Application Note. Increasing the activity of monoclonal antibody isoforms by MCSGP. Summary

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1 Application Note Increasing the activity of monoclonal antibody isoforms by MCSGP Category Matrix Method Keywords Countercurrent chromatography, FPLC Antibodies MCSGP FPLC, Biobetters, MCSGP, countercurrent chromatography, biological activity, mab purification Analytes Monoclonal Antibody isoforms, Erbitux, Avastin, Herceptin, Bevacizumab, Trastuzumab, Cetuximab ID VBS0040N_C_E Summary This application note describes how the specific activity of pharmaceutical monoclonal antibodies can be increased by separating more active isoforms from less active isoforms using Multi-column countercurrent solvent gradient purification (MCSGP). The KNAUER Contichrom system which is offered in a cooperation of KNAUER and ChromaCon is the only system on the market that can be used in MCSGP mode for difficult bio-separations. 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 chromatographic separation of different isoforms of monoclonal antibodies is very challenging since they have very similar adsorptive properties. Using preparative batch chromatography the different isoforms elute very closely and overlap at least partially leading to very low purities and/or yields for the most active isoform. This trade-off between yield and purity can be resolved by MCSGP. 2,3

2 Introduction Monoclonal antibodies (mabs) represent a large fraction of newly developed and approved drugs. The overwhelming majority of mab drugs on the market belong to the IgG subclass. The suitability of IgGs as therapeutics depends on their biological activity, pharmacokinetics and tissue targeting. While pharmacokinetics are related mainly to the molecule size, which in the case of mabs varies around 150 kda, tissue targeting and biological activity depend strongly on the molecule structure and amino acid sequence. During product development, an important analytical task is the detection of mab isoforms that exhibit low activity or even negative effects and may have to be potentially regarded as impurities. Despite significant differences in activity, current production processes do not separate mab isoforms. This can be attributed to the widespread use of Protein A chromatography that is not capable of separating mab isoforms, as Protein A binds to the Fc region of all mab isoforms. Nevertheless, the product obtained with Protein A chromatography is frequently used to set the specification for the mab isoform pattern for the final product since it matches the one generated in the fermentation process. Charged mab isoforms have rather similar adsorptive properties on cation-exchange materials but even small differences of one charge unit can be exploited to separate them using small-particle analytical ion-exchange stationary phases and gradient chromatography. In preparative scale however strong overlapping due to mass transfer limitations can be generally observed. This leads to a yield purity trade-off. The product cannot be obtained in high yield and high purity simultaneously. Using the MCSGP process this problem can be circumvented and allows to separate charged antibody isoforms with high yield and purity. In this application note the charged monoclonal antibody (mab) isoforms of the commercially available therapeutics Avastin, Herceptin and Erbitux were separated by ion-exchange gradient chromatography in batch and in MCSGP mode. The results were compared with respect to yield, purity and productivity. Figure 1 shows the analytical chromatogram of Herceptin as example. This therapeutic mab contains three main isoforms with different activities. To increase the specific activity of this therapeutic the isoform with the highest activity should be separated from the other ones. 3,4 Fig. 1 Analytical chromatogram of Herceptin Figure 1 shows an analytical chromatogram of Herceptin. It contains 3 main IgG isoforms with different activities between 10 and 140%. 3 VBS0040N_C_E Page 2 of 7

3 Experimental: Sample preparation The mab therapeutics were obtained from the pharmacy: Avastin and Herceptin from Roche (Basel, Switzerland) and Erbitux from Merck-Serono (Darmstadt). Avastin contains the IgG 1 Bevacizumab, Herceptin contains the IgG 1 Trastuzumab and Erbitux contains the IgG 1 Cetuximab. Prior to use, all mabs were diluted to a concentration of 0.4 g/l using the binding buffer. Knauer recommends to filter samples through a 0.2 µm filter before using. Preparative method for transfer to MCSGP Before starting a MCSGP process, a reference batch chromatography run has to be performed using a preparative cation exchange column. For the batch purification of Avastin Fractogel SO 3 (S) with a particle size of 30 µm (Merck) was used. The material was packed into PEEK columns (7.5 x 100 mm) according to the manufacturer s instructions. The following elution conditions were used for the batch run 4 : Batch purification of Avastin Buffer A 25 mm phosphate, ph 6.0 Buffer B 25 mm phosphate, 250 mm NaCl, ph 6.0 Gradient Time [min] % A % B Flow rate Injected mass 1.1 ml/min 2.3 mg Column temperature 25 C System pressure Detection Run time 280 nm 70 min For the batch purifications of Herceptin and Erbitux BioPro SP-10 with a particle size of 10 µm (YMC) was used. The material was packed into PEEK columns (7.5 x 150 mm with titanium frits, YMC (Kyoto, Japan) according to the manufacturer s instructions. For Herceptin the following elution conditions were used for the batch run 4 : Batch purification of Herceptin Buffer A 25 mm phosphate, ph 6.5 Buffer B 25 mm phosphate, 150 mm NaCl, ph 6.5 Gradient Time [min] % A % B Flow rate Injected mass 1.2 ml/min 1.5 mg Column temperature 25 C System pressure Detection Run time 214 nm 70 min VBS0040N_C_E Page 3 of 7

4 Batch purification of Erbitux For Erbitux the following elution conditions were used for the batch run 4 : Buffer A 25 mm phosphate, ph 6.0 Buffer B 25 mm phosphate, 250 mm NaCl, ph 6.0 Gradient Time [min] % A % B Flow rate 1.2 ml/min Injected mass 1.6 mg Column temperature 25 C System pressure Detection Run time 214 nm 70 min The concentrations of all mab isoforms in this runs were obtained by offline analysis using analytical cation-exchange liquid chromatography (CIEX-HPLC). The generic problem in the chromatographic purification of biomolecules can be simplified to the chromatogram as shown in Figure 2. The chromatogram can be divided into five fractions as indicated by the numbers on the time axis: 1= weakly adsorbing impurities 2= Product contaminated by weakly adsorbing impurities 3= Product 4= Product contaminated with strongly adsorbing impurities 5= strongly adsorbing impurities. The aim of an ideal purification process is to collect fraction 3, to drain fractions 1 and 5, and to recover the product contained in fractions 2 and 4. Fig. 2 Principle of the MCSGP Process Schematic chromatogram of the generic chromatographic purification problem: W: weak adsorbing, early eluting impurities; P: product; S: strong adsorbing, late eluting impurities. VBS0040N_C_E Page 4 of 7

5 Automatic transfer from Batch to MCSGP The definition of the fractions 1 5 and therefore the operating parameters of the MCSGP process are based on the experimental data from the offline fraction analysis of the preparative batch chromatogram. 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 operating software of the Contichrom system. For the MCSGP process the two columns are interconnected to recycle fractions 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. Figure 3 shows the analytical chromatograms of the feed mixtures (left side) compared to the analytical chromatograms of the purified products that were obtained using the MCSGP process. Spiked feeds with different isoform patterns were purified using the same MCSGP operating parameters. As shown in Figure 3, this does not lead to any differences in the MCSGP purification, which demonstrates the potential of MCSGP to straighten out the isoform patterns of upstream processes with relatively high variability. Feed (variable isoform content) Product (MCSGP purified) Fig. 3 Analytical chromatograms of Feed and product purified by MCSGP. From top to bottom: Avastin (Bevacizumab), Herceptin ((Trastuzumab) and Erbitux (Cetuximab). The left side shows the analytical chromatograms for the feed solution with variable isoform contents (dashed and solid line). The right side shows the analytical chromatograms of the product, purified using MCSGP. Figure 3 (Herceptin ) shows that the less active isoforms (see Figure 1) could be depleted by MCSGP. This results in a product with higher specific activity (activity / gram of mab product) potentially allowing dosage reduction and the development of a biobetter drug. Table 1 compares the results of the batch runs to the results of the MCSGP process. For the batch runs the described purity-yield trade-off is obvious. The results for a high purity and a high yield batch pool are shown. Using MCSGP product can be obtained with high purity and high yield at the same time. VBS0040N_C_E Page 5 of 7

6 Tbl. 1 Comparison of the results from batch and MCSGP processes. Bevacizumab Trastuzumab Cetuximab Yield Purity Yield Purity Yield Purity Feed MCSGP Batch high purity pool Batch high yield pool Conclusion The Contichrom system in MCSGP mode can be used to increase the activities of pharmaceutical antibodies by separating more active isoforms from less active isoforms. As stationary phases different cation-exchange resins were used with very poor separation under preparative conditions resulting in a yield-purity trade-off. Using MCSGP, in all cases the main mab isoform could be obtained with high yield and purity. MCSGP product quality was largely independent from the isoform pattern of the feed mixture. 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. Harris RJ, Kabakoff B., Macchi FD, Shen FJ, Kwong M., Andya JD, Shire SJ, Bjork N., Totpal K, Chen AB : J. Chromatography B, Vol. 752(2) : T. Müller-Späth, M. Krättli, L. Aumann, G. Ströhlein, M. Morbidelli : Biotechnology and Bioprocess, Vol. 107, Number 4, (2010). 4. M. Krättli, F. Steinebach, M. Morbidelli : Journal of Chromatography A, Vol. 1293, (2013). Authors Dr. Friederike Sander, Columns and Applications Department, KNAUER. Dr. Thomas Müller-Späth, Chromacon AG. VBS0040N_C_E Page 6 of 7

7 Physical properties of recommended column materials 1. Column (recommended for the purification of Avastin ) Stationary phase Fractogel EMD SO3 (S) (Merck) Particle size 30 µm Matrix Crosslinked polymethacrylate Functional groups SulfonylpH range Column (recommended for the purification of Herceptin and Erbitux ) Stationary phase BioPro SP-10 (YMC) Particle size 10 µm Matrix hydrophilic polymer bead Functional groups SulfonylpH range 2-12 Recommended instrumentation The recommended system for this separation is the KNAUER Contichrom MCSGP mode. system in 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 [email protected] Berlin, Germany Internet: Chromacon AG Tel: Technoparkstraße 1 [email protected] 8005 Zürich, Switzerland Internet: VBS0040N_C_E Page 7 of 7