Figure 3. Diagram illustrating the
dual column operation. Condition indicates pre elution phases such as
equilibration and wash. Regen indicates the combination of the
post-elution phases: strip, sanitization and re-equilibration. DBC
stands for dynamic binding capacity. Grey shade illustrates protein
loaded into the column.
Virus Inactivation
The viral inactivation strategy implemented in this study used a plug
flow reactor made from a 4.4 cm diameter x 20 cm height SEC (Size
Exclusion Chromatography) column with Sephadex G-25 Coarse® resin to
achieve a target residence time of 15 minutes. Acidification was
performed before the SEC column and neutralization was performed post
column. The capture product was titrated to an acidification target of
pH 3.5 with an allowable range of ± 0.1. Acid was added in-line with the
product line and the acidified product immediately flowed through the
flow kit’s static mixer and pH probe. A feedback PID controller is used
to continuously adjust the acid pump flow rate to ensure the pH is
within the desired range. After the pH is within the acidification
target range for an appropriate amount of time, the product is directed
through the SEC column which was equilibrated at pH 3.5. Post SEC
column, an in-line addition of base is continuously fed to neutralize
the acidic product to a target of pH 7.2 with an allowable range of ±
0.2. The viral inactivation procedure during this process began on day 8
of the 14-day run. The start day for this step is determined based on
(1) product quality impact from hold time at capture product conditions
and (2) system flow rate lower limit to allow VI to start and run for
extended duration. For this study VI was run in three sub-batches. For a
future optimized process, we aim to operate without pausing.
Anion Exchange
Chromatography
The product from VI step flowed through a filter train comprised of two
Opticap® Sterile 0.2µm filters on either side of a Mustang Q® Membrane
Capsule. This filter train was prepped offline, prior to its connection
to the system according to the vendor specifications. This prep work was
done the day before the filter train was connected to the system (day
7). The filter train was connected on day 8; this timeline coincided
with the start of the viral inactivation step. From here, material was
collected in a final 50L product collection bag. This final polishing
chromatography step was performed with a single-use membrane in a
flowthrough mode. The membrane was not reused. The 0.2µm filter
downstream of the Mustang Q membrane ensured that no potential bioburden
reached the final product collection bag.
Bioburden Sampling and Testing
Strategy
Samples were taken from four points in the process: (1) the cell culture
harvest 10L vessel, (2) Protein A product 10L vessel, (3) VI product
flow kit vessel and (4) final product collection bag. Samples were taken
from sample ports that were specifically installed during set-up and
were clamped closed when not in use to prevent potential exposure to the
environment. Samples were taken aseptically using 10 mL syringes, about
24 hours apart. Samples were aliquoted into individual, labelled tubes
under a biosafety cabinet in order to preserve sterility. Once in the
tubes, the samples were stored at 2-8°C until they were ready to be
submitted for analysis (see section Analytical Testing
for more details). This sampling method was repeated every day of the
run, for each sampling point. The protein A bindable testing was done in
order to record the daily titer at each stage. The HCP and DNA
concentration assays tested the impurity level at each stage and
therefore see how effective each unit operation was at removing the
impurities. The SE-HPLC testing was done to determine the monomer purity
at each stage and therefore determine daily product quality across the
process. The samples for testing bioburden were taken and plated onto
culture plates under a biosafety cabinet using L-shaped spreaders. Once
done, the plates were stored inside of an incubation chamber running at
35 °C. The plates were monitored every day until 72 hours had passed in
order to ensure the sterility of the system. These sampling and
bioburden plating methods were repeated every day for each sampling
point, through the end of the run.
Analytical Testing
Size exclusion-high performance liquid chromatography
(SE-HPLC)
SE-HPLC was performed using a Waters 515 pump, a Waters 2487 Dual
Absorbance Detector (Waters) and a Rheodyme 77,251 injector and a TSK
Gel G3000 SWXL column (300 mm × 7.8 mm, Tosoh Biosep). The volume of
injection was adjusted according to each sample concentration in order
to inject 250 µg of mAb1, and separation was performed at a flow rate of
1.0 mL/min. The running buffer was composed of 100mM sodium sulfate,
100mM sodium phosphate dibasic, pH 6.8. UV detection was performed at
280 nm. The results were reported as monomer peak percentage and high
molecular weight (HMW) content percentage of the total integrated area.
CHO DNA Real-Time PCR
Assay
The assay is performed in a 96-well plate format using a forward and
reverse primer set specific for the Chinese hamster short interspersed
repetitive element (SINE) DNA sequence. A unique 64 base-pair product is
generated from CHO genomic DNA calibrators and test samples within 40
cycles of the PCR. DNA is extracted from test samples and an extracted
control using the DNA Extractor Kit® from Wako. Test samples, CHO
genomic DNA calibrators and controls are added to a reagent master mix
containing primers and SYBR Green. Cycling is performed in three steps
as follows: (1) 10 minutes at 95°C for polymerase activation; (2) 40
cycles of 15 seconds at 95°C for denaturation; (3) 40 cycles of 60
seconds at 60°C for annealing of primers and extension. Quantities of
DNA in the samples above LLOQ are regressed from an assay specific
standard curve generated by the Sequence Detection Systems (SDS)
software.
Gyros CHO Host Cell Protein (HCP)
Assay
Standard curve, samples and controls are diluted in a proprietary Gyros
buffer and loaded into sample microplates. In-house prepared
biotinylated sheep anti-CHO HCP capture antibody and fluorescent labeled
sheep anti-CHO HCP detection antibodies are diluted and loaded into a
reagent microplate. The microplates are loaded on the Gyrolab instrument
and automated execution of the assay is initiated. Once all reactions
are complete in the CDs are read by the instrument using a fluorescence
detector at 647 nm. HCP quantities are regressed from the standard curve
generated by the software and corrected for dilution factor. Reported
sample concentrations are the mean concentration of the dilutions
yielding spike recovery values of 75-125%.
HPLC Protein A bindable Assay for mAb
concentration
Protein A bindable HPLC is used to determine IgG concentration utilizing
Protein A affinity chromatography separation technique. Test sample is
injected onto a PA Immuno-Detection Sensor Cartridge (2.1 mmD x 30 mmL)
with PBS buffer as a mobile phase. The sample flows through the
cartridge where the target IgG specifically binds to the Protein A
immobilized on the cartridge and non-target components are washed out.
The bound IgG is eluted with 0.1% phosphoric acid in PBS, and detected
using UV absorbance at 280 nm. The IgG content is then quantified using
linear regression analysis of the standard curve prepared from the
corresponding calibration standards. Results are reported in µg/mL.