Based on the higher cell densities, BL21(DE3) fermentation broths had
greater turbidity. All BL21(DE3) clones had high reduction of turbidity,
at least 94%, except B<oFabx>, which was also
the one that had the highest impact on growth. Notably, the highest
reduction was achieved for B<dFabx>, which did
not express any Fab and grew unaffected. In general, the HMS174(DE3)
clones had lower centrifugation efficiency, reflecting the higher impact
on cell growth.
MS analysis
LC-ESI-MS was used to evaluate the Fab products. We determined the
molecular mass of both intracellular and extracellular Fab. All of FTN2
(100%) was the correctly folded full Fab both intracellularly and
extracellularly, except for B<dFTN2>, in which a
minimal amount (~1%) presented a mass shift of +148 Da.
In the case of BIWA4, all clones produced only the correct Fab, except
for H<dBIWA4>, which had 70% with +2 Da in the
extracellular fraction, and B<oBIWA4>, which had
a mass shift of -178 Da for approximately 5% of the intracellularly
retained Fabs. BIBH1 had a more pronounced mass shift, as the ratio of
correctly processed Fab varied from 40% to 75% for both fractions. The
observed mass shift was -17 Da for all clones. For
B<dBIWA4>, the intracellular fraction had a
mixture of -16 and +100 Da. Due to the low amounts that were gained for
Fabx, there was only one MS result, which indicate a ratio of 30% -17
Da in the extracellular fraction of B<oFabx>.
Wide variations were observed in terms of the ratio of free LC and LC
fragments of total Fab. The ratio ranged from 0% for
B<oBIWA4> up to 74% for
H<oFTN2>; nevertheless, all combinations showed
similar ratios in intracellular and extracellular fractions (Table 2).
In general, the ranking for these impurities was
FTN2>BIBH1>BIWA4, independent of the
investigated host and leader combination. Due to the very low expression
levels of Fabx, this point was only investigated for one exemplary
combination.
Discussion
In this study, we investigated how the combination of host strain,
expressed Fab, and translocation pathway interact with and influence
process efficiency. One of the main results was that the selected
variations and their combined application led to scattered product
yields and cell growth, as well as significant differences in DSP
operability. The greatest influence was from the Fab molecule that was
expressed, though all four candidates had the same backbone and only the
CDA region varied. The highest product concentrations for all
combinations were measured for FTN2, followed by BIWA4, BIBH1, and Fabx,
with few exceptions. For all Fab-leader combinations, HMS174(DE3) was
superior to its BL21(DE3) counterparts in terms of specific Fab titers.
However, the BL21(DE3) host responded less strongly to the expression of
the Fabs, and cells were generally more vital with better growth
behavior. This was also confirmed by the DSP investigations, in which
considerably less DNA was quantified in the supernatant, resulting in
lower viscosity and better clearance (Fig. 3A and B). Considering the
values from Table 1, linearity between clearance efficiency, viscosity,
and DNA content is clearly apparent if the values for
B<oFabx> are left aside. The least influence on
process and yield was observed for the leader sequence. No general
overriding behavior could be identified, only that
OmpASS led to slightly higher expression levels in
both strains in most cases. Taken together with DSP operability, the
chosen procedure in the USP, with early induction and a long production
phase independent of strain and Fab, resulted in very unfavorable
distribution ratios of intracellular and extracellular Fabs, in the
range of 60:40 to 40:60. One of the fractions would have to be omitted
in DSP, which is not acceptable in view of the generally low product
titers. Although high extracellular fractions were observed in both
strains, there was a significant difference in extracellular DNA
content, which was much lower in BL21(DE3), indicating increased release
of Fab into the supernatant independent of cell lysis (Fig. 2A and Fig.
3C and D). This finding is clearly a positive characteristic
attributable to the strain with high impact on process design and
efficiency.
The traditional approach for process development focused on USP yield
would favor H<oFab> clones as expression hosts
due to the higher specific total Fab titers. However, the high cell
lysis rates, high extracellular DNA concentrations, and high viscosities
would negatively affect the DSP performance and pose great challenges
for process development. DSP would be faced with cell broths containing
high levels of extracellular product fraction and increased DNA and
endotoxin levels, in combination with low centrifugation efficiencies
due to the elevated viscosity. Therefore, a classical purification
process for intracellular products would not be economic and would lead
to high product loss. This could be circumvented by homogenizing the
cell broth before harvesting. However, a drawback would be that DSP
would be confronted with a broth containing media residue and
extracellular metabolites that are normally removed by harvesting the
cells. These impurities would need to be removed during subsequent unit
operations.
These results highlight the importance of a holistic approach for
process optimization, including integrating the upstream and downstream
aspects. The parameters selected for the evaluation of DSP performance
were found to be a good choice. These parameters allow for a
comprehensive process evaluation and provide relevant information for
integrated process optimization. In our specific case, due to the
observed growth and product leakage kinetics, shifting the induction and
harvest time points could be a starting point for optimization. Later
induction would shorten the expression period and, therefore, could
reduce cell lysis and product leakage.
The MS analysis showed that, in almost all cases, the correct regular
Fab was expressed and the extracellular fraction was not subjected to
changes. Only BIBH1 presented a significant mass shift caused by the
cyclization of N-terminal glutamine to pyroglutamate, which is a
well-described phenomenon in the literature for the production of
recombinant antibodies [38, 39]. This represents another issue
affecting DSP operability, as separation of product variants is
challenging due to their small differences in charge. Furthermore, in
all combinations except B<oBIWA4>, remarkable
amounts of free LC and LC-fragments were found, which can impede DSP by
limiting Protein L column capacities, as these impurities will compete
with the product for binding [34].
Thus, we showed that strong impairment of growth by Fab expression leads
to a high extracellular product fraction and contamination, resulting in
high viscosity and low centrifugation efficiency. The chosen host and
expressed Fab product exerted the main influences on growth, titer, and
DSP operability. In particular, HMS174(DE3) clones had higher
extracellular product fractions, DNA and endotoxin contents, and
viscosities, and lower final CDM.
Our results indicate significant interdependencies between the two
disciplines, and that decisions made at the USP level are especially
important for the performance of the first steps of DSP. Therefore, the
classical yield-oriented USP development approach would create major
challenges for DSP development, as higher extracellular impurities,
product localization, and poor manufacturability would lead to low
overall product yields and high manufacturing costs. This underlines the
need for a holistic and integrated process development approach to
establish reliable and economical production processes, and to shorten
the overall time to market. Our results provide the basis for further
research into integrated and holistic process development. By monitoring
the impact on growth and product localization caused by changes in the
USP strategy, we are now able to better predict fluctuations in DSP.