We generated our cell culture harvest in-house using the XD cell culture technique (1) with PER.C6 cells producing a representative MAb (IgG₁, pI = 8.3, 150 kDa). These are E1-immortalized human embryonic retinal cells described elsewhere (14). Using the XD technique, they typically reach a total density of 150–170 × 10⁶ cells/mL (1) in 14–21 days.

Table 1 summarizes the IEX matrices we used. They were prepared by washing with four to five volumes of PBS. We used GIBCO Dulbecco's PBS 1× (catalog #14190) purchased from Invitrogen (www.invitrogen.com) as received or diluted to the desired conductivity with MilliQ water from Millipore (catalog #ZMQS6V00Y, www.millipore.com). The particle density (dry grams/mL) was determined pycnometrically. Matrices were added as a 50% (v/v) slurry in buffer unless otherwise noted.

For depth filtration we used two-stage filter trains from Millipore and Cuno (www.cuno.com). The Millipore train involved different grades of Millistak+HC media: a LabScale Pod primary filter containing 540 cm² of D0HC media (catalog #MD0HC054H1) and a LabScale Pod secondary filter containing 540 cm² of X0HC media (catalog #MX0HC054H1). Millipore depth filtration was followed by sterile filtration with two parallel AcroPak 20 filters from Pall Corporation (www.pall.com) containing 20 cm² of 0.8/0.2-µm Supor membrane.

NOMENCLATURE
APAC: analytical protein A chromatography
CM: carboxymethyl
DEAE: diethylaminoethyl
DNA: deoxyribonucleic acid
ECS: enhanced cell settling
ELISA: enzyme-linked immunosorbent assay
g: gravitational force
HCP: host cell protein
IEX: ion exchange
MAb: monoclonal antibody
ND: not determined
PBS: phosphate-buffered saline
PEI: polyethyleninimine
pI: isoelectric point
rt-PCR: real-time polymerase chain reaction
SDS-PAGE: sodium dodecylsulfate polyacrylamide gel electrophoresis
Si-PEI: Bakerbond wide-pore PEI
TBE: Tris borate ethylenediaminetetraacetic acid (EDTA)
TFF: tangential-flow filtration
TMP: transmembrane pressure
TP: Toyopearl brand
%V: percent viability
V_pool: volume of pooled supernatant and washes
V_work: working volume of bioreactor
XD: extreme-density (perfusion bioreactor)
X_t: total cell density

We chose the area of each filter based on the volume of partially clarified media to be processed and operated the filters in series, with the TMP across each filter monitored by pressure transducers. First we flushed the depth filters with ≥100 L of reverse-osmosis water for each square meter of filter area at 600 L/m²/h to wet the filter media and flush out extractables, after which we attached the sterile filters. Then we loaded partially clarified harvest at 50 L/m²/h until the TMP across any one filter reached 15 psig (or until the partially clarified harvest was fully processed). The filter trains were then flushed with 40 L of 7-mS/cm PBS for each square media of filter area and blown down with air to recover the hold-up volume.

We measured antibody titers in cell culture media samples using APAC with a PA immunodetection cartridge (catalog #2-3001-00) from Applied Biosystems (www.appliedbiosystems.com) run on a Waters 2695 separations module (www.waters.com). The mobile phase was 10 mM sodium phosphate from EMD Chemicals (catalog #SX0720, www.emdchemicals.com), 150 mM sodium chloride from Mallinckrodt (catalog #7581, www.mallinckrodt.com) at pH 8.5. The elution buffer was 12 mM hydrochloric acid from Sigma (catalog #H1758, www.sigmaaldrich.com) with 150 mM sodium chloride. HCP levels were determined with a proprietary ELISA at the quality control department of DSM Biologics (www.dsm.com). We determined DNA levels using rt-PCR with customized primers from Applied Biosystems on a StepOne instrument from the same company (catalog #4376600).

We performed several small-scale experiments to develop a method for clarifying XD cell culture by ECS. Unless otherwise noted, 20 mL of cell culture broth was added to a 50 mL conical tube. In most cases, the XD culture was diluted to a cell density of 70–80 × 10⁶ cells/mL using 5- to 7-mS/cm PBS. We added the IEX matrix and gently mixed the suspension end-over-end for 30 seconds. After the tubes settled for 0.5–2.0 hours, we analyzed aliquots of the supernatant by APAC, HCP ELISA, and rt-PCR. Cell densities and viability were measured with a Vi-Cell XR instrument from Beckman-Coulter (www.beckmancoulter.com). Then the remaining supernatant was decanted and cell pellets washed two times with isotonic PBS.

We pooled the initial supernatant and supernatants from both washes for analysis by APAC, HCP ELISA, and rt-PCR. The percent product recovery was determined using Equation 1, the percent reduction of HCPs with Equation 2, and the percent reduction of DNA by Equation 3. Because of the high cell density of the culture, it was necessary to correct MAb, HCP, and DNA concentrations for the percent biomass. We determined biomass through centrifugation of a known volume of culture at 10,000g for five minutes and taking the biomass volume from the pellet volume after centrifugation.