1D SDS-PAGE with Coomassie blue and silver stain to detect total
residual proteins in API samples
Unlike ELISA that relies on specific antibody-antigen binding to
quantify protein amount, SDS-PAGE resolves all proteins according to
their molecular weight, and its detection sensitivity depends on the
staining method used, with Coomassie blue staining has a detection
sensitivity of ~10-100 ng/band and silver staining has a
detection sensitivity of ~0.1-1 ng/band (Weiss W.,
2009). To estimate the amount of residual protein levels in the API
samples, Prep. Lab batch API at different concentrations, along with a
bovine serum albumin (BSA) standard obtained from micro BCA protein
assay kit (Pierce Biotechnology, Rockford, IL) at different
concentrations, were loaded and separated on 4-12% Bis-Tris gel as
described in Materials and methods. Duplicate gels were either stained
with Coomassie blue (Imperial stain) or silver stain. As shown in Figure
5, Coomassie blue stained gel (Figure 5a) can barely detect BSA at
~ 0.4 µg/mL load (~4.8 ng) and silver
stained gel (Figure 5b) is able to detect BSA at as low as 0.05 µg/mL
(~0.6 ng). In 1 mg/mL API, no protein band is detectable
by Coomassie blue staining and a few bands are barely detectable with
silver staining. When increasing the concentration of API, more proteins
become detectable in both Coomassie blue and silver-stained gels.
Although different proteins can have different response factors when
stained with Coomassie blue (Congdon, Muth, & Splittgerber, 1993; ”The
European Agency for the Evaluation of Medicinal Products Human Medicines
Evaluation Unit
”, 1997; Tal, Silberstein, & Nusser, 1985), the amount of proteins
detected in the API samples on the gel can be estimated using the
intensity of protein bands observed and BSA as a reference standard
(data analysis performed using the ImageQuant IQTL 8.0 software, GE
healthcare (Piscataway, NJ). Since there is barely any protein band
detectable in API at 1 and 2 mg/mL load, and BSA band is detectable at
0.4 µg/mL, assuming all proteins have similar detection sensitivity as
BSA, then no individual protein in the API is above 200 ng/mg (0.4
µg/mL/2 mg/mL). The bands observed in 4, 10, and 30 mg/mL API were also
used to estimate the total protein amount in those bands according to
the calibration curve generated by BSA standard using IQTL8.0 software.
A second gel was run and Coomassie blue stained to assess the
variability of this estimate (gel image not shown). The ng/mg difference
calculated between the two gels are included in Table 2. Across three
different API concentrations, the average protein amount is estimated to
be 764 ng/mg, ranging from 621 to 884 ng/mg. Of note, since Coomassie
blue non-specifically binds all proteins through ionic interactions
between dye sulfonic acid groups and positive protein amine groups as
well as through Van der Waals attractions, in this method, all proteins
over the detection limit should be visible on the gel. The increased
appearance of protein bands along with the increase of loading
concentration indicates that many protein species is at an abundance
level lower than the detection limit when API is loaded at low
concentrations, while the dominant proteins were shown as 6 major bands
in Figure 5 and 6. Although using SDS-PAGE gel with Coomassie blue
staining for protein quantitation can have high variations due to gel
loading volume variability, the differences in response factors of
staining between BSA and other proteins, the time difference on staining
and destaining steps etc., the results from gel analysis indicates the
presence of proteins at levels much higher than that quantified by
Cygnus kit, which is also indicated in our previous publication using
ImageJ software coupled with densitometry analysis on 1D SDS-PAGE
gel(Smith et al., 2022). To further identify those proteins and
investigate whether they can be detected by the Cygnus kit antibody, we
excised those bands and did in-gel digestion coupled with nanoLC-MS/MS
protein identification.