Identification and quantification of HCPs by LC-MS
Total HCPs measurements of HCCF from different modes of cell culture
processes, FB, SS and NSS are listed in Table 1. Samples from both
unpurified (i.e. HCCF) and Protein A purified materials were analyzed.
For the perfusion systems, HCP counts across the harvest duration
provides insights to the HCP profile changes from early to late growth
stage.
During the early stages of cell culture growth, there is a significant
lag between central metabolism and post-translational modification. A
strategy was developed to exclude harvest product from the early days
(1-5 days) of production to ensure stable product quality. Hence, the
mAb1 quality and composition of HCPs were less varied depending on
culture days but maintained significant changes with the different
culture processes. The level of HCPs in HCCF were greater than
~105 ppm for FB and NSS perfusion but
approximately 104 ppm for SS perfusion. Low cell
viability for both FB and NSS perfusion culture processes can mean high
levels of HCPs released in the feed stream due to cell lysis.
General patterns of HCP distribution was visualized in 2D-PAGE shown
Figure 2. The loading of the gel is normalized by the amount of product.
Different culture processes show a comparable spot pattern and intensity
with mAb1 spots. For HCP comparison, SS shows less overall HCP
population. Day 5 perfusion product also shows less HCPs detected
compared to perfusion day 14. Interestingly, NSS perfusion demonstrates
additional HCPs in the HMW area (across pI range) while the FB shows
additional HCPs across the MW and PI ranges. There are several HCP
species (spots) and abundance (spot volume) varied among the culture
processes. These minor variations in HCP composition and abundance
between different processes suggest influence of cultivation process on
HCP in cell culture supernatants.
ELISA and 2D-PAGE have limited capacity to detect HCPs that are in low
abundance or poorly immunogenic. Orthogonal detection and monitoring
technique using MS offers the opportunity not only to arrive at an
accurate quantification of total HCPs but also provides the ability to
identify the individual protein species with high sensitivity (Levy et
al., 2014). Samples were analyzed by high-resolution LC-MS and HCPs were
identified by SEQUEST search against the CHO K1 proteome database (GCF
000223135.1 CriGri 1.0 from
www.ncbi.nlm.nih.gov) through Thermo
Scientific Proteome Discoverer v2.2. Table 1 summarizes the group number
of HCPs in HCCF in the cultures with at least 2 identified unique
peptides. A total of 1310 HCPs were identified in FB culture
supernatants. Perfusion SS contained fewer detected HCPs
(~300) and NSS perfusion shows comparable numbers of
HCPs (~1700) as FB that were likely due to the high cell
concentration. Approximately 83% of fed -batch, 70% NSS perfusion,
92% SS perfusion were commonly identified with other culture processes
using. HCP composition was significantly more diverse in the FB and NSS
perfusion culture than in the SS perfusion culture. These data confirm
similar trends in HCP population using ELISA.
The HCCF from the three types of cultures were later purified by ProA
chromatography and analyzed for HCP population. As indicated in Table 1,
results from both MS and ELISA method show drastic decrease on the HCP
level for both perfusions, but HCP levels remain relatively high for FB.
This result indicates that it might be easy to remove host cell protein
from perfusion process by ProA column. To investigate any significant
differences in product quality from FB, SS and NSS and their potential
impacts on methodologies used in the study, product quality indicators
such as aggregation, fragmentation, N-linked glycosylation patterns and
charge variants were evaluated after ProA purification (Supplementary
Table 1). Both aggregation and fragmentation were slightly reduced in
perfusion cultures compared to fed-batch. N-glycosylation showed similar
pattern for both types of culture. Perfusion NSS has been observed a
simpler glycosylation (G0F) increased as the runs progress and cell
densities go up due to the decrease in nutrient availability in all
culture processes. At the same time, high mannose (Man5) of NSS
perfusion increased and a similar correlation has also been reported in
other publications, which indicate that this could be a general
phenomenon (Pacis et al., 2011).
The charged variants analysis of mAb1 shows that FB has 24.4% acidic
peaks, and 8.4% basic peaks (Figure 3). With perfusion processes, the
abundance acidic species is significantly reduced to 13.6% for SS and
15.8% for NSS while more basic forms are produced to 14.6% for SS and
13.4% for NSS compared to FB. Other mAbs also exhibited similar trends
in charged variants with perfusion processes due to the increase
C-terminal lysine and/or less light chain N-terminal glutamine
cyclization (Gomez et al., 2020; Walther et al., 2019). The general
consensus according to a number of scientific publications and various
conventions is that C-terminal lysine and N-terminal glutamine
cyclization, are likely non-CQAs (critical quality attributes), which
may not have substantial effects on antigen binding affinity, efficacy
and safety of the antibody product (Du et al., 2012; Sing h et al.,
2016). However, charged variants can alter the composition of HCP
impurity profile if HCPs are binding to mAbs.
The relatively minor charge differences have been shown to have a large
impact on the total HCP association due to the single abundant charge
species that bind substantially more strongly to HCPs (Levy et al.,
2014). For ProA chromatography, post load wash is a critical step to
reduce HCPs. In the mAb1 purification process, neutral pH (pH 7.4) was
employed for the capture and wash step. Since HCPs tend to have lower
isoelectric points (pI) (Lavoie et al., 2019), the majority of HCPs are
anionic whereas mAb1 (pI 8.5) is cationic at the neutral pH. Thus,
strong hydrophobic and electrostatic interactions can form between mAb1
and HCPs. Based on our proteomic analysis after ProA purification, FB
culture contains HCPs that have theoretical pI below neutral pH, pI
< 6 ≈ 50%, pI < 7 ≈ 74%, and pI < 8 ≈
82%. This result showed that these low isoelectric points tend to bind
with acidic species of mAb1 in FB culture. Our work brings up the
awareness of potential differences of charged variants between fed-batch
and perfusion and the needs for further characterization of the product,
as well as HCPs. In general, the differences we have observed in all
product qualities tested, i.e. aggregates, N-glycan, charged
variants, are not expected to have a noticeable impact on the
methodologies used in this work.