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ß 2017 Wiley Periodicals, Inc. Biotechnology and Bioengineering, Vol. 9999, No. xxx, 2017 1
nutrients for additional growth, as we will show oftentimes cell limiting cells take up lactic acid and the bulk culture pH rises, when
division will continue for another 24–48 h. Since the process ends as glucose is in excess cells make and likely secrete lactic acid which
a fed-batch with a single discrete harvest, conventional purification suppresses pH. In perfusion culture at this point a pump is activated
trains designed for legacy fed-batch upstream processes can still be that delivers perfusion medium containing a high concentration of
employed provided they are scaled sufficiently to deal with the glucose directly to the culture. Since the culture is a perfusion
increase in solids removal and overall productivity. Additionally, it reactor with a cell retention system, the volume of the bioreactor is
is likely that at least during the phase of culture in which the maintained constant through the simultaneous activation of a pump
majority of the protein is produced, such a hybrid perfusion/fed- that removes cell-free culture from the bioreactor system (level or
batch process would result in culture conditions similar in many weight-based control systems, or merely linked to the medium
aspects to a conventional fed-batch, and therefore, less likely to addition pump). As with the HIPDOG control scheme, the HIPCOP
produce protein with a product quality profile dramatically different control system can operate over the time span of minutes.
from legacy fed-batch processes (Meuwly et al., 2006). Therefore, even though the perfusion medium addition and cell-free
While undergoing rapid cell division, particularly in the early harvest pumps are turned on and off repeatedly, the culture is still
stages of batch growth in nutrient rich medium, mammalian cells effectively operating in a continuous perfusion mode. As the cell
commonly exhibit aerobic glycolysis; meaning that a large fraction densities increase, the cells smoothly increase their own perfusion
of the glucose consumed by the cells is converted to lactic acid via rate with a continuous feedback control based on pH, without the
the glycolysis pathway in spite of high availability of oxygen (Miller need for significant operator intervention, or timed finite step
et al., 1987, 1988). In environmentally controlled bioreactors, the changes in perfusion rate. Additionally, since the rate of addition of
lactic acid formed and secreted into the bulk culture fluid is perfusion medium is directly dependent upon the concentration of
neutralized with basic titrant to maintain a stable pH, and the glucose in the perfusion medium when the HIPCOP scheme is
resulting accumulation of lactate and increase in osmotic strength operating, simply decreasing the concentration of glucose in the
can negatively affect cell growth and productivity (Gagnon et al., perfusion medium will increase the volume of perfusion medium
2011; Hassell et al., 1991; Yoon et al., 2006). We have previously delivered to the culture for any particular cell density, and therefore
described a method of high-end pH-controlled delivery of glucose more effectively flush other inhibitory metabolites from the culture.
(HIPDOG) which when used in fed-batch cultures of CHO cells As with HIPDOG in fed-batch cultures during the growth phase,
completely suppresses the accumulation of lactate during the when HIPCOP is operating in a continuous perfusion culture,
growth phase (Gagnon et al., 2011). When HIPDOG is operating depending upon the metabolic characteristics of the cell line, the
and glucose concentrations drop to a low level, cells “signal” their levels of lactate in the culture can fall while the control system is
need for additional glucose by taking up lactic acid (as the acid functioning. When the HIPDOG control scheme is operating in a
form) from the bulk culture and thereby cause the pH to rise. When fed-batch culture it is possible that the drop in concentration of
the high-end pH setpoint is reached, a small amount of lactate might be due in a roundabout way to other cell-produced
concentrated glucose is added which pushes pH down as lactic acids secreted into the media which have a minor suppressive effect
acid is formed again. on pH. These additional metabolic acids serve to partially
The HIPDOG technology can be considered to be a form of “cell- circumvent the normally expected control scheme in which every
controlled” glucose addition. In the current work we have extended molecule of lactic acid that is produced when glucose is
and adapted the basic premise of this technology to continuous momentarily in excess is stoichiometrically reabsorbed when
perfusion culture to allow cells to self-regulate their own rates of glucose is momentarily limited. Since the HIPDOG control scheme
delivery of perfusion medium. The new control scheme can be operating in a fed-batch culture keeps pH constant throughout the
described as high-end pH control of perfusion (HIPCOP). Figure 1 growth phase, the downward “pressure” on pH generated by these
is a pictorial representation of the hypothesized sequence of events other cell-produced acids manifests itself as a slowly decreasing
occurring in the bulk cell culture fluid when the HIPCOP control level of lactate. When the HIPCOP technology is controlling the
scheme is operating. In perfusion culture at fast growth rates cells perfusion rate of a continuous-perfusion bioreactor the phenome-
respond in a similar manner as in fed-batch; when glucose becomes non of lactate levels falling is exacerbated by the perfusion process
itself flushing lactate out of the bioreactor system. Recognizing that
very low levels of lactate in the bulk fluid of a perfusion bioreactor
might cause the HIPCOP control system to cease functioning
properly, we found it advantageous for these initial experiments to
add a low concentration (1–3 g/L) of purified sodium-L-lactate
directly to the perfusion medium.
Figure 4. Timecourse of various nutrients, metabolites, and cell culture conditions over the course of the experiments. A: Residual glucose. B: Lactate. C: Ammonium. D:
Osmolality.
Cell line/project
Parameter A B C D E
Total volume of perfusion medium used (reactor volumes, based on final day-12 bioreactor volume) 1.6 1.4 1.4 1.8 1.0
Total volume of perfusion medium used (reactor volumes, based on bioreactor volume during perfusion phase) 2.5 2.2 2.2 2.4 1.6
Peak perfusion rate (volume/reactor volume/day in last 24 h of perfusion based on bioreactor volume during perfusion) 1.1 1.2 1.0 1.3 0.7
Cell specific perfusion rate from days 1–4 (picoliters/cell/day) 25 33 34 24 27
Hybrid process integrated viable cell density from days 4–12 (e6 cell day/mL) 397 400 407 426 490
Optimized fed-batch integrated viable cell density (12-day culture, e6 cell day/mL) 216 318 223 217 181
Hybrid process total feed volume used (percent of final reactor volume) 35 36 37 34 36
Hybrid process final day-12 titer (g/L) 11.8 11.3 12.0 6.1 8.2
Hybrid process percent titer improvement over optimized fed-batch process 151 95 71 22 70
Specific productivity of hybrid process (picograms/cell/day, days 4–12) 27 26 27 14 16
Specific productivity of optimized fed-batch process (picograms/cell/day) 22 18 31 23 27
Hybrid process final day-12 cell viability (%) 86.7 97.3 94.4 92.4 96.4
Specific productivities for the hybrid process are calculated using the change in titer during the time in fed-batch and the integrated viable cell densities over the same span of
time.
affected cell growth rates near the time the cell densities peaked groups. In the presence of excess ammonium, mammalian cells will
around day 5–6. While not detailed in the current work, on-going also oftentimes transaminate pyruvate to form alanine which will
experimental investigations into CHO fed-batch culture in our accumulate in the culture (Altamirano et al., 2001). In the current
laboratory suggest that numerous low molecular weight metabolites experiment, generally when ammonium levels in the culture were
accumulating in culture serve as inhibitors of cell growth. elevated, the levels of alanine were also elevated (data not shown).
After the HIPCOP portion of the bioreactor experiment, no The timecourse of osmolality of the cultures is shown in
attempt was made to continue glucose limitation for any of the Figure 4D. Within the first 24 h for most cultures, the osmolality
bioreactors. As a result, due to cell line metabolism variability, the increases slightly as some fraction of glucose is converted to lactate
lactate level for some bioreactors rose modestly from day 4 to 5, and and then neutralized by base. This osmolality increase is
then generally fell throughout the remainder of the fed-batch compensated to some extent as the glucose and amino acids
(Fig. 4B). One cell line, known from previous conventional fed- present in the medium are consumed. After the perfusion is self-
batch experiments to be particularly prone to lactic acid production initiated by the cells between 24 and 48 h after inoculation, the
when glucose is freely available, peaked at a lactate level of osmolality of the culture stabilizes and trends slightly lower for all
4.7–4.8 g/L on days 5 and 6. cultures until the perfusion stage is terminated on day 4. From an
While no attempt was made to limit glucose availability beyond analysis of residual amino acids, we estimate that between 40 and
day 4, in some cases due to the rapidly increasing cell densities from 60% of the amino acids entering in the perfusion medium are being
days 4 to 6 and the difficulty in predicting the metabolism during consumed (data not shown). To minimize high osmolality late in
this transition state, some cell lines drove the glucose concentration
down to below 1 g/L transiently (Fig. 4A). One bioreactor was
slightly overfed with concentrated glucose (cell line D days 5–7,
Fig. 4A). After completing the experiments with cell lines A and B,
because the residual lactate levels were getting to low values just
prior to the termination of perfusion, there was some concern that
the HIPCOP control scheme might be on the edge of failure. If the
culture could no longer signal a need for additional glucose through
a rise in bulk pH, the perfusion rate might no longer continue to
ramp up. For this reason the sodium-L-lactate concentration in the
perfusion medium was increased from 1.4 to 2.5 g/L for
experiments performed with cell lines C, D, and E (Table I).
Ammonium levels in the bioreactors were generally below about
6 mM until the last several days of culture. Some accumulation of
amino acids in the culture occurred due to a slight excess of
concentrated feeds (data not shown). The excess availability of
amino acids may have contributed to the slight rise in ammonium
levels from days 10–12 for some cultures as sometimes mammalian Figure 5. Final day-12 harvest titer of HIPCOP hybrid perfusion fed-batch culture
cells will utilize excess amino acids as a carbon source for the Kreb’s compared to that of the optimized fed-batch process designed for each cell line/
product.
cycle, which can lead to an accumulation of the residual amine
Cell line/project
All bioreactor samples underwent a simplified purification process consisting of drip column proA purification. High and low molecular species were quantitated by size
exclusion chromatography; fragment was quantitated by reduced capillary gel electrophoresis; N-linked glycan analysis was by the N-linked Oligosaccharide method as
described in materials and methods. Elevated high molecular mass species in bioreactor samples from project C may be due to the truncated purification process. The reference
standards for projects B and E were created using clonal cell lines that were abandoned in later stage development partly due to elevated high molecular mass species levels. All
reference standard material was generated at 500 or 2,500 L scale with a complete purification process. Blanks in the table indicate the assay was not performed.
culture, after the experiments with cell lines A and B it was decided exceeded 375 mOsm/kg, well within a reasonable range for CHO
to slightly decrease the overall osmotic strength of the perfusion cells in culture.
medium for the experiments with cell lines C, D, and E, so that the The final day-12 cell densities in the current experiments were
osmolality of the culture is as low as possible when the fed-batch between 31 and 48 106 viable cells/mL (Fig. 3A), but it is possible
portion of the experiment begins. that a significant number of cells were lost to shear damage during
The timecourse of product titer, the concentration of the culture and therefore calculating total cell mass using the
recombinant humanized IgG antibody molecule, is plotted in percent viability measurement might be somewhat misleading. For
Figure 3C. Samples of permeate were also tested for product titer this reason careful measurements of total solids were performed by
during the perfusion stage of the experiment and the results centrifuging culture samples. This analysis showed that the percent
showed that no significant product retention occurred across the solids were between 10% and 12% by volume on the final day of
microfiltration hollow fiber unit. For this reason, during the culture which might be close to the limit for some solids removal
perfusion stage most IgG antibody is flushed out of the bioreactor technologies typically utilized for large-scale harvest operations.
system, which explains the very low antibody concentration in the
bioreactor around days 4–5. Calculations performed utilizing the
Comparison of Product QualityHybrid Perfusion
flow rates and concentrations of product during the perfusion
Fed-Batch to Optimized Fed-Batch
phase allow us to estimate that only 9–11% of total antibody
produced during the entire 12-day culture is lost to the permeate Product quality analytics were performed on some of the samples
stream. from the experiments, the results are shown in Table III and
Although extensive optimization experiments utilizing this Figure 6. For many of the parameters, there are no significant
hybrid perfusion/fed-batch approach have not been performed, a differences between the day-12 harvest sample of the hybrid
comparison can be made to the late-stage optimized fed-batch perfusion fed-batch process when compared to the reference
processes for all of the cell lines/projects utilized in the current standard that was generated at large scale (500 or 2,500-L) using an
experiments. With the possible exception of cell line D (which optimized fed-batch process and fully purified. The acidic and basic
exhibited a higher cell specific productivity in its legacy basal species quantitation illustrated by Figure 6 shows that while there
medium and used a very high inoculation density in the optimized may be differences in earlier time-point samples, usually by the
fed-batch process), Figure 5 and Table II show that substantial day-12 harvest the profiles are quite similar. A more detailed
improvements in titer were achieved, some as high as 2.5 times the analytical investigation would be required to determine the precise
fed-batch productivity. It is likely that using the hybrid perfusion/ nature of the changes to the molecule that are causing the shifts in
fed-batch approach even higher peak cell densities would likely have acidic and basic species, and the reasons for the shifts might also
been achievable if the perfusion was extended beyond day 4. Since vary from antibody to antibody. From prior work and knowledge of
the cell density typically peaks 1–2 days after the perfusion phase, hold time stability experiments performed on proteins under
and the majority of the protein of interest is produced during the development, we know that changes in acidic and basic species
fed-batch portion of the culture, optimizing the feed composition levels are common in antibodies produced in animal cell culture
and feeding rate during this stage is particularly critical. It is and can be due to chemical or enzymatic modification of molecules
interesting to note that despite using a feed solution with an osmotic already secreted, or as a result of shifts in the properties of the
strength above 1,300 mOsm/kg and a total feed volume of 37% for molecules being secreted at different times in culture. For the
cell line C, the bulk osmotic strength of the bioreactor never majority of the antibodies studied in this work we see an increase in
the acidic species with time in culture, which is sometimes the original process with no increase in length of culture. The
indicative of increasing levels of amino acid deamidation (Du et al., HIPCOP technology functioned effectively without significant
2012). While less pronounced, we also see a slight increase in the manual intervention for all five cell lines, allowing the cells to self-
level of basic species with time in culture for the majority of the control their perfusion rates as the cell mass increased. The total
molecules. Basic species increases are often due to increases in volumes of perfusion medium utilized were modest and because the
the levels of carboxy-terminal lysine in the heavy chain, or increases perfusion takes place only for a short duration and occurs when the
in amidation of carboxy-terminal proline. The levels of high- or low- cellular viability is high, scale-up of the cell retention system with
molecular mass species and fragments are also not significantly currently available technology and equipment is also likely to be
increased when compared to the reference standards for each achievable. Potentially of more concern regarding scale-up might be
project (Table III). the control of environmental parameters when viable cell densities
exceed 80 106 cells/mL. Design modifications of bioreactor
sparging and agitation systems may be required to supply sufficient
Conclusions
oxygen and even more importantly strip adequate carbon dioxide at
The hybrid perfusion/fed-batch process demonstrated significant large scale.
increases in productivity when compared with a more conventional This hybrid perfusion/fed-batch approach provides an opportu-
optimized fed-batch process for five different CHO cell lines/ nity to increase productivity significantly without moving to a fully
projects. In one case (cell line A) the final harvest titer was 2.5 times continuous and long-duration upstream process. The hybrid