Characterization of a New Alpha1-proteinase Inhibitor from Human Plasma—Prolastin®-C


process, four virus-removal steps (cold ethanol fractionation, PEG precipitation, depth filtration and 15nm nanofiltration) and one virus-inactivation step (S/D treatment) were modeled. Three virus-removal steps (cold ethanol fractionation, PEG precipitation, and depth filtration) and one virus-inactivation step (pasteurization) were modeled for the Prolastin manufacturing process. The virus-reduction capacity of each step was evaluated by spiking process intermediates with model viruses and the results were quantitated using infectivity-based assay systems. Viruses used included the enveloped bovine viral diarrhea virus and the non-enveloped porcine parvovirus.


The overall virus-reduction capacity of each manufacturing process (global virus reduction factor) was calculated by summing virus


reduction factors from individual process steps achieving a log10 reduction ≥1. Where reduction to the limit of detection was achieved, results are reported as ≥ the global reduction factor. A greater reduction could potentially be achieved if a higher titer of virus could be used as the spike without disrupting the process model.


Prion Reduction


The prion reduction capacities of the Prolastin-C and Prolastin manufacturing processes were quantitated using validated bench-scale models of relevant processing steps. Three of the manufacturing steps (that are common to both Prolastin-C and Prolastin processes and are upstream of process modifications) are capable of removing TSE infectivity: cold ethanol fractionation, PEG precipitation, and depth filtration. The capacity of these steps to reduce TSE infectivity was evaluated by spiking process intermediates with a TSE agent preparation containing the pathogenic form of prion protein (PrPSc). The levels of TSE agent in the intermediates and resulting fractions were analyzed. Two methodologies were used to evaluate TSE removal:


• •


the reduction of TSE infectivity was assessed using an animal bioassay; and


the removal of a marker for TSE infectivity (the protease-resistant form of the prion protein, PrPRes) was measured using a Western blot assay.


The animal bioassay is typically 1–2 log10 more sensitive than the Western blot assay.


Results Protein Characterization


The additional steps in the Prolastin-C manufacturing process result in excellent purity (see Figure 4). Protein characterization studies were carried out in order to compare Prolastin-C produced by the modified process with Prolastin. The characterization studies support the manufacturing process changes. Prolastin-C was shown to have the


same active drug substance (alpha1-PI) as Prolastin. Capillary gel electrophoresis showed that Prolastin-C appeared as a single-band


alpha1-PI. This demonstrated a high degree of purity (see Figure 4). Isoelectric focusing revealed several protein bands in Prolastin-C (see Figure 4). These bands represented the naturally occurring isoforms


of alpha1-PI. Alpha1-PI exists as an acidic glycoprotein with two primary and other minor glycoisoforms. SE-HPLC showed monomeric alpha1-PI as the primary peak. Higher-molecular-weight protein


US RESPIRATORY DISEASE


456789101112 Time (min)


Prolastin-C SE-HPLC


CGE = capillary gel electrophoresis; IEF = isoelectric focusing; SE-HPLC = size-exclusion high-performance liquid chromatography.


impurities and polymerized protein (oligomers and aggregates of


alpha1-PI) with larger hydrodynamic volumes of elute appear prior to the monomer peak (see Figure 4). Aggregates were below detectable levels in Prolastin-C. The Prolastin-C formulation had an increased


concentration of alpha1-PI (50mg/ml) and had approximately twice the functional activity compared with Prolastin (54.3 versus 26.7mg/ml, respectively). This provides for lower infusion volumes and potentially shorter infusion times.


Pathogen Reduction


The pathogen-reduction capacity of the Prolastin-C manufacturing process was demonstrated. The global virus-reduction factors for


Prolastin-C were ≥10.2 to ≥25.5 log10 for enveloped viruses and ≥12.7 to ≥13.7 log10 for non-enveloped viruses. In addition to this, a minimum of 6.0 log10 reduction of TSE infectivity was achieved.


25 Prolastin


92 CGE


Figure 4: Prolastin-C as Characterized by CGE, IEF and SE-HPLC


Prolastin Prolastin-C s


38 36 34 32 30 28 26 24 22 20


IEF Prolastin Prolastin-C


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