Various precipitation techniques have been used in the industrial purification of proteins for many years. Precipitation processes
can be separated into two main categories: impurity precipitation and product precipitation. Impurity precipitation is operationally
simpler but carryover of the precipitants can challenge subsequent unit operations. Product precipitation may have a higher
risk of damage to the target molecule, but in addition to purifying the product, product precipitation also enables buffer
exchange and concentration during the resolubilization step. An antibody precipitation step has been developed using a recombinant
antibody produced in PER.C6 cells and statistical design of experiments to optimize product yield and host cell protein (HCP)
removal. After appropriate precipitation conditions were developed, two methods to capture the antibody pellet were evaluated:
depth filtration and microfiltration. A wash step was incorporated in both methods to reduce soluble impurities. The final
process resulted in a product yield of 90% and HCP reduction of approximately 1 LRV.
The method of pellet capture was shown to have a significant impact on the purity of the redissolved product. The precipitation
step is readily scalable and fits a fully disposable downstream process.
Efforts are ongoing to identify alternatives to packed bed chromatography to reduce the time and cost of processing high-titer
product streams. Although many efforts focus on membrane adsorbers that directly replace columns of the same or similar chemistries,
some older technologies are beginning to gain ground in recombinant protein manufacturing. One attractive alternative to chromatography
is precipitation, which has been used in the plasma protein industry for many years.1 A simple method of precipitation involves titrating the process fluid to the isoelectric point of the protein that is to
be precipitated.2 Lyotropic salts, such as ammonium sulfate, also have a long history of use in precipitation processes.3
Short-chain fatty acids, such as caprylic acid, are well known for their ability to precipitate DNA and host cell proteins
(HCPs).4 Polyionic species also are useful precipitants for capturing a product of interest or removing contaminating proteins.5,6
Polyethylene glycol (PEG) has been used for product and impurity precipitation.7,8 It also can be combined with isoelectric precipitation to improve the efficiency of the separation.9,10 After precipitation, centrifugation or filtration can be used to perform solid–liquid separation.11 Although centrifugation is a well-established method to achieve this separation, washing the product pellet to remove impurities
could be problematic, and it is not suited to a single-use process. Filtration—normal- or tangential-flow—requires more development,
but washing the pellet is simpler, and it is readily adaptable to a single-use process.
In the present work, a product precipitation step was developed using PEG to recover a monoclonal antibody (MAb) from clarified
PER.C6 cell culture media. Appropriate precipitation conditions were identified through the use of full factorial experimental
designs. Two filtration steps were evaluated for the capture and washing of the precipitated product, and the superior method
was scaled-up 10-fold. The total precipitation process resulted in yields of approximately 90% and HCP reduction of 1 LRV
with no significant increase in the aggregate level of the redissolved MAb. Finally, the impact of the precipitation step
on the subsequent cation exchange (CEX) capture step was investigated.
Materials and Methods Reagents
USP grade salts, Tween 20, hydrochloric acid, acetic acid, and sodium hydroxide were purchased from JT Baker (Phillipsburg,
NJ). PEG was of reagent grade and purchased from JT Baker or EMD Chemicals (Gibbstown, NJ). All buffers were prepared using
MilliQ-grade water (Millipore, Billerica, MA) and were filtered by 0.22-Ám filtration before use.
A human MAb (IgG1, pI = 8.3, 150 kDa) was produced at Percivia, LLC using a PER.C6 cell line. PER.C6 cells are human embryonic retinal cells
immortalized by the adenovirus E1 gene, as described in US patent 5,994,128.12 The cells were cultured in a standard fed-batch process or the XD process, both using chemically defined media.13,14 The fed-batch media were clarified by sedimentation and depth filtration, and the XD media were clarified by the enhanced
cell settling (ECS) method followed by depth filtration.15 During ECS, Silica-PEI resin was used to enhance cell settling and also reduce DNA and HCP.
Precipitation Condition Optimization
The conditions used to precipitate the MAb—PEG molecular weight, PEG concentration, and pH—were optimized by full factorial
experimental designs using Minitab software (State College, PA). The pH of the clarified XD media was adjusted to the desired
level with 2-M Tris in a 15-mL conical tube. The PEG was added as a 40% (w/w) stock solution to the desired final concentration.
The tube was then centrifuged at 1,000g and the supernatant decanted. Finally, the pellet was redissolved in phosphate-buffered saline (PBS).
Pellet Capture by Depth Filtration or Microfiltration
Depth filtration was performed with various grades of filter media. Millistak+HC D0HC, C0HC, and X0HC were purchased from
Millipore Corp. (Billerica, MA), and ZetaPlus 60SP02A was purchased from Cuno (Meriden, CT). Precipitation was carried out
using a 40% (w/w) stock solution of PEG-3350 and the precipitated media was loaded at a feed flux of 50 L/m2 /h until all of the material was loaded or the transmembrane pressure (TMP) was 15 psid. The filters were then washed with
20–30 L/m2 of 20 mM Tris pH 8.5 + 14.4% (w/w) PEG-3350. After washing, 80 L/m2 of resolubilization buffer was passed through the filters at 100 L/m2 /h, and the permeate was recirculated through the device at 600 L/m2 /h until the A280 of the permeate pool was stable indicating complete MAb dissolution. Finally, any held-up product was recovered
with a 20 L/m2 buffer flush and air blowdown of the filter module. In some tests, the filter media was subsequently washed with 85-mM acetate
pH 5.3 followed by 1-M NaCl. Pressure and flow data were collected using a custom engineered system from ARC Technology Services
Microfiltration was performed with a 0.22-Ám hollow fiber membrane from GE Healthcare Life Sciences (Piscataway, NJ). The
PEG-3350 was added as a 40% (w/w) stock solution for the small-scale experiment and in powder form for the scale-up work.
The feed flux was 710 L/m2 /h and the retentate and permeate were unrestricted. The precipitate was first concentrated 10- to 14-fold and then washed
with three diafiltration volumes of 20 mM Tris pH 8.5 plus 14.4% (w/w) PEG-3350. Finally, the precipitate was redissolved
in 85 mM sodium acetate pH 5.3 or 20 mM Tris plus 50 mM NaCl pH 7.5. Pressure, flow, and conductivity data were collected
using a Slice 200 benchtop system (Sartorius, Gottingen, Germany) for small-scale testing and a SciPro system (SciLog, Middleton,
WI) for the scale-up experiment.
Cation Exchange Chromatography
Toyopearl GigaCap S-650 was procured from Tosoh Bioscience (Montgomeryville, PA) in the Toyoscreen 5-mL format. This resin
has been previously demonstrated as a high capacity capture step for MAbs.16 The column was equilibrated with 74-mM sodium acetate pH 5.3 and loaded to 90–95 mg-MAb/mL-resin using either clarified
media or clarified and PEG-treated material, each adjusted to the same pH and conductivity as the equilibration buffer. The
column was then washed with equilibration buffer and the antibody eluted with 50 mM sodium acetate pH 5.3 plus 90 mM NaCl.