In most downstream purification

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1 V E N D O RVoice A Salt-Tolerant Anion-Exchange Chromatography Sorbent for Flexible Process Development Jérôme Champagne, Aleksandar Cvetkovic, Guillaume Balluet, Sylvio Bengio, Magali Toueille, and René Gantier In most downstream purification processes designed for biopharmaceutical drug production, dilution and diafiltration sequences are unavoidable. Such operations are routinely used to adjust a feedstock or chromatographic fraction to the optimal conditions required for best process performances. Nevertheless, those steps are often time, water, and labor consuming without participating directly in final product purification. Because biopharmaceutical production is increasingly driven by cost reduction, a possible means for enhancing process economics is to streamline purification by eliminating these unit operations before or between chromatography steps as much possible. Anion exchangers quaternary amine (Q) or diethyl-amino-ethyl (DEAE ) sorbents are commonly used first purification steps in biomanufacturing. However, use of conventional anion exchangers requires low to moderate ionic strength to achieve sufficient capacity (1, 2). Therefore, integrating Pr o d u c t Fo c u s : All biologicals Pr o c e s s Fo c u s : Downstream Wh o Sh o u l d Re a d: Analytical manufacturing, process development Ke y w o r d s : Purification, salt tolerance, dynamic binding capacity, CHO, human serum albumin Le v e l: Intermediate Figure 1: Dynamic binding capacity (DBC, mg/ml) against and conductivity of anionexchange sorbents: contour plots from response surface-modeling analysis; DBC at 1% BT measured in 25 mm Tris-HCl adjusted at the corresponding and conductivity using a 5 mg/ml solution prepared in equilibration buffer; (left) and Rigid Q agarose (right) chromatography sequences with anionexchanger sorbents often requires dilution or diafiltration to adapt feed conductivity to sorbent limitations (3). Implementation of a salt-tolerant anion exchanger would allow direct capture from undiluted feedstock and improve process economics signifcantly (4). The new salttolerant anion exchanger from Pall Life Sciences is designed to provide high dynamic binding capacity (DBC) at moderate to high conductivities over a wide range of values and at short residence times. The salt tolerance is based on a primary amine ligand, immobilized on a robust, industryscalable HyperCel matrix. To assess the performance of this new sorbent, we first evaluated DBC and selectivity using model proteins and compared them with those of a conventional anion exchanger. The sorbent was then challenged in three different real-case applications to address the impact of salt tolerance in full purification processes. We evaluated sorbent in bind elute mode for capture of from Chinese hamster ovary (CHO) cell culture supernatant and human serum albumin (HSA) in cryo-poor plasma. Evaluations in negative mode tested the elimination of CHO contaminant proteins to purify recombinant human interleukin 7 (rhil7). Materials and Methods Chemicals and Equipment: Sigma Aldrich provided analytical-grade reagents and. Millipore provided bovine serum albumin (). Human plasma and CHO cell culture supernatant (CCS) for application was produced within Pall 5 BioProcess International 11(6) Jun e 13

2 Figure 2: Selectivity separation of model protein mixture with linear salt gradient on two anion exchangers; columns equilibrated in 5 mm Tris-HCl 8. adjusted at 2 ms/cm (left) or 1 ms/cm (right) with NaCl; load is 1-µL protein mixture (cytochrome c 2 mg/ml, transferrin 1 mg/ml, 1 mg/ml) prepared in the corresponding equilibration buffer; wash in equilibration buffer followed by 3 CV gradient up to 5 ms/cm; HyperCel STAR AX sorbent is in blue and Rigid Q agarose in purple Cyt C Cyt C R S = 1.5 α = 1.25 Trsf 11 ms/cm Trsf 18 ms/cm R S = 1.85 α = ms/cm 37 ms/cm ms/cm Cyt C Cyt C + Trsf R S = 1.9 α = 1.58 Trsf 17 ms/cm 36 ms/cm 18 ms/cm ms/cm ml ml 1 Figure 3: DBC of on HyperCel STAR AX sorbent and Rigid Q agarose using undiluted and diluted CCS; DBC at 1% BT measured at 1 min residence time using CCS spiked at.5 mg/ml undiluted (12 ms/cm), diluted twofold (8 ms/cm) and fourfold (5 ms/cm); columns were equilibrated in 25 mm Tris-HCl 7.5 adjusted with NaCl at the corresponding conductivity. DBC (mg/ml) Rigid Q Conductivity (ms/cm) facilities. CHO CCS for the rhil7 application was kindly provided by Cytheris (Issy-les-Moulineaux, France). Pall provided S HyperCel and HyperCel STAR AX sorbents, each packed according to manufacturers instructions into glass columns from Kronlab. We used Äktaexplorer 1 and Äkta avant 25 systems from GE Healthcare for all chromatographic runs. Methods: We used several quantification assays to estimate recovery and purity of target proteins (sidebox Analytical Assays ). Recovery and purity of and HSA were calculated as shown in the Equations box. Runs used 1 ml columns (.5 5. cm) at 2 min residence time except where otherwise stated (Table 1). Equations: Calculation of recovery and purity for and HSA recovery (% of load) = purity (%) = Albumin recovery (% of load) = To optimize conditions, we used highthroughput screening on 96-well microplates as described by Toueille et al. using the range of conditions listed in Table 2 (5). We planned and analyzed a design of experiments (DoE) study using Minitab statistical software (Minitab Inc.). For our process economics analysis, we used BioSolve cost of goods (CoG) analysis software (Biopharm Services) and performed real-case applications developments as listed in Table 3. Results and Discussion Salt Tolerance (Influence of and Conductivity on DBC): We evaluated the effects of a wide range of (7. 8.5) and conductivity (3 ms/cm) on the DBC for of sorbent and compared results with those of a standard Q anion-exchanger. Figure 2 shows that the contour plots evidenced different profiles directly linked to the chemistry used to functionalize each sorbent. Standard Q anion exchangers provide in elution (mg) 1 in load (mg) (mg) 1 CHOP (mg) + (mg) Albumin in elution (mg) 1 Albumin in load (mg) Albumin (mg) 1 Albumin purity (%) = Ig (mg) + Trf (mg) + Albumin (mg) 1 An a ly t ic a l Ass ay s Us e d in Real Case Studies Total Proteins Bradford assay kit and bicinchoninic acid (BCA) assay kit (Pierce Thermo Scientific) Human Serum Albumin Bromocresol green colorimetric assay (Fisher Diagnostics) Transferrin, CHO Host Cell Protein (CHOP) ELISA assay kit (Cygnus Technologies) Immunoglobulins Protein A HPLC column (Applied Technologies) Ceralpha colorimetric assay (Megazyme) Recombinant human interleukin 7 (rhil7) ELISA assay kit (Cell Sciences) SDS-PAGE NuPAGE 4 12% Bis-Tris gels and Coomassie SimplyBlue (Invitrogen) 52 BioProcess International 11(6) June 13

3 Figure 4: Optimization of elution conductivity on Rigid Q agarose (left) and (right) sorbents with twofold diluted CCS as loading feed; after equilibration in 25 mm Tris-HCl 7.5, 8 ms/cm a volume of twofold diluted cell culture supernatant (CCS) equivalent to 6% of the DBC 1%BT was loaded at 1 min residence time. After a wash in equilibration buffer, a 3 CV NaCl gradient up to 1 M NaCl was applied followed by a strip in the same buffer containing 2 M NaCl. 2,8 2,4 2, 1,6 1, ms/cm Load CCS 7.5, 8 ms/cm, 26 mg /ml sorbent Eq. Flow-through NaCl gradient 1M OD28 nm Conductivity Strip 2M NaCl Nucleic acids 5 ms/cm OD26nm 2,8 2,4 2, 1,6 1, ml Strip 2M NaCl Load CCS 7.5, 8 ms/cm, 24 mg /ml sorbent Eq. Flow-through NaCl gradient 1M 38 ms/cm Conductivity OD28 nm OD26nm ml Nucleic acids >8 ms/cm Figure 5: Dynamic binding capacity of and Rigid DEAE agarose sorbents for human serum albumin (HSA); sorbents were equilibrated in 5 mm Tris-HCl 7.6 adjusted with NaCl at the corresponding conductivity and loaded with cryo-poor plasma undiluted (11 ms/cm), 1.6- fold diluted (7 ms/cm) and threefold diluted (4 ms/cm). Albumin was quantified in flow through fractions to determine 1% breakthrough. DBC for HSA (mg/ml) HyperCel STAR AX 4 ms/cm 7 ms/cm 11 ms/cm Rigid DEAE high DBC at low to moderate conductivity (6). Logically, rigid Q agarose sorbent reaches high capacity (>1 mg/ ml) at moderate conductivity but shows no salt tolerance as DBC drops when conductivity increases (4 6 mg/ml at 15 ms/cm). For the sorbent, the primary amine chemistry ligand we used allows both high DBC and high conductivity. As with other modern anion exchangers, the material delivers very high DBC ( 18 mg/ml) at low conductivity ( 5 ms/cm). It keeps capacity >1 mg/ml at conductivities 15 ms/cm. Even under the least favorable conditions for capture, DBC is 6 mg/ml, and performance of the Table 1: Chromatographic runs parameters Run Sample Load Wash Elution Strip CIP DBC 1%BT Pure protein evaluation buffer (1 CV) solution or feed stock Gradient elution Step elution Figure 6: Contour plots from response surface modeling analysis; optimization of wash and elution conditions for human serum albumin purification on sorbent; effect of wash conditions on purity, elution at 4, conductivity 2 ms/cm (left); effect of elution conditions on yield, wash 7.5, conductivity ms/cm (right) Wash Conductivity buffer (1 CV) buffer (1 CV) Pure proteins mixture; feedstock at 6% of DBC Feedstock at 6% of DBC Purity (%) >98 Optimal wash conditions Elution Conductivity Wash Elution sorbent is maintained at residence times as low as 1 min (not shown). Therefore, it displays capacity equivalent to advanced anion exchangers but significantly extends the conductivity operation range. So buffer (1 CV) buffer (1 CV) or alternative buffer specified % buffer B gradient (3 CV); buffer B: equilibration buffer + 1 M NaCl Salt elution: equilibration buffer + NaCl at specified conductivity (1 CV); elution: buffer specified (1 CV) Yield (%) < >95 Optimal elution conditions High salt buffer (5 CV) 5 mm Tris-HCl M NaCl (5 CV) 1 M NaOH (5 CV) 1 M NaOH (5 CV) integration of sorbent in purification processes offers the possibility of directly capturing protein from undiluted feedstock, thereby eliminating the need for dilution or diafiltration. Jun e 13 11(6) BioProcess International 53

4 Selectivity (Separation of a Model Protein Mixture with a Linear Salt Gradient): Selectivity is an important parameter in the choice of an ion exchanger for a specific process and should be evaluated case by case. For example, we compared selectivity with that of a rigid Q agarose sorbent by using a mixture of three model proteins. The proteins separated through a linear salt gradient after loading at conductivities of 2 or 1 ms/cm. Figure 3 shows that cytochrome c was not retained on the anion-exchange sorbents because it is positively charged at 8.. The other two proteins were bound and separated from sorbent with good resolution (Rs) and selectivity (α) performance maintained at different conductivities. Rigid Q agarose sorbent showed a similar separation pattern at low load conductivity, although resolution was lower. That pattern was not maintained when load conductivity increased. For this Figure 7: Human serum albumin (HSA) capture on sorbent run in optimal conditions as described in Table 5; sorbent was equilibrated in 5 mm Tris-HCl 7.6 adjusted with NaCl at 11 ms/cm. Nondiluted plasma corresponding to 6% of the DBC 1%BT for HSA was loaded. Wash 1 in equilibration buffer and wash 2 in equilibration buffer adjusted to ms/cm by adding NaCl. Elution buffer was 5 mm Na acetate; chromatogram (left) and SDS-PAGE gel (right) (FT is flowthrough; W1 is Wash 1; W2 is Wash 2; E is elution, and P is plasma load) 2,5 2, 1,5 1, 5 Elution ( 4., 2 ms/cm) E FT from neat Wash 2 plasma load Wash 1 ( 7.5, ms/cm) Albumin FT W1 W2 1 ml IgG Transferrin Albumin Figure 8: Purification processes compared using process economics analysis Cryo-Poor Plasma 7.6, conducivity 11 ms/cm FT W1 W2 E P case, transferrin did not bind, thus leading to a modification of the separation capabilities on rigid Q agarose depending on loading conductivity. Note that elution of transferrin and from HyperCel STAR AX sorbent needs higher salt concentration because of its salt tolerant properties. However, all bound proteins are completely eluted in the 1 M NaCl gradient as with other standard ion exchangers (not shown). Selectivity data at different conductivities highlight the robustness of the separation performance of HyperCel STAR AX sorbent independently of conductivity. That provides flexibility in process design and the possibility of Figure 9: Cost comparison between and rigid DEAE agarose processes; cost analysis was performed considering a concentration of 5 g/l HSA in plasma and 2,-L batches, with an average number of batches per year (4, L of plasma per year). The HSA recovery for dilution steps was considered to be 1%. The flow rate, column bed height, and number of cycles were kept constant at cm/h, cm, and cycles, respectively. Cost Difference, Salt tolerant vs Standard AEX ( %) Cost of Goods ($US/g) Capital Materials Consumables Water Use (m 3 /batch) 89% yield, DBC 3 mg/ml Elution in 5mM Na acetate, 4 Cation Exchange (CEX) 95% yield, DBC 6 mg/ml Elution in 5 mm Na phosphate,.15 M NaCl, 7 Rigid DEAE 73% yield, DBC 11 mg/ml Elution in 5mM Na acetate, 4 Cation Exchange (CEX) 95% yield, DBC 6 mg/ml Elution in 5 mm Na phosphate,.15 M NaCl, 7 Table 2: Test conditions for high-throughput screening optimization of wash and elution conditions of human serum albumin capture step on AEX sorbents Sorbent HyperCel STAR AX Rigid DEAE Load Wash Wash Conductivity (ms/cm) Elution Elution conductivity (ms/cm) Cryo-poor plasma Threefold diluted cryo-poor plasma Figure 1: Dynamic binding capacity (DBC) for contaminant proteins and cell culture supernatant (CCS) dilution on HyperCel STAR AX and Rigid Q agarose sorbents; DBC at 1% BT was measured at 1 min residence time after equilibration in 25 mm Tris-HCl 8. adjusted with NaCl at the corresponding conductivity using CHO CCS (rhil7:.2 mg/ml, total proteins:.4 mg/ml) undiluted (16 ms/cm) and twofold diluted (8 ms/cm). Breakthrough of bound proteins was measured in flow-through fractions with total protein assay. Protein Contaminants DBC (mg/ml) Crude CCS (16 ms/cm) Twofold Diluted CCS (8 ms/cm) HyperCel STAR AX Crude CCS (16 ms/cm) Twofold Diluted CCS (8 ms/cm) Rigid Q 54 BioProcess International 11(6) Jun e 13

5 accommodating feed variations without modifying a separation profile. Capture of from CHO CCS: We evaluated the performance of HyperCel STAR AX sorbent for protein capture from a CHO CCS using as a model. To address the effect of feedstock dilution, we evaluated DBC for sorbent and rigid Q agarose with CCS containing.5 mg/ml and adjusted by dilution at different conductivities. sorbent provided the highest capacity at mg/ml with crude feedstock (Figure 3), thereby confirming its salt tolerance. The highest capacity for both sorbents was obtained with fourfold dilution (5 ms/cm), but this dilution would be too high for favorable process scale-up economics. Therefore, capture using only crude and twofold diluted feedstock was further investigated. Figure 4 shows results of standard elution optimization using a NaCl gradient up to 1 M. We observed two peaks for both sorbents during salt elution. The activity was detected only in the first peak, whereas the second probably corresponds to nucleic acids (high ratio of A 26 nm to A 28 nm ). For sorbent, however, the second peak was well separated from the peak because it eluted only during the high-salt strip. Therefore, thanks to its strong affinity for nucleic acids, sorbent provides efficient separation of such contaminants. On the basis of the elution profiles, we chose elution conductivities for purification runs as 48 ms/cm on HyperCel STAR AX sorbent and 34 ms/cm on rigid Q agarose sorbent. We then screened different wash conditions to improve purity using the above elution conditions for both sorbents (not shown). Data indicated that a wash step combining low and low conductivity ( 4.5, 2 ms/ cm) improved CHOP removal for both sorbents while maintaining activity and recovery yield (Table 4). Overall, our study demonstrated that the salt-tolerant anion-exchange sorbent can efficiently capture and purify biologically active enzymes from both crude and diluted CHO feedstock with equivalent productivity. Thus it can bring process flexibility and robustness to purification processes (Table Table 3: Real-case applications development plan (CCS is cell culture supernatant) Capture of Capture of human serum albumin (HSA) Flowthrough mode purification of rhil7 Feedstock CHO CCS,.5 mg/ml Cryo-poor human plasma, HSA 42 mg/ ml CHO CCS, rhil7.2 mg/ml Figure 11: Purification sequences compared for process economics analysis; dynamic binding capacity (DBC) specified for CHO contaminants proteins (CCS is cell culture supernatant) >9% yield in FT DBC 8.5 mg/ml Scenario 1 Scenario 2 rhil7 CHO CCS 8., 16 ms/cm Rigid Q >9% yield in FT DBC 3. mg/ml 5). By contrast, using undiluted feedstock on conventional rigid agarose can decrease productivity by about four times (not shown). Process Economics for Bind Elute Mode: To address the effects of salt tolerance of sorbent on process economics, we developed a twostep process for HSA purification from plasma using anion exchange as capture. Performance of this sorbent was evaluated in parallel with that of a DEAE agarose sorbent, a commonly used type of sorbent in plasma fractionation. We determined the effect of feed conductivity on sorbent capacity by evaluating DBC of both sorbents using plasma at different dilutions (Figure 5). DBC of sorbent was maintained around 3 mg/ml with load at conductivities ranging from 4 to Steps 1. Evaluation of dynamic binding capacity (DBC) versus CCS dilution 2. Optimization of elution conductivity on column 3. Wash conditions optimization on column 4. Evaluation of overall performance in optimized conditions 1. Evaluation of DBC versus plasma dilution 2. Optimization of purification parameters for capture step on AEX by design of experiments in AcroPrep 96-well filter plate/data analysis by response surface modeling and contour plot 3. Transfer of optimal conditions to column 4. Optimization of second step on cation-exchanger following same approach (step 1 3) 5. Analysis of process economics of complete two-step processes 1. Evaluation of DBC for contaminant proteins versus CCS dilution 2. Process economics analysis of the contaminant removal step >9% yield in FT DBC 8.5 mg/ml rhil7 CHO CCS 8., 16 ms/cm Twofold dilution in DI Water Rigid Q >9% yield in FT DBC 6. mg/ml 11 ms/cm (dilution one- to threefold). By contrast, an increase of load conductivity significantly decreased DBC of the rigid DEAE agarose sorbent. Those data confirm the salt-tolerant behavior of sorbent and its ability to directly capture HSA with high capacity from nondiluted plasma. Compared with the standard sorbent, the DBC of sorbent was more than twofold higher and maintained in a broad range of conductivities. That brings greater process robustness and flexibility, by accommodating potential variations in the ionic strengths of feedstocks. We optimized wash and elution conditions using DoE on 96-well filter plates (Table 2). We determined optimal wash and elution conditions for purity and yield by using response surface modeling Jun e 13 11(6) BioProcess International 55

6 Table 4: Optimization of wash for the purification of on AEX sorbents Rigid Q agarose Feed Wash Yield Purity Yield Purity Loaded Conditions (mg/ml sorbent) (%) (%) (mg/ml sorbent) (%) (%) Comments Twofold Same as For both sorbents: low wash improves CHOP diluted CCS equilibration 4.5, 2 ms/cm elimination and maintains high yield; similar purity, slightly higher yield for Crude CCS Same as equilibration 4.5, 2 ms/cm : Improvement of purity with low wash Rigid Q agarose: Good purity but low yield; very low DBC with crude CCS does not allow consideration of a productive scalable process in these conditions * Initial purity of in CCS was 62%. Columns were loaded after equilibration in 25 mm Tris-HCl 7.5 adjusted to the corresponding load conductivities at 6% of DBC determined in respective conditions (see legend of Figure 4). Wash uses the same buffer or 5 mm Na acetate 4.5, 2mS/cm. Elution uses equilibration buffer adjusted with NaCl to 34 ms/cm for runs on rigid Q agarose sorbent and to 48 ms/cm for runs on sorbent. Table 5: Performance of sorbent for capture of under optimized conditions (CCS is cell culture supernatant) Feedstock DBC 1%BT (mg/ml) Yield Purity Productivity* (g/l/h) Crude CCS (12 ms/cm) 21 96% 94% 7.9 Twofold diluted CCS (8 ms/cm) 4 94% 93% 8.4 * Values obtained with loads of samples at 6% DBC in runs performed as provided in Table 4, applying a wash at 4.5 and 2 ms/cm Table 6: Optimal conditions for human serum albumin (HSA) capture step on anion-exchange sorbents by high-throughput screening optimization; results obtained during column runs Sorbent HyperCel STAR AX Rigid DEAE Load Wash Conditions Elution Conditions HSA Yield* (%) HSA Purity* (%) Cryo-poor plasma 7.5, 4, 2 ms/cm 9 99 ms/cm Threefold diluted cryo-poor plasma 7.5, 9 ms/cm analysis. Contour plots of HyperCel STAR AX sorbent data revealed that HSA purity was significantly affected by wash conditions (Figure 6 left). Increase in wash conductivity positively affected HSA purity, with an optimal zone for wash conductivity >15 ms/cm. The optimal elution conditions zone was at conductivities of 2 27 ms/cm (Figure 6 r i g h t). We chose optimal combinations of wash and elution conditions providing estimated yield and purity 9% according to model predictions (Table 6). We applied optimized conditions to column runs yielding highly pure HSA and satisfying yield for both sorbents (Table 6). The possibility of eluting from sorbent using only a drop at low conductivity (Figure 7) provides the opportunity to directly load elution from this sorbent on a cation exchange sorbent (orthogonal step). However, the same elution conditions tested on rigid DEAE agarose resulted in good purity but only partial elution (73% yield) (not shown). 4, 25 ms/cm 9 98 * Data obtained during runs performed on column with a load at 6% of DBC as shown in Figure 7. We used HSA purified on HyperCel STAR AX and rigid DEAE agarose sorbents as loading feed to optimize the second purification step of HSA on S HyperCel cation exchanger. Optimization on 96-well plates (data not shown) allowed determination of the best elution conditions at 7., 15 ms/cm, providing HSA with 95% recovery and >99% purity. With process economic analysis, we compared the two two-step purification processes for HSA using either HyperCel STAR AX or rigid DEAE agarose sorbents as a first step and S HyperCel as a second step (Figure 8). The -driven elution from the capture column provided the most streamlined purification scheme, which appeared promising for plasma fractionation. We compared the salttolerant and the standard anionexchanger sorbents using those conditions. A cost comparison showed that a process using rigid DEAE agarose as its capture step can increase costs by 67% compared with processes using HyperCel Figure 12: Cost comparison between and rigid Q agarose; cost analysis performed considering a concentration of.2g/l rhil7 in cell culture supernatant (CCS) and 5-L batches, with average of 1 batches per year; ril7 recovery for dilution steps was considered to be 1%. Scenario 1 is crude CCS (16 ms/cm); Scenario 2 is twofold-diluted CCS (8 ms/cm) Cost Difference, Salt tolerant vs Standard AEX ( %) Cost of Goods ($US) Capital Materials Scenario 1 Scenario 2 Consumables Water Use (m 3 /batch) STAR AX sorbent (Figure 9). The lower DBC obtained at capture with undiluted plasma necessitates more runs to process the same amount of HSA. That results in a higher buffer consumption, water use, and material/tankage costs. In addition, lower throughput in kg/year ( 22%) was achieved as a result of the poorer elution yield from rigid DEAE agarose with low salt buffer. We also simulated an alternative scenario for a process using optimal loading and elution conditions for capture on rigid DEAE agarose. This case integrated a threefold plasma dilution to maximize HSA capture and applied optimal elution conditions of 4. and 25 ms/cm to increase yield. Despite better performances for the conventional anion-exchanger, the global cost of this process was still 14% higher than for a process using sorbent. That is a result of preliminary plasma dilution, followed by a second dilution of 56 BioProcess International 11(6) Jun e 13

7 the first-step elution required for the second purification step on the cationexchanger (data not shown). This second case study highlights again the performances of sorbent for purification of proteins in high-conductivity environments and illustrates the positive effects of salt tolerance on process economics. Process Economics for Flow- Through Mode: Our study included early contaminant removal for the purification of a recombinant human Interleukin 7 expressed in CHO cells. Anion-exchange sorbents are often operated in negative (or flow-through) mode to capture contaminants, which leaves a target protein in the flow through. We evaluated performance of sorbent for capture of contaminants from a CHO CCS during a recombinant human Interleukin 7 (rhil7) purification process and performed a process economics analysis. We determined the DBC for contaminant proteins on HyperCel STAR AX sorbent compared with that of rigid Q agarose sorbent at different conductivities using crude and twofolddiluted CCS. As Figure 1 shows, the highest DBC was obtained with crude CCS for sorbent and with twofold diluted CCS for rigid Q agarose. As expected, the standard anion exchanger requires lower conductivity to capture the largest number of contaminants. For sorbent, the salt tolerance allows efficient binding of contaminants at high conductivity. We obtained high recovery yields in the flowthrough for rhil-7 (>9%) in all conditions tested for both sorbents. Results showed that lower conductivity decreases contaminant DBC for the sorbent. The lower protein concentration in twofolddiluted CCS could negatively affect the adsorption equilibrium of contaminants, thus decreasing total protein capacity. Contaminant DBC of the HyperCel STAR AX sorbent is equivalent to that of rigid Q agarose using twofold-diluted CCS. We used two different processes for process economics analysis that included either crude CCS (16 ms/cm) or twofolddiluted CCS (8 ms/cm) (Figure 11). When compared with rigid Q agarose, HyperCel STAR AX sorbent always provided significant cost reduction and water savings, regardless of the chosen scenario (Figure 12). The largest decrease of CoG (23%) was shown through comparing the use of both sorbents loading crude CCS (scenario 1). CoG reduction came mostly from potential savings on capital equipment (25%), because a lower capacity of contaminants on rigid Q agarose would require larger column diameter, larger pumping system, and larger vessels for buffer preparation. The savings from using sorbent were still significant even when loading twofold-diluted CCS (second scenario). We scaled up using a 1 ml (2.5 cm 22 cm) column of sorbent and positioning it at the first step of a complete purification process for rhil7 from crude CCS. The trial confirmed the efficiency of CHO contaminant protein removal (not shown). Our studies illustrate that HyperCel STAR AX sorbent provides strong economical benefits compared with a standard ion-exchange sorbent when used in negative mode. Next-Generation Tool The new salt-tolerant anion-exchange sorbent facilitates the protein capture (target or contaminants) from moderate- to high-conductivity feedstocks, limiting dilution or ultrafiltration/diafiltration (UF/DF) requirements. We demonstrated the sorbent s ability to maintain robust DBC over a large range of conductivity and values using model proteins as well as different types of real feedstocks (plasma, CHO CCS) with different target proteins. The new sorbent provided a significant advantage for streamlined process development compared with conventional anion-exchange sorbents. It allows for elimination of initial dilution or diafiltration steps usually required to accommodate feedstock for operation on standard anion exchangers. That is possible while keeping purification and recovery performance as well as optimization of operational conditions in line with those of the existing range of conventional anion exchangers. In realcase applications, salt tolerance led to significant savings compared with conventional sorbents (decreased CoG for operation of the sorbent in bind elute mode and in flow-through mode). The sorbent appears to be a next-generation chromatography tool for capture steps as well as contaminant removal in various purification sequences. Altogether, the data herein illustrate its functional and economical performance and demonstrate its benefits toward allowing development of more streamlined and cost-effective processes. Re f e r e n c e s 1 Bengio S, et al. Improving IEX Throughput and Performance with Differentiated Chromatography Sorbents. BioProcess Int. 8(5) 1: Harinarayan C, et al. An Exclusive Mechanism in Ion-Exchange Chromatography. Biotechnol. Bioeng. 95(5) 6: Arunakumari A, Wang J. Purification of Human Monoclonal Antibodies: Nonprotein A Strategies in Process Scale Purification of Antibodies, Process Scale Purification of Antibodies, Gottschalk U. Wiley Online, 9. 4 Han C, et al. Process Development s Impact on Cost of Goods Manufactured (COGM). BioProcess Int. 8(3) 1: Toueille M, et al. Designing New Monoclonal Antibody Purification Processes Using Mixed-Mode Chromatography Sorbents. J. Chromatogr. B 879(13 14) 11: Staby A, Jensen I, Mollerup I. Comparison of Chromatographic Ion-Exchange Resins: I. Strong Anion-Exchange Resins. J. Chromatogr. A 897(1 2) : Jerome Champagne, PhD, is senior R&D scientist, chromatography applications at Pall Life Sciences (France). Aleksandar Cvetkovic, PhD, is principal R&D engineer, chromatography applications at Pall Life Sciences (USA). Guillaume Balluet is associate R&D scientist, chromatography applications; Sylvio Bengio, PhD, is scientific communications manager, global chromatography; and Magali Toueille, PhD, is principal R&D scientist, chromatography applications, all at Pall Life Sciences (France). Corresponding author, René Gantier, PhD, is senior R&D manager, Biopharm Applications at Pall Life Sciences, 5 Bearfoot Road, Northborough, MA, 1532; ; rene_gantier@pall.com. The following are registered trademarks: HyperCel (Pall Corporation); Minitab (Minitab Inc.), ÄKTAexplorer and ÄKTA avant (GE Healthcare), and NuPage (Invitrogen). Jun e 13 11(6) BioProcess International 57