Alpha1-antitrypsin Deficiency


Figure 3: Chromatogram Showing the Wash, Elution and Strip Profile for a Typical AIEX Run


2,500 2,000 1,500 1,000 500 0


0


100mM NaCl (control)


Elution Strip


Figure 3. The older Prolastin process included an AIEX step, but a second PEG precipitation step was necessary to concentrate the process stream before loading onto the column. Due to improvements in chromatography media, the upstream concentration step is no longer needed and has been eliminated in the Prolastin-C process. Using the conditions determined for the Prolastin-C AIEX step, anion eluates with high purity were obtained during scale-up from bench (~30ml column volume) to clinical and commercial scale (see Table 1).


Wash Flow-through 100 200 AIEX = anion exchange chromatography.


Table 1: Development Process Scales in Relation to Commercial Scale


Process Scale Bench


Macrobench Preclinical Clinical


Commercial


Relation to Commercial Scale 1/5,000 1/400 1/20 1/10 1/1


Table 2: Results from a Study of Alpha1-PI Purity and Potency Recovery from Cation Chromatography done as a


DoE Evaluation of pH and Conductivity (Sodium Chloride Concentration) in Column Load


Sodium Chloride in pH* Column Load (mM)


Control Increase


Increased


Alpha1-PI % Purity Alpha1-PI % (SE-HPLC)


Minimal effect Increased slightly Decreases


Recovery** Decreases Decreases


*pH of both column equilibration buffer and column load. **As measured by the potency assay. DoE = design of experimentation; SE-HPLC = size-exclusion high-performance liquid chromatograph.


screened for alpha1-PI-binding capacity and purification efficiency, as well as for stability, leachables, and toxicity. Due to similarities in their surface


biochemistry, both alpha1-PI and albumin bind to the resin, so process conditions were designed to selectively wash off as much albumin as


possible while recovering alpha1-PI with high purity.


Design-of-experiment studies showed that column load pH, wash buffer pH and conductivity, and elution buffer conductivity were important parameters impacting output quality and potency yield. Robust eluate purity and potency recovery can be achieved for load conditions of


2–14g alpha1-PI/l of resin when using a phosphate wash buffer. Changing the pH of the wash buffer decreases the amount of albumin


washed from the column and decreases alpha1-PI purity in the eluate. The amount of wash used also affects eluate purity. Washing to baseline


A280 is necessary for complete albumin removal prior to elution of alpha1-PI. A typical chromatogram showing the wash albumin peak, elution alpha1-PI peak and impurity peak in the column strip is shown in


24


300 Volume (ml)


400 500 600


The other chromatography step in the Prolastin-C process is cation exchange chromatography (CIEX). CIEX is used as an additional purification step to remove impurities, such as albumin and IgA. Impurity levels vary between batches due to heterogeneity that can be traced back to the starting material (fraction IV-1 paste). This step was developed to be performed at a pH where positively-charged impurities in the feed stream bind to the negatively-charged CIEX column,


while negatively-charged alpha1-PI passes through and is collected in the flow-through. Again, several types of cation exchange media were tested for their ability to efficiently remove the protein impurities albumin and


IgA while maintaining high recovery of alpha1-PI. A series of buffer systems were also tested to reach acceptable purity and yield levels.


The effects of pH and flow rate on alpha1-PI potency and purity across the CIEX step were examined and used to determine the operating ranges for conductivity, equilibration buffer pH and column load pH. There was a general trend for potency to decrease (from 95.7 to 64.5%) as the pH of the column load decreased. Likewise, analysis by size- exclusion high-performance liquid chromatography (SE-HPLC) showed a decrease in the percentage of monomer and an increase in high-molecular-weight aggregates as the pH decreased. The results from a design-of-experiment study on the effects of column load pH and conductivity (modeled using sodium chloride) are presented in Table 2. Scale-up from bench scale (~8ml) to clinical scale (~35lml column


volume) was accomplished while achieving an alpha1-PI purity of >93% (n=46) and a potency recovery of >91% (n=46). These chromatography steps are followed by 15nm nanofiltration. Virus-retentive filtration has been developed in both normal-flow filtration and tangential-flow filtration formats. Normal-flow filtration was the preferred mode due to its ease of operation, low water usage and lower capital costs. The 15nm nanofilter was selected because it effectively cleared pathogens, tolerated variations in feed streams and provided high product yields


(mean recovery in alpha1-PI potency of 98.1%). After the nanofiltration step, an additional ultrafiltration/diafiltration step follows to concentrate the protein and remove buffer components. Finally, buffers are added to the concentrated nanofiltrate, followed by sterile filtration and lyophilization to produce the final sterile bulk.


Prolastin-C and Prolastin Characterization Studies Protein characterization of Prolastin-C and Prolastin was conducted using capillary gel electrophoresis, isoelectric focusing and SE-HPLC.


Pathogen Reduction Studies Virus Reduction


The virus-reduction capacities of the Prolastin-C and Prolastin manufacturing processes were quantitated using validated bench-scale models of relevant processing steps. For the Prolastin-C manufacturing


US RESPIRATORY DISEASE


mAU


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