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


Figure 2: SE-HPLC of Prolastin-C Showing the Defined Regions of Monomer, Oligomer and Aggregate


Monomer


Figure 3: Western Blot of Isoelectric Focusing Gel Comparing Alpha1


with Prolastin-C 1234 –


-PI Isoforms of Normal Human Plasma


Aggregate Oligomer +


5101520 Time (min)


SE-HPLC = size-exclusion high-performance liquid chromatography.


Table 2: Molecular Distribution of Prolastin-C by Size Exclusion High-performance Liquid Chromatography (Mean±1 SD)


Test Monomer (%)


Average monomer mass Oligomer (%)


Oligomer mass range Aggregate (%)


Aggregate mass range Purity Six different lots of Prolastin-C were evaluated by CZE. The mean


alpha1-PI purity by CZE was measured at 97%. SDS-PAGE and SDS-CGE showed similar purity levels with a prominent alpha1-PI band at approximately 50kDa and 29 seconds, respectively


(see Figure 1). Under non-reducing conditions, SDS-PAGE showed a faint band with a molecular weight of approximately 100kDa. Based on


its molecular weight and detection with an antihuman alpha1-PI antibody probe in Western blots, the faint band represents a low level


of alpha1-PI dimer. SE-HPLC separated Prolastin-C into monomer (50kDa), oligomer (100–500kDa), and high-molecular-weight aggregate (>500kDa) regions (see Figure 2). The molecular masses of these three regions were defined by light scattering. The six Prolastin-C lots showed an average monomer level of 88.8% and contained a relatively low aggregate level of ≤1% (see Table 2).


Mass spectrometry using MALDI confirmed the purity of Prolastin-C by not detecting the presence of other plasma proteins. The reported


molecular weight of m/z 50001 for alpha1-PI was an average of all isoforms.


Glycan Profile


IEF of Prolastin-C revealed the two primary isoforms of alpha1-PI (M6 and M4), together with a faint band representing the minor isoform (M2). This


US RESPIRATORY DISEASE 50,000 51,070 51,000 Mass RP-HPLC = reversed-phase high-performance liquid chromatography. IEF profile of Prolastin-C is comparable to that found in normal human


plasma, as confirmed by Western blotting using an anti-alpha1-PI antibody probe (see Figure 3). Impurities


Deconvolution of the electrospray mass spectrometry data collected after RP-HPLC of Prolastin-C showed a closely spaced pattern of


molecular masses representing the multiple glycoforms of alpha1-PI (see Figure 4). The primary glycoforms of alpha1-PI are M6 and M4, with the higher-mass species likely representing the naturally-occurring fucosylated glycoforms of M6 and M4 (M6 + fucose, M4 + fucose).


The presence of the naturally-occurring single cysteine in Prolastin-C was proven by intact protein mass spectrometry (using electrospray ionization) after processing under reducing and non-reducing conditions (see Figure 4). The reduced profile is shifted to the left compared with the non-reduced profile. This reflects the decrease in


29


Prolastin-C (n = 6 lots) 88.8 ± 1.7 ~50kDa


10.5 ± 1.6


100–500kDa ≤1.0


>500kDa 50,000 51,000 Mass 50,918 M6 M4 51,569 51,720 52,000 52,000 25 30


Western blot of Isoelectric focusing gel comparing alpha1-PI isoforms of normal human plasma (lanes 1 and 2) with Prolastin-C (lanes 3 and 4). Each sample type was loaded at two alpha1-PI concentrations (~2 and ~0.5µg).


Figure 4: Deconvolution of the Electrospray Mass Spectrum Following RP-HPLC of Prolastin-C under Non-reducing (Top) and Reducing (Bottom) Conditions


51,051 M6 51,188 50,943 51,569 M4 51,701 51,852


M6 M4 M2


Response


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