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152 Biotechnology and Bioengineering, Vol. 104, No. 1, September 1, 2009 2009 Wiley Periodicals, Inc.
processes are affected by diffusion limitations, particularly
with macromolecular substrates like IgG. Furthermore,
immobilized enzymes are advantageous only in applications
where the enzyme is significantly more expensive than the
substrate, for example, antibiotic intermediate production
using Penicillin G Acylase (Bruggink et al., 1998; Kallenberg
et al., 2005).
Membrane bioreactors could be broadly classified into
two types depending on the nature of enzyme retention
(Gekas, 1986; Giorno and Drioli, 2000; Heath and Belfort,
1990). In one type, the soluble enzyme is retained by an
ultrafiltration membrane while in the other type the enzyme
is immobilized on a membrane. In both types, the enzyme
is sequestered within the reactor while the reactant is
allowed to flow through for bioconversion. The first type is
suitable only for low molecular weight products. With
the second type, further purification of the fragments
from undigested IgG and byproducts is required. In our
membrane bioreactor the substrate, that is, the antibody is
reversibly immobilized on a stack of environment respon-
sive microporous membranes as in membrane chromato-
graphy followed by flow through enzymatic reaction. To the
best of our knowledge, this is the first report describing such
a reactant adsorptive membrane bioreactor separator
system, which henceforth shall be abbreviated as RAMBS.
The main advantages of membrane chromatography
over competing separations techniques result from the
predominance of convective mass transport. Hydrophobic
interaction membrane chromatography (HIMC) with
Figure 1. Schematic diagram for integrated hIgG purification from human
environment responsive membranes has been shown to serum, its enzymatic fragmentation followed by Fab purification using a RAMBS
be suitable for antibody purification (Ghosh, 2001, 2005). system.
The current work deals with an HIMC based RAMBS where
three functions are integrated into one unit operation as
shown in Figure 1: (1) human immunoglobulin G (hIgG)
purification from human serum, (2) hIgG fragmentation conjugated antibody (A8542), anti-hIgG (Fc-specific) alka-
using papain, and (3) separation of the Fab fragment from line phosphatase conjugated antibody (A9544), BCIP1/
undigested hIgG and byproducts such as the Fc. hIgG was NBT-purple liquid substrate for membranes (B3679),
first selectively adsorbed on to hydrophilized polyvinylidene TWEEN1 20 (P5927), L-cysteine (30089), disodium EDTA
fluoride (PVDF) membrane by hydrophobic interaction salt dihydrate (E4884), iodoacetamide (16125), sodium
in the presence of salt while other serum proteins phosphate (monobasic) (S0751) and sodium phosphate
(mainly human serum albumin or HSA) were obtained in dibasic (S0876), ammonium sulfate (A4418), citric acid
the purification flow through. Papain solution was then (C0759) and sodium citrate (S4641) were purchased from
introduced into the RAMBS system to digest the hIgG into Sigma-Aldrich (St. Louis, MO). High quality deionized
Fab and Fc. Fab being less hydrophobic than either Fc or water (18.2 MV cm) obtained from a Barnstead DiamondTM
intact hIgG was obtained in the digestion flow through. The NANOpure (Dubuque, IA) water purification unit was used
system could then be regenerated by eluting the undigested to prepare all the solutions and buffers. Hydrophilized
hIgG and Fc fragment using the salt-free buffer. The RAMBS PVDF membrane (0.22 mm; GVWP) was purchased
process for producing pure Fab was systematically opti- from Millipore (Billerica, MA). HybondTM-ECLTM nitro-
mized and effects of operating and solution conditions on cellulose membrane was purchased from GE Healthcare
efficiency of both separation and catalysis were examined. Bio-Sciences (Piscataway, NJ).
circulator and an AKTA Prime liquid chromatography Protein-A Based Separation of Fab and Fc
system (the last two from GE Healthcare Bio-Sciences).
A 1 mL Protein-A HP column (GE Healthcare Bio-Sciences)
The stacked membrane bioreactor consisted of a membrane
was used to separate Fab from Fc and undigested hIgG
stack provided with a heating water jacket for temperature
using 20 mM sodium phosphate (pH 7.0) as binding buffer
control. The buffers, reactant and enzyme solutions were fed
and 100 mM sodium citrate (pH 3.0) as eluting buffer,
into this membrane bioreactor through a heat exchanger
at 1 mL/min flow rate. The reaction mixture was diluted
by the AKTA system. The effluent from the membrane
1:10 in binding buffer prior to loading onto the column. The
bioreactor was continuously monitored using the UV
flow through and eluate samples were collected and analyzed
detector and conductivity and pH sensors of the AKTA
by SEC and SDSPAGE.
system and the data were logged into a computer using
Prime View software (GE Healthcare Bio-Sciences).
Digestion of Pure hIgG and Purification
of Fab Using RAMBS
Thirty PVDF membrane discs having 18 mm diameter
Liquid Phase Enzymatic Digestion of Human IgG
were stacked within the membrane bioreactor to provide
This was carried out at 37 8C for 1 h in 1.5 mL plastic tubes 0.95 mL bed volume. The eluting buffer used was 20 mM
containing 1 mg pure hIgG and 0.05 mg papain in 1 mL sodium phosphate (pH 7.5) while the binding buffers were
digestion buffer. The starting digestion buffer consisted of prepared by adding appropriate amounts of ammonium
20 mM pH 7.0 sodium phosphate buffer, 10 mM disodium sulfate to the eluting buffer. The membrane bioreactor
EDTA and 10 mM L-cysteine. The reaction was terminated was first equilibrated using binding buffer and the effluent
by adding 50 mL of 0.6 M iodoacetamide into the reaction temperature was maintained at 37 0.5 8C. hIgG solution
mixture. The effects of pH and ammonium sulfate (0.2 mg/mL) prepared in the appropriate binding buffer was
concentration on papain activity were studied. The reaction then fed into membrane reactor followed by washing
mixtures were analyzed by Size Exclusion Chromatography with binding buffer until a stable UV absorbance baseline
(SEC) for determining hIgG conversion (calculated was obtained. One milligram of hIgG was used in the
based on reduction in IgG peak area using standard IgG experiments for studying the effect of salt concentration
calibration) and by SDSPAGE for qualitative assessments. while 4 mg was used in those for studying the effect of
HSA.
Figure 10a shows the Western blot obtained by anti-
hIgG Fab immunoprobing. hIgG present in the feed (lane 1) corresponding to intact hIgG, Fc, and half Fc respectively.
and Fab present in the digestion flow through (lanes 23) Some faint bands close to the 130 kDa MW marker
could be clearly identified. The eluate sample (lane 4) (corresponding to actual MW of ca. 100 kDa due to atypical
contained undigested hIgG and some amount of Fab. The shape) were detected with both anti-hIgG Fab and anti-hIgG
results obtained with samples from the human serum Fc (lanes 4 and 8 of both blots). Papain digestion of hIgG
digestion experiment were consistent with expectations. is known to generate, albeit in small quantities, three
Figure 10b shows the blot obtained by anti-hIgG Fc molecular entities having 100 kDa molecular weight: F(c)2,
immunoprobing. All Fab containing samples were free from F(ab)c, and F(ab)2 (Michaelsen and Natvig, 1972, 1973).
Fc. The eluate samples from the pure hIgG digestion The conversion of pure hIgG in liquid phase reaction
experiment (lane 4) and the human serum digestion carried out at pH 7.5 and 37 8C for 1 h using an enzyme/
experiment (lane 8) each showed three major bands substrate mass ratio of 1:20 was 85.1%. In the corresponding
Figure 10. Western-blot analysis of sample obtained during digestion of IgG and human serum using RAMBS (a) anti-hIgG-Fab detection, (b) anti-hIgG-Fc detection
(lanes: M, marker; 1, pure hIgG; 2, flow through from hIgG digestion experiment; 3, same after 30 kDa centrifugal ultrafiltration; 4, eluate from hIgG digestion experiment; 5, human
serum; 6, flow through obtained during purification of hIgG from human serum; 7, flow through from human serum digestion experiment; 8, eluate from human serum digestion
experiment).