Application Note. Purifying common light-chain bispecific antibodies using MCSGP. Summary

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1 Application Note Purifying common light-chain bispecific antibodies using MCSGP Category Matrix Method Keywords Analytes ID Continuous chromatography, biochromatography Antibodies MCSGP Bispecific antibody, MCSGP, continuous chromatography, biological activity, mab purification Monoclonal Antibody variants VBS0038N Summary In this application note the purification of a common light-chain bispecific antibody using Multicolumn countercurrent solvent gradient purification (MCSGP) is described. The KNAUER Contichrom system that is offered in cooperation of KNAUER and Chromacon is the only system on the market that can be used in MCSGP mode for difficult bioseparations. MCSGP is a countercurrent chromatographic process that is particularly suited for applications in the field of bioseparations 1. MCSGP is suitable for three-fraction chromatographic separations and able to perform linear gradients. It is superior to batch chromatography in terms of buffer consumption, yield, purity and productivity due to the countercurrent movement of the liquid and the solid phase. The separation of the product and product-related impurities in the downstream process is a very challenging task. In normal batch chromatography product-related impurities elute very closely to the product of interest leading to very low purities and/or yields of the product. This trade-off can be resolved by MCSGP 2.

2 Introduction Bispecific antibodies (bsabs) are recombinant proteins composed of conjugated chains of two different monoclonal antibodies (mabs). IgG type bispecific antibodies are produced in mammalian cell culture. The difficulty in producing bifunctional antibodies bsabs is that two independently expressed different monoclonal antibody mab heavy chains have to dimerize to form a bispecific antibody 3. Depending on the protein sequence, also homodimeric forms are formed as two identical mab heavy chains dimerize 2,3. In this application note, the purification of IgG type bispecific bsabs with identical light chains is described. In this antibody format the IgG light chains are identical for both antibody variable regions and two different heavy chain types (A and B) generate the different specificities 2. A, B and the light chains are expressed independently in the host cells and are assembled into three IgG types- AA, AB and BB. Without genetic engineering the assembly is random and the three types are produced close to the statistical ratio of 1:2:1 (AA, AB, BB; AB and BA are equivalent). Since the downstream process is a major cost factor in bispecific antibody production a high product yield is crucial. In the investigated case the cell culture supernatant containing the product of interest (AB), contained also high amounts of the unwanted mabs AA, BB and their isoforms. The difficulty in the chromatographic purification of AB is that the AA and BB isoforms elute closely to AB in linear gradient chromatography, leading to an overlap of the product peak AB with impurity peaks. Therefore, in batch chromatography, only a small product fraction corresponding to a low yield can be isolated with sufficient purity. In this application note a high purity and high yield purification of a bispecific Immunoglobulin G (IgG) antibody from its homodimeric forms using cation-exchange MCSGP is shown. A comparison with a reference batch chromatography showed better performance of MCSGP with respect to AB purities and yields 2. Fig. 1 Analytical chromatogram of the three mab variants Analytical chromatogram of the three antibody types AA, AB and BB. The content of the bispecific antibody AB is 36%. The separation challenge is the recovery of AB from the homodimeric antibodies AA and BB with a purity of at least 98% 2. VBS0038N Page 2 of 6

3 Experimental Sample preparation The cell culture harvest containing the common light-chain bispecific antibody mixture was supplied by Merus (PER.C6) 2. The overall antibody concentration was 1.7 g/l of AA, AB and BB together 2. The cell culture harvest was filtered using Sartoban 300 cartridges from Sartorius Stedim Biotech prior to use 2. Preparative method for transfer to MCSGP Before starting the MCSGP process, a reference batch chromatography run was performed using a preparative cation exchange column (Fig.2). Column Poros HS 50 µm, 150 x 50 mm (Life Technologies) Buffer A phosphate buffer (ph 6.0) Buffer B Gradient Flow rate Injection volume Column temperature System pressure phosphate buffer (ph 6.0), M NaCl Time [min] % A % B ml/min 12 ml ambient Detection UV at 280 nm (1 Hz, s) Run time 90 min Fig. 2 Preparative batch chromatogram Squares (blue), full diamonds (red), triangles (green) indicate AA, AB and BB concentrations, obtained by offline analytical CIEX-HPLC, whereas empty diamonds (purple) indicate the computed purity. The yellow line indicates the minimal purity of 98%. The blue transparent rectangle pointed out with an arrow represents the product collection interval selected for MCSGP using the MCSGP wizard of the ChromIQ software. VBS0038N Page 3 of 6

4 The concentrations of AA, AB and BB were obtained by offline analysis using CIEX-HPLC. The results showed that the bispecific antibody could only be found in a very small elution window with the desired purity. This leads to a very low yield of only 37% in batch chromatography. Fig. 3 Principle of the MCSGP process Schematic chromatogram of the generic chromatographic purification problem S: strong adsorbing, late eluting impurities; P: product; W: weak adsorbing, early eluting impurities. The generic problem in the chromatographic purification of biomolecules can be simplified to the chromatogram as shown in Figure 3. The chromatogram can be cut into five fractions as indicated by the numbers on the time axis: 1 = Strongly adsorbing impurities 2 = Product contaminated by strongly adsorbing impurities 3 = Product 4 = Product contaminated with weakly adsorbing impurities 5 = Weakly adsorbing impurities. The aim of an ideal purification process is to collect fraction 3, to drain fractions 1 and 5, and to recycle fractions 2 and 4. Automatic transfer from batch to MCSGP The definition of the fractions 1 to 5 and therefore the operating parameters of the MCSGP process are based on the experimental data from offline analysis of the preparative batch chromatogram (Fig. 2). For the MCSGP process the same stationary phase as for the batch experiments is used. The transfer from the results obtained in a batch process to a MCSGP method can be automatically done using the Chrom IQ software of the Contichrom system. For the MCSGP process the two columns are switched in series to recycle fraction 2 and 4 from one column to the other and they are switched to batch mode to elute the impurity fractions 1 and 5 and the pure product fraction 3. VBS0038N Page 4 of 6

5 Figure 4 shows the analytical chromatogram of the unpurified feed mixture (blue) compared to the chromatogram of the target compound, the bispecific antibody AB after the purification using the Contichrom system in MCSGP mode. Offline analysis showed that the MCSGP process led to a purity of more than 99.5% (with respect to AA and BB main isoforms) with a yield of 87%. Fig. 4 Analytical chromatograms of mab variants. Blue: feed mixture; red: mab variant F1 purified with MCSGP process. Conclusion The Contichrom system in MCSGP mode was applied for the purification of an intermediately eluting bispecific Immunoglobulin (IgG) antibody on a preparative cation exchange resin. With batch chromatography in linear gradient mode a yield of 37% was obtained. Using the MCSGP process that exploits the advantage of linear gradients and countercurrent movement of the stationary and mobile phase, the difficulty of the separation was overcome and the bispecific antibody was purified with a yield of 87%. References 1. Müller-Späth, T., Aumann, L., Melter, L., Ströhlein, G., Morbidelli, M.: Biotechnology and Bioengineering, Vol. 100, Number 6, (2008). 2. Müller-Späth, T., Ulmer, N., Aumann, L.Ströhlein, G. Bavand, M., Hendriks, L., de Kruif, J., Throsby, M., Bakker, BioProcess International, Vol. 11(5), (2013). 3. Implementation of Advanced Chromatography techniques to Mitigate Purification Concerns in Bispecific Monoclonal Antibody Manufacturing Aloke Das, Senior Research Analyst (Biologics), Beroe Inc. Authors Dr. Friederike Sander, Columns and Applications Department, KNAUER. Dr. Thomas Müller-Späth, ChromaCon AG. VBS0038N Page 5 of 6

6 Physical properties of recommended columns Stationary phase Poros HS 50 (Life Technologies) Pore size Å Particle size 50 µm Matrix Crosslinked Poly(styrene divinylbenzene) Functional groups Sulfopropyl- Pressure limit 100 bar Working range ph 1-14 Dimensions 150 mm x 50 mm ID Recommended instrumentation The MCSGP process was performed on a KNAUER Contichrom system in MCSGP mode. Description Order No. Contichrom Lab 10 System C Contichrom Prep 100 System C Contact information Wissenschaftliche Gerätebau Tel: Dr. Ing. Herbert Knauer GmbH Fax: Hegauer Weg 38 info@knauer.net Berlin, Germany Internet: ChromaCon AG Tel: Technoparkstraße 1 sales@chromacon.ch 8005 Zurich, Switzerland Internet: VBS0038N Page 6 of 6