ANALYTICAL

BIOCHEMISTRY

190,317-320

(1990)

Quantification of Monoclonal Antibodies Mixtures by Protein G High-Performance Liquid Affinity Chromatography Gregory

S. Blank’

Department

Received

and David

Vetterlein

of Recovery Process R & D, Genentech,

May

Inc., 460 Point San Bruno Boulevard,

Academic

Press,

Inc.

The ability to rapidly quantify MAbs is advantageous in areas such as hybridoma preparation, tissue culture, fermentation, and process recovery. These MAb samples are often in the form of complex mixtures such as ascites fluid, serum, cell culture fluid, or buffers and are typically quantified by using ELISA,’ RIA, or IRMA techniques or immunoelectrophoresis, all of which are time consuming. Although able to detect very small levels of IgG in test samples, these methods often require 2-5 h to complete. HPLAC offers an easy, rapid, and highly specific assay by combining the selective power of affinity recognition with the speed of HPLC (l-3). With HPLAC, analysis is simplified by the two-compo-

i To whom correspondence should he addressed. * Abbreviations used: ELISA, enzyme-linked immunospecific assay; RIA, radioimmunoassay; IRMA, immunoradiometric assay; HPLAC, high-performance liquid affinity chromatography; SDSPAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; BSA, bovine serum albumin; CCF, cell culture fluid. 0003-2697/90

Copyright All rights

0 of

$3.00 1990

South San Francisco,

California

94080

18, 1990

High-performance liquid affinity chromatography (HPLAC) utilizing Protein G as a ligand has been evaluated for rapid quantification of monoclonal antibodies (MAbs) in various solutions. The results obtained by HPLAC agreed to within 10% of a standard enzymelinked immunospecific assay (ELISA). A standard curve was prepared by injection of known amounts of a purified murine IgG, with the elution peak area analyzed by computer integration software. Accuracy of quantification was independent of the injection volume, solution composition, or mouse IgG subclass. A method is described for using Protein G HPLAC to determine murine IgG levels in various complex mixtures within 15 min, compared to the ELISA which required 5 h. 0 1990

in Complex

by Academic Press, Inc. reproduction in any form reserved.

nent nature of affinity chromatography, consisting of an unbound (flow-through) portion followed by elution of an affinity bound component. HPLAC of immunoglobulins has been described utilizing immobilized Protein A as a group-specific ligand (4-7). Protein A is a cell-surface protein from staphylococcal bacteria which binds various IgG subclasses from several species (8). However, Protein A does not bind equally well to all IgGs, and in particular binds poorly to the principal subclass of MAbs, murine IgG, (9). Previous work using Protein A HPLAC (4-7) has not demonstrated quantification of IgGs from complex mixtures or has required the addition of high salt to ensure binding of murine IgG, to Protein A. In contrast to Protein A, Protein G (from streptococcal bacteria) shows a wider range of binding to IgGs, and in particular to murine IgG,, than does Protein A (10,ll). When Ohlson et al. (12) compared the binding of various antibodies to Protein A and Protein G, they found that a murine IgG,, a rat IgG,, and a rat IgG,, bound well to Protein G but bound poorly to Protein A. Since Protein G may be a better HPLAC ligand for general MAb quantification, a sensitive and rapid procedure was developed and tested utilizing Protein G HPLAC for quantification of murine antibodies (IgG,, kG,, > and IgG,) from a variety of complex mixtures. This Protein G HPLAC technique was validated by comparison to a standard murine IgG ELISA method.

MATERIALS

AND

METHODS

The HPLAC column contained Gammabind G Protein G (Genex) attached to HiPAC silica in a 4.6 X 100 mm column from ChromatoChem (Missoula, MT). Chromatography was carried out with a Waters 600 HPLC system, a Waters 490 detector, and an AUFS setting of 1.0. Data were acquired and elution peaks analyzed using the Maxima chromatography system (Dy317

BLANK

AND

VE1’TERLEIN

g 0 1.0 2 mo 9

: 4

8

Time

4

12

(min)

FIG. 1. Protein G HPLAC of a purified IgG, used to produce the standard curve. Several of the chromatograms used to generate the standard curve are shown overlaid. The curves are from the injection of 0.1,0.75, 1.5,2.25,3.00, and 3.50 mg of MAb 354. Not all the chromatograms used to generate the standard curve are shown. The inset shows the linear relationship between peak area and amount of IgG loaded onto the column. Details of the Protein G HPLAC program and the generation of the standard curve are described under Materials and Methods.

namic Solutions; Ventura, CA) on a NEC IV personal computer. Samples were filtered (0.2 pm) prior to injection. Following injection, chromatography was carried out as follows: Buffer A (0.01 M sodium phosphate, 0.15 M NaCl, pH 7.4) for 4 min at 2 ml/min, Buffer A for 2 min at 4 ml/min, Buffer B (0.1 M glycine, 2% acetic acid, pH 3.0) for 4 min at 4 ml/min, Buffer C (20% acetic acid) for 1 min at 5 ml/min, and Buffer A for 4 min at 5 ml/min. The column was stored in Buffer A plus 0.02% sodium azide at 4°C. A standard curve was constructed by injecting 0.1 to 3.5 mg of a purified murine IgG, (MAb 354 in 0.03 M Tris, 0.15 M NaCl, pH 8.5) in a constant volume. MAb 354 used as standard was purified by Protein A and anion-exchange chromatography. The integrated area of the IgG elution peak was determined and plotted against the amount injected and the regression line determined (Cricket Graph, Cricket Software, Malvern, PA). This standard curve was used for all subsequent quantifications. MAb samples were quantified by injecting various sample volumes onto the Gammabind G column. IgG levels in the eluted peak were determined from the integrated peak area by using the standard curve described above and the IgG concentration determined by normalizing on the volume injected. The purity of the elution peaks was determined by SDS-PAGE (13). Ascites, culture fluid, and serum samples were run on Protein G HPLAC and the elution peaks dialyzed, reduced with dithiothreitol, and run on 10% SDSPAGE. The gel was Coomassie blue stained. Molecular

8

Time FIG. 2. Chromatogram ml of 20-fold concentrated tein G HPLAC column Methods.

12

(min)

of cell culture fluid containing MAb 354. 1 cell culture fluid was injected on the Proand run as described under Materials and

weight standards were from Bio-Rad Laboratories (Richmond, CA). HPLAC results were compared to those obtained with a murine IgG specific sandwich ELISA (14,15). Rabbit anti-murine IgG heavy and light chain specific antibody (Zymed) was coated onto microtiter plates overnight at 4°C with all remaining steps at room temperature. The plates were washed with PBS/ Tween, blocked for 1 h with PBS/BSA/Tween, and washed. Samples, standards, and controls were added and incubated for 2 h. After the plates were washed rabbit anti-mouse horseradish peroxidase (Cappel) was added (2 h), and the plates were washed again. o-Phenylenediamine substrate solution was added and the reac-

12

34 kDa - 97.5 - 99.2 - 42.7

FIG. 3. SDS-PAGE of the eluted IgG peak fluid, and serum. Lane 1, ascites; lane 2, culture lane 4, molecular weight markers.

from ascites, culture fluid; lane 3, serum,

CHROMATOGRAPHIC TABLE Quantification

of MAb

354”

QUANTIFICATION

1

by Protein

G HPLAC

and

ELISA

Concentration (mg/ml) Condition

Injection volume (ml)

Cone CCF High

1.0 2.0 0.5 1.0 20

salt

CCF CCF CCF a Subclass

Amount eluted (mg)

1.41 1.23 0.03 0.06 1.47

HPLAC

ELISA

1.41 0.62 0.06 0.06 0.07

1.49 0.66 0.06 0.06 0.06

IgG,.

tion stopped after 20 min by addition of H,SO,. The absorbances were read at 492 nm with a Titertek plate reader (Flow Labs). Sample concentrations were interpolated from a standard curve on the basis of MAb 354. RESULTS

AND

DISCUSSION

Protein G HPLAC was used to quantify MAbs from a variety of complex mixtures. A standard curve was constructed by loading increasing amounts of a known concentration of a purified MAb (354) on the column and determining the peak areas. The MAb 354 elution peak was quite reproducible with a peak maximum at 9.3 * 0.1 min (Fig. 1). The maximum capacity of the column was determined to be 4.6 mg of MAb 354 (data not shown) and a linear relationship (c? = 0.999) between amount loaded and peak area was obtained from 0.1 to 3.5 mg of 354 MAb (Fig. 1, inset). Using this standard curve, we quantified MAb 354 from several complex mixtures and compared the results to quantification by ELISA. For example, MAb 354 in 20-fold concentrated CCF (cell culture fluid) was quantified by injecting a known volume of harvest fluid and measuring the area of the integrated elution peak

OF MONOCLONAL

(Fig. 2). The homogeneity of the eluted peak was verified by SDS-PAGE (Fig. 3) which showed that greater than 98% of the protein in the elution peak was IgG heavy chain (50 kDa) and IgG light chain (25 kDa). Sample concentration was determined by dividing the amount of IgG eluted by the injection volume. Injection of 1.0 ml of concentrated CCF gave a peak area corresponding to 1.41 mg of MAb 354, or 1.41 mg/ml (Table 1)) while quantification by ELISA gave a concentration of 1.49 mg/ml, within 10% of the value determined by HPLAC. MAb 354 was also measured within 10% of the ELISA value in a high salt buffer (1.5 M glycine, 3.0 M KC& pH 9.0) and in unconcentrated CCF (Table 1). Thus, with a standard curve based on MAb 354, we were able to quantify MAb 354 in various complex mixtures to within 10% of ELISA values. In addition, IgG quantification by HPLAC proved independent of load volume since injections of 0.5, 1.0, and 20 ml CCF respectively gave 0.03,0.06, and 1.47 mg IgG eluted (Table 1). When normalized against the load volume, the results were in good agreement with ELISA values. Quantification by Protein G HPLAC would be simplified if a single standard curve could be used for other MAbs. We tested this by measuring several additional MAbs of three subclasses present in serum, ascites, or CCF and compared the results to values obtained by ELISA (Table 2). For all MAbs tested, the results from HPLAC and ELISA agreed within 10%. The homogeneity of the elution peak as evaluated by SDS-PAGE was greater than 98% IgG (heavy and light chain) whether the load was ascites, serum, or CCF (Fig. 3). The assay we describe was designed to have a relatively broad range with amounts as small as 30 pugand as large as 3.14 mg &g/ml to mg/ml range) accurately detected. Smaller amounts of IgG could be measured by increasing the load volume, or increasing uv detector sensitivity and establishing a new standard curve. Although the limits of this method were not determined,

TABLE Quantification

of MAbs

319

ANTIBODIES

by Protein

2 G HPLAC

and ELBA Concentration

MAb

3A8 3A8 AP2 D3 03 612 619 633 4FlO 5c1

Subclass

I&, Id& 18% kG, I&, W, I@, kG, kG,, kG,a

Condition Ascites Ascites Cone CCF Purified

CCF Serum

Amount eluted (mg)

HPLAC

ELISA

0.1

0.67 3.14 0.060 1.91 0.218 0.797 0.456 0.053 0.34 0.04 0.21

6.68 6.29 0.60 2.54 0.11 7.97 5.06 0.71 3.38 0.44 0.43

6.44 6.44 0.55 2.51 0.12 8.32 5.22 0.13 3.30 0.43 0.43

0.5 1.0

0.75 2.0 0.1

Serum

0.09

Serum

0.07 0.1

Ascites Ascites

(mg/ml)

Injection volume (ml)

0.1

0.5

320

BLANK

AND

further optimization such as microbore HPLAC should permit rapid quantification in the nanogram range. Protein G HPLAC quantification of murine MAbs proved to be about 20 times faster than the ELBA with equal accuracy and sensitivity. A standard curve based on one IgG, MAb was used to quantify several murine IgGs representing different subclasses, obviating the need to develop a new standard curve for each antibody to be quantified. Protein G HPLAC could also be useful for quantification of other antibodies that bind poorly to Protein A. ACKNOWLEDGMENTS We thank Brian Fendly for the serum and ascites derived monoclonal antibodies, and Gian Polastri and Judy Canham for the cell culture derived monoclonal antibodies. Vince Anicetti and Eric Patzer developed the murine ELISA assay.

2. Roy, S. K., Weber, togr.

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B., Brodin,

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VETTERLEIN

J. O., and Pen, L. B. (1988)

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