 2

Analysis of Size Distribution Following Process and Formulation Changes

Figure 2. AUC–SV analysis for the drug substance lots of the KLH conjugate. AUC–SV was performed at a centrifugation speed of 45,000 rpm in the respective formulation matrices. Data were analyzed using the Sedfit program. The sedimentation coefficients were converted to the standard condition at 20 C in water in the Sedphat program. The profiles are the average of two replicates.
AUC–SV analysis provides a size distribution profile based on separation by gravitational force in homogeneous solutions, and therefore, is orthogonal to size exclusion chromatography (SEC) when used to evaluate and characterize aggregates. A single speed commonly is used in AUC–SV to separate molecular species. Gravitational sweep AUC, however, applies varying speeds in a single run to expand the dynamic range of analysis to possibly 1.2 μm in the diameter of particles.8 Here, we present an example of applying both approaches to evaluate the comparability of highly heterogeneous samples. Figure 2 shows a profile of single speed AUC–SV for a protein conjugate of ~500 kDa in monomeric form, analyzed using the Sedfit program.9 The conjugate contains a carrier protein, keyhole limpet hemocyanin (KLH), and a peptide, cross-linked to KLH at multiple sites through a specific linker. One drug substance lot (Lot 2) produced by a later process and in a new formulation matrix, is compared to Lot 1 from an early process. The overall profiles of the single-speed AUC–SV are grossly similar, with two main peaks corresponding to the monomer and the dimer. There also are larger species, but in much lesser amounts. However, the sedimentation coefficients of the monomer and dimer in Lot 2 shifted to lower values compared to those in the early lot. The smaller sedimentation coefficients suggest that the monomer and dimer in Lot 2 could be further extended, resulting in larger hydrodynamic radii. This conformational change is likely a response to the changes in formulation conditions, and no impact on efficacy was found in the subsequent preclinical tests.

Gravitational sweep AUC has been shown to include much larger species in one sedimentation run.8,10 Although it is still difficult to obtain quantitative information, the method is proven to be valuable for qualitative assessment of size distribution, which also provides an estimate of the appropriate speed to be used in a single speed run for highly heterogeneous samples. Subjected to speeds ranging from 3,000 to 45,000 rpm, the two lots of the KLH conjugate, before and after the process changes, were analyzed using the wide distribution analysis of the SedAnal program, and exhibited similar profiles with regard to very large aggregates (Figure 3).11 There seem to be three pools of large species with the sedimentation coefficients centered at ~120 S, ~400 S, and ~3,000 S, respectively. These peaks are broad, so they may contain a very heterogeneous population of aggregates. The lower limits of the hydrodynamic radius and the molecular weight can be calculated, assuming they are compact spheres, using the following equations:

Figure 3. Gravitational sweep AUC analysis for the KLH conjugate. The centrifugation speed varied from 3,000 to 45,000 rpm. Data were analyzed using the wide distribution analysis in SedAnal.
in which D is the diffusion coefficient, k is the Boltzmann constant, T is the temperature in Kelvin, h is the viscosity of the solvent, Rs is the radius of anhydrous particle, s is the sedimentation coefficient in Svedberg, M is the molecular weight, v bar is the partial specific volume, r is the solvent density, R is the gas constant, and N0 is Avogadro's number. The calculated parameters for the three aggregate populations are shown in Figure 3. It is important to point out that the calculated numbers represent underestimated values. That is because the change in rotor speed can broaden the peaks, and therefore, the diffusion coefficient no longer corresponds to the peak width. For the same reason, the relative abundance cannot be determined by peak area either. However, it is obvious that the very large species have very low quantities in the drug substance.

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