Cation exchange frontal chromatography for the removal of monoclonal antibody aggregates

A low ligand density cation exchange (CEX) chromatography resin, Eshmuno® CP-FT resin, was investigated for the removal of aggregates from monoclonal antibody (mAb) feeds using a continuous loading process. Removing mAb aggregates with a CEX resin using continuous loading is advantageous relative to a bind/elute loading process.

Journal of Chromatography A, 1599 (2019) 152–160 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Cation exchange frontal chromatography for the removal of monoclonal antibody aggregates Matthew T Stone ∗ , Kristen A Cotoni, Jayson L Stoner EMD Millipore Corporation, United States a r t i c l e i n f o Article history: Received 26 November 2018 Received in revised form April 2019 Accepted April 2019 Available online April 2019 Keywords: Cation exchange chromatography Frontal chromatography Continuous loading chromatography Overloaded chromatography Monoclonal antibody aggregates a b s t r a c t ® A low ligand density cation exchange (CEX) chromatography resin, Eshmuno CP-FT resin, was investigated for the removal of aggregates from monoclonal antibody (mAb) feeds using a continuous loading process Removing mAb aggregates with a CEX resin using continuous loading is advantageous relative to a bind/elute loading process, because the resin can use nearly its full capacity to bind the aggregates ® enabling much higher loadings The removal of mAb aggregates with Eshmuno CP-FT resin using a continuous loading process was found to be consistent with a frontal chromatography mechanism where the mAb monomer initially binds to the column and is subsequently displaced by dimers and higher molec® ular weight aggregates The removal of mAb aggregates with Eshmuno CP-FT resin using a continuous loading process was compared with six other commercially available strong CEX chromatography resins and found to correlate with their ionic densities, but not their mAb static binding capacities The influence ® of pH, conductivity, residence time, and mAb concentration on the removal of aggregates with Eshmuno CP-FT resin using a continuous loading process was also investigated Finally, the percentage of aggre® gates in a mAb feed was varied to examine the effect on the removal of aggregates with Eshmuno CP-FT resin using a continuous loading process © 2019 The Authors Published by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Introduction It is critical to remove aggregates during the downstream purification of monoclonal antibodies (mAbs) as they can increase the risk of an immunogenic response in patients [1–4] Unlike other impurities, such as host cell proteins and DNA, mAb aggregates contain an Fc binding domain and are not typically separated from the monomeric product during a protein A chromatography capture step [5,6] The chromatographic separation of mAb aggregates from the monomer is particularly difficult, as they have nearly identical isoelectric points and hydrophobicities Aggregates are most commonly removed with bind/elute chromatography processes using ion exchange, mixed-mode, hydrophobic interaction, or hydroxyapatite media [5,7,8] However, there is interest in the development of CEX chromatography processes that use continuous loading for the removal of mAb aggregates rather than bind/elute loading [9–12] CEX chromatography using continuous loading allows the resin to be loaded with the mAb feed until it is completely occupied by the aggregates, which is significantly higher than bind/elute ∗ Corresponding author E-mail address: matthew.stone@emdmillipore.com (M.T Stone) processes where the resin must bind both the monomer and aggregates For instance, if a mAb feed containing 10% aggregates can be loaded up to 50 g/L with a bind/elute loading process using a CEX resin, then it has the potential to be loaded up to 500 g/L by a continuous loading process assuming the CEX resin has a similar capacity for both the mAb monomer and aggregates Higher loadings of the CEX resin are advantageous because they require significantly smaller volumes of both resin and buffer shrinking the footprint of the mAb downstream purification process Continuous loading processes using CEX media to purify mAb feeds have been previously reported as overloaded chromatography [9,10,13] We suggest the more precisely defined term frontal chromatography as has been described by Rachinskii [14], Jonsson [15], Hill et al [16], and Ahuja [17] to describe the mechanism of separation observed in these processes Frontal chromatography is characterized by the continuous loading of the column under conditions where all the components of a mixture will bind with the resin [17] This mechanism separates the components of a mixture into fronts based on their relative strength of interaction with the resin [14,15] The weakest interacting component will elute from the column first in a pure form The next front eluted from the column will be composed of the weakest interacting component plus the next strongest interacting compo- https://doi.org/10.1016/j.chroma.2019.04.020 0021-9673/© 2019 The Authors Published by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4 0/) M.T Stone et al / J Chromatogr A 1599 (2019) 152–160 nent Successive fronts will exit the column as mixtures including all components of the previously eluted fronts This process will continue until the column has reached its full capacity for the most strongly interacting component Frontal chromatography is not commonly used for preparative scale purification of proteins, however Hill et al demonstrated that it could be applied for the preparative purification of the weakest interacting component within a mixture [16] The removal of mAb aggregates with a CEX resin is an opportunity for the preparative scale application of frontal chromatography because the mAb monomer interacts less strongly with the CEX resin relative to the aggregates Thus, the monomer will elute from the CEX column first as a single component and the column can continue to be loaded with the mAb feed until it is completely occupied by aggregates In practice, some monomer is likely to still be retained by the column after the aggregates begin to elute However, a good monomer recovery can still be achieved by this method, because at higher loadings the amount of monomer retained is only a small percentage of the total monomer processed Herein, we report our investigation into a low ionic density CEX ® resin, Eshmuno CP-FT resin, for the removal of mAb aggregates using a continuous loading process To understand if the removal ® of aggregates with Eshmuno CP-FT resin using continuous loading was consistent with a frontal chromatography mechanism we measured the composition of both the eluted and retained components as the loading of the resin was varied The removal of ® aggregates from a mAb feed with Eshmuno CP-FT resin using a continuous loading process was then compared with six other commercially available strong CEX chromatography resins and the percentage of aggregates in the elution pool were compared to the ionic density and mAb static binding capacity of the resins In addition, the influence of several different process conditions including pH, conductivity, residence time, and mAb feed concentration were examined to understand how each of these factors influenced the removal of aggregates with Eshmuno® CP-FT resin using a continuous loading process Finally, we examined how the removal of mAb ® aggregates with Eshmuno CP-FT resin using a continuous loading process was influenced by the percentage of aggregates in the mAb feed Experimental 2.1 Materials 2.1.1 Enrichment of mAb05 and mAb02 feeds with aggregates by high pH hold A high pH hold process was used to induce aggregates for both the mAb05 and mAb02 feeds The process was modified from the procedure reported by Potty and Xenopoulos [18] First, internally generated mAb-containing Chinese hamster ovary cell cultures were clarified and subjected to protein A capture chromatography The resulting protein A elution pool was then adjusted to a neutral pH The mAb concentration of the elution pool ranged from 10 g/L to 20 g/L The neutralized mAb solutions were gently stirred and M sodium hydroxide was added dropwise until the solution pH reached 11.0 Care was taken to avoid increasing the solution pH above 11.0, which could cause significant degradation of the mAb protein The mAb solution was held at pH 11.0 for 30 and then 1.0 M hydrochloric acid was added dropwise until the solution was reduced to pH 5.0 The percentage of aggregates in the resulting solution was determined by analytical size-exclusion chromatography The pH cycling procedure was repeated up to four times until the desired percentage of aggregates was obtained The resulting mAb solution was then dialyzed into the desired buffer This process was found to generate variable percentages of aggregates The per- 153 centage of aggregates in an overly enriched mAb feed was lowered to the desired level by combining with an untreated mAb feed 2.1.2 Resins The following CEX chromatography resins were used in this ® investigation including Eshmuno CP-FT resin (500 mL, catalog number: 1200930500, EMD Millipore Corporation, Burlington, ® MA, 01803, USA), Eshmuno CPX resin (500 mL, catalog number: 1200830500, EMD Millipore Corporation, Burlington, MA, 01803, ® USA), Poros XS Strong Cation Exchange Resin (50 mL, catalog number: 4404338, Thermo Fisher Scientific Inc., Waltham, MA, 02451, ® USA), Poros 50 HS Strong Cation Exchange Resin (50 mL, catalog number: 1335906, Thermo Fisher Scientific Inc., Waltham, MA, ® 02451, USA), Toyopearl Gigacap S-650 M (100 mL, part number: 0021833, Tosoh Corporation, Minato-Ku, Tokyo, 105–8623, Japan), ® Capto S ImpAct (100 mL, product number: 17371702, GE Healthcare Bio-Sciences AB, Uppsala, Sweden), and SP SepharoseTM Fast Flow (300 mL, product number: 17072901, GE, Healthcare BioSciences AB, Uppsala, Sweden) 2.2 Methods 2.2.1 Standard procedure for removal of aggregates using frontal chromatography For all experiments, a glass chromatography column (Omnifit Benchmark Column 6.6 mm/100 mm, 6.6 mm diameter, 100 mm length, SKU: 006BCC-06-10-AF, Diba Industries, Danbury, CT 06810, US) was packed to a height of cm with 1.0 mL of the CEX chromatography resin Continuous loading chromatography experiments were performed using a GE Healthcare Life Sciences ÄKTA avant 25 Before each experiment, the columns were equilibrated with the same buffer as that of the mAb feed solution for 10 CV Before a column was reused for additional experiments it was first washed with the loading buffer for 10 CV, stripped with the loading buffer also containing 1.0 M sodium chloride for 15 CV, cleaned with 0.5 M sodium hydroxide for CV, and equilibrated with loading buffer for 15 CV 2.2.2 Analytical size exclusion chromatography Analytical size-exclusion chromatography of proteins was performed using a Waters 2695 Separation Module, a Waters Dual ␭ Absorbance Detector, and a Tosoh Biosciences TSKgel G3000SWxl column (part number: 08541, column size: 300 × 7.8 mm, Tosoh Bioscience LLC, King of Prussia, PA, USA) The isocratic mobile phase was a solution of 50 mM sodium phosphate and 150 mM sodium chloride at pH 7.0 The column was run at a flow rate of 1.00 mL/min for 20 and the UV detector was set to a wavelength of 280 nm The percentage of aggregates was calculated based on the areas of the HPLC peaks 2.2.3 UV spectroscopic analysis of protein concentration UV spectroscopic analysis of protein solution concentration was performed with a Thermo Scientific GENESYS 10S UV–vis Spectrophotometer The concentration of the mAb05 and mAb02 fractions was determined by measuring their absorbance at 280 nm in a disposable plastic cell having a 1.0 cm path length The absorbance was divided by the extinction coefficient of the mAbs (mAb05 = 1.419 mL·g−1 ·cm−1 , mAb02 = 1.467 mL·g−1 ·cm−1 ) and the pathlength to calculate the protein concentration The monomer recovery was calculated based on the concentration of the mAb in a fraction as determined by UV spectroscopic analysis and the percentage of monomer in a fraction as determined by analytical size-exclusion chromatography 154 M.T Stone et al / J Chromatogr A 1599 (2019) 152–160 ® Fig SEC chromatograms of individual fractions of a mAb05 feed processed through a 1.0 mL packed column of Eshmuno CP-FT resin at a residence time of at various loadings (left) The mAb05 feed for this experiment examining the composition of proteins in the elution fractions had a concentration of 15 g/L with 10% total aggregates (5% dimers) and was dialyzed into a buffer composed of 100 mM sodium acetate at pH 5.0 and a conductivity of 5.0 mS/cm SEC chromatograms of high salt elutions from a ® 1.0 mL packed column of Eshmuno CP-FT resin loaded with various amounts of the mAb05 feed at a residence time of (right) The mAb05 feed used for this experiment examining the composition of proteins that were retained by the column had a concentration of 16 g/L with 11% total aggregates (5% dimers) and was dialyzed into a buffer composed of 100 mM sodium acetate at pH 5.0 and a conductivity of 5.0 mS/cm 2.2.4 Determination of CEX resin ionic density A mL portion of gravity settled CEX resin in 20% ethanol was measured in a small plastic column To remove the ethanol from the CEX resin it was three times suspended in mL of water and then the water was removed by suction from the column The sulfonate group on the CEX resin was converted to a sulfonic acid group by three times suspending the resin in mL of 1.0 M hydrochloric acid for and then removing the hydrochloric acid by suction To remove the remaining hydrochloric acid the resins were suspended in mL of water that was then removed by suction and this process was repeated until the pH of the water removed was neutral The CEX resin was then transferred into a 200 mL glass beaker To the glass beaker was also added 80 mL of 1.0 M sodium chloride and a 1.0 mL solution of phenolphthalein at a concentration of 1% by weight in ethanol The solution was titrated with a 0.01 M solution of sodium hydroxide and the end of the titration was indicated when the solution changed to a pink color The ionic density of the CEX resin was calculated by the following formula: ionic denisity = VNaOH × CNaOH Vresin In this equation VNaOH is the volume of sodium hydroxide titrated into the suspension of CEX resin, CNaOH is the concentration of sodium hydroxide, and Vresin is the volume of the resin that was titrated The ionic density of the CEX resins is the average of two separate measurements to centrifuge and a portion of the supernatant mAb05 solution was diluted 20-fold The UV absorbance of the 20-fold diluted solution at 280 nm was determined as described in Section 2.2.3 The process of preparing a 20-fold dilution and measuring the UV absorbance of the solution at 280 nm was performed in triplicate for each tube and the average of the three measurements was used to calculate the mAb05 concentration The static binding capacity of the CEX resin for mAb05 was then determined by the following formula: static binding capacity = (V control × Ccontrol ) − (V resin treated × Cresin treated ) Vresin In this equation Vcontrol and Vresin treated are the total volume of the mAb05 control solutions and the total volume of the mAb05 solutions treated with the resin, respectively The Ccontrol and the Cresin treated are the concentration of mAb05 in the control tubes after they were rotated for h and the concentration of mAb05 in the tubes treated with the resin after they were rotated for h, respectively Vresin is the volume of the resin added to the resin treated solutions The static binding capacity of the CEX resins for mAb05 is the average of two separate preparations of the resin slurry that were each measured in triplicate Results 3.1 Mechanism for the removal of aggregates 2.2.5 Determination of CEX resin static binding capacity for mAb05 A mL portion of gravity settled CEX resin in 20% ethanol was measured in a small plastic column To remove the ethanol from the CEX resin it was three times suspended in an acetate buffer composed of 100 mM sodium acetate at pH 5.0 and 5.0 mS/cm and then the acetate buffer was removed by suction The CEX resin was added to a 50 mL centrifuge tube To the centrifuge tube was also added mL of the acetate buffer to give a 10% resin slurry A 1.0 mL portion of the 10% resin slurry was added to a 15 mL centrifuge tube To the 15 mL tube was also added 1.5 mL of the acetate buffer and 2.5 mL of a mAB05 solution at a concentration of 10 g/L with 0.4% aggregates that was dialyzed into the acetate buffer The resulting slurry contained 0.1 mL of CEX resin and mAb05 at a concentration of g/L Controls omitting the resin were prepared by adding 2.5 mL of the acetate buffer and 2.5 mL of the dialyzed mAB05 solution to 15 mL tubes The 15 mL tubes containing CEX resin and the control tubes were rotated for h The 15 mL tubes were then subjected ® We investigated the removal of aggregates with Eshmuno CPFT resin using a continuous loading process to determine if the separation of the mAb monomer from the aggregates was consistent with a frontal chromatography mechanism A mAb05 feed containing 10% aggregates was dialyzed into a 100 mM sodium acetate buffer at pH 5.0 and mS/cm then loaded onto a column of ® the Eshmuno CP-FT resin The composition of the fractions eluted from the column were analyzed by size exclusion chromatography (Fig 1, left) Initially, only the monomer was observed to elute from the column At a loading of 600 g/L, dimers were also detected in the elution However, no higher molecular weight aggregates were observed up to a loading of 1000 g/L where the experiment was ended These results are consistent with a frontal chromatography mechanism in which pure monomer eluted in the first front and then the dimers coeluted with the monomer in a second front The ® Eshmuno CP-FT resin was not loaded with a sufficient amount of the mAb05 feed to observe a third front that would be com- M.T Stone et al / J Chromatogr A 1599 (2019) 152–160 155 Fig The cumulative percentage of aggregates in the elution pool as a function of mAb05 loading (left) and the cumulative percentage of aggregates in the elution pool as a function of the cumulative mAb05 monomer recovery in the elution pool (right) The mAb05 feed was processed through a 1.0 mL packed column of a CEX chromatography resin at a residence time of The mAb05 feed had a concentration of 15 g/L with 11% total aggregates (5% dimers) and was dialyzed into a buffer composed of 100 mM ® ® ® ® sodium acetate at pH 5.0 and a conductivity of 5.0 mS/cm CEX media legend: C1 - Eshmuno CPX resin, C2 – Poros XS, C3 – Poros 50 HS, C4 - Toyopearl Gigacap S-650 M, ® C5 – Capto S ImpAct, C6 – SP SepharoseTM Fast Flow The percentages included on the plots in black font indicate the cumulative monomer recovery at the last fraction before the cumulative percentage of aggregates exceeded 1% posed of the higher molecular weight aggregates along the with the monomer and dimers Next, we examined the components that were retained by the ® Eshmuno CP-FT resin after it was loaded with various amounts of a mAb05 feed containing 11% aggregates that was dialyzed into a 100 mM sodium acetate buffer at pH 5.0 and mS/cm After a specific loading of the column was completed the components of the mAb05 feed retained by the column were eluted in a subsequent step using a high salt buffer We found that as the loading of the mAb05 feed was increased, the amount of monomer retained by the column decreased and the amount of aggregates increased (Fig 1, right) For example, at a loading of 200 g/L, the composition of mAb05 feed that was retained by the column consisted of 71% monomer while at a loading of 1000 g/L, the percentage of monomer retained by the column decreased to 9% The composition of the aggregates that were retained by the column included dimers and higher molecular weight aggregates These results are consistent with a frontal chromatography mechanism, in which the mAb monomer is initially retained by the column and subsequently displaced by aggregates Table Ionic density and static binding capacity for mAb05 determined for the CEX resins Note that the percentage of aggregates in the elution pool corresponds to a resin loading of approximately 1000 g/L However, there were variations in the final loading of the CEX resins which ranged from 967 g/L to 1000 g/L 3.2 Removal of mAb aggregates by continuous loading of various CEX chromatography resins tion may indicate an influence of the base bead because Capto S ImpAct and SP SepharoseTM Fast Flow have agarose resin base beads while the other CEX resins are composed of polymeric base beads Next, we measured the ionic density and the mAb05 static binding capacities of these seven strong CEX resins to determine if either of these attributes correlated with the efficient removal of aggregates using a continuous loading process (Table 1) The ionic density of a CEX resin was determined by converting the sulfonate groups to sulfonic acids and then titrating the acidified resin with sodium hydroxide in the presence of a phenolphthalein indicator The static binding capacity of the CEX resins was measured using a mAb05 feed dialyzed into 100 mM sodium acetate at pH 5.0 and mS/cm We plotted the cumulative percentage of aggregates in the elution pool at a loading of approximately 1000 g/L as a function of both variables (Fig 3) We found that there was a rough correlation between the ionic density and the percentage of aggregates in their elution pool For ® instance, Eshmuno CP-FT resin had the lowest ionic density at 37 ␮eq/mL and had the lowest percentage of aggregates in the cumu® ® lative elution pool at 1.9% Eshmuno CPX resin, Poros XS, and ® Poros 50 HS had intermediate ionic densities of 70–81 ␮eq/mL and very similar percentages of aggregates in the elution pool of ® 7.5–7.8% Toyopearl Gigacap S-650 M and SP SepharoseTM Fast Flow had the highest ionic densities of 188–210 ␮eq/mL and the highest percentages of aggregates in the elution pool of 8.8–9.7% ® The ability of Eshmuno CP-FT resin to remove aggregates using a continuous loading process was compared with six commerciallyavailable strong CEX chromatography resins having sulfonate ligands We selected a solution pH of 5.0 and a conductivity of mS/cm because a sulfonate CEX resin will typically have a high capacity for the mAb monomer and aggregates under these solution conditions A mAb05 feed containing 11% aggregates was dialyzed into 100 mM sodium acetate at pH 5.0 and mS/cm, loaded onto the CEX column, and the compositions of the elution fractions were determined Under these solution conditions five of ® ® the CEX resins including Eshmuno CP-FT resin, Eshmuno CPX ® ® ® resin, Poros XS, Poros 50 HS, and Toyopearl Gigacap S-650 M showed a gradual breakthrough of the aggregates as is consistent ® with a frontal chromatography mechanism (Fig 2) Eshmuno CPFT resin is an outlier of these five as it removed significantly more aggregates with a better monomer recovery The elution pool for ® Eshmuno CP-FT resin had 0.8% aggregates with a 92% monomer recovery at a loading of 741 g/L By contrast, the elution pool for the other CEX chromatography resins all exceeded 1% aggregates ® before their monomer recoveries reached 50% Capto S ImpAct TM Fast Flow did not show a gradual increase in and SP Sepharose the cumulative percentage of aggregates in the elution pool as is expected for a frontal chromatography mechanism This observa- CEX resin ® Eshmuno CP-FT resin ® Capto S ImpAct ® Eshmuno CPX resin ® Poros 50 HS ® Poros XS ® Toyopearl Gigacap S-650M SP SepharoseTM Fast Flow ionic density (␮eq/mL) static binding capacity for mAb05 (g/L) aggregates in elution at loading of ˜ 1000 g/L 37 69 1.9% 64 89 8.9% 70 71 7.5% 78 81 188 50 68 100 7.8% 7.8% 9.7% 210 66 8.9% ® 156 M.T Stone et al / J Chromatogr A 1599 (2019) 152–160 Fig The cumulative percentage of aggregates in the elution pool at a loading of approximately 1000 g/L as a function of the ionic density of the CEX resin (left) and the cumulative percentage of aggregates in the elution pool at a loading of approximately 1000 g/L as a function of the static binding capacity of the CEX resin for mAb05 (right) The CEX static binding capacity was measured with a mAb05 feed at a concentration of g/L with 0.4% aggregates that was dialyzed into a 100 mM sodium acetate buffer at pH 5.0 and a conductivity of 5.0 mS/cm Fig The cumulative percentage of aggregates in the elution pool as a function of mAb05 loading (top left) or mAb02 loading (bottom left) and the cumulative percentage of aggregates in the elution pool as a function of the cumulative mAb05 monomer recovery in the elution pool (top right) or the cumulative mAb02 monomer recovery in the elution pool (bottom right) The feeds were processed through a 1.0 mL packed column of Eshmuno® CP-FT resin at a residence time of The mAb05 feed had a concentration of 12 g/L with 10% total aggregates (6% dimers) and was dialyzed into a buffer composed of 50 mM sodium acetate at pH 4.5, pH 5.0, pH 5.5, or pH 6.0 The mAb02 feed had a concentration of 13 g/L with 6% total aggregates (4% dimers) and was dialyzed into 50 mM sodium acetate at pH 4.0, pH 4.5, or pH 5.0 The conductivities of the acetate buffers for the mAb05 and mAb02 feeds were adjusted to 5.0 mS/cm and 2.5 mS/cm respectively by the addition of sodium chloride The percentages included on the plots in black font indicate the cumulative monomer recovery at the last fraction before the cumulative percentage of aggregates exceeded 1% Capto® S ImpAct does not follow this trend as it has an intermediate ionic density of 64 ␮eq/mL, but still has a highest percentages of aggregates in the elution pool of 8.9–9.7% However, it might not ® be appropriate to compare the dependence of Capto S ImpAct and SP SepharoseTM Fast Flow if they are not operating according to a frontal chromatography mechanism as is suggested by shape of their curves in Fig We did not find a correlation between the mAb05 static binding capacity of the CEX resins and the percentage of aggregates in ® their elution pool For instance, SP SepharoseTM Fast Flow, Poros ® ® XS, Eshmuno CP-FT resin, and Eshmuno CPX resin all had very similar mAb05 static binding capacities of 66 g/L, 68 g/L, 69 g/L, and 71 g/L respectively, but varied significantly in the percent® age of aggregates in their elutions Neither Poros 50 HS that ® had the lowest static binding capacity of 50 g/L or Toyopearl Gigacap S-650 M that had the highest static binding capacity of 100 g/L showed the lowest percentage of aggregates in their elution pools 3.3 Influence of solution pH The influence of solution pH on removal of aggregates with ® Eshmuno CP-FT resin using a continuous loading process was investigated with both a mAb05 feed and a mAb02 feed A mAb05 feed containing 10% aggregates and a mAb02 feed containing 6% aggregates were dialyzed into acetate buffers that varied in pH We observed for both the mAb05 and mAb02 feeds that as the solution pH was lowered more aggregates were removed with a higher monomer recovery (Fig 4) Based on their isoelectric points, both mAb05 (pI = 8.1) and mAb02 (pI = 8.24) are more strongly charged at a lower solution pH Thus the removal of aggregates was most efficient at a lower solution pH where the electrostatic interactions M.T Stone et al / J Chromatogr A 1599 (2019) 152–160 157 Fig The cumulative percentage of aggregates in the elution pool as a function of mAb05 loading (top left) or mAb02 loading (bottom left) and the cumulative percentage of aggregates in the elution pool as a function of the cumulative mAb05 monomer recovery in the elution pool (top right) or the cumulative mAb02 monomer recovery in ® the elution pool (bottom right) The feeds were processed through a 1.0 mL packed column of Eshmuno CP-FT resin at a residence time of The mAb05 feed had a concentration of 12 g/L with 11% total aggregates (6% dimers) and was dialyzed into a buffer composed of 50 mM, 100 mM, 150 mM, or 200 mM sodium acetate at pH 5.0 having a conductivity of 2.8 mS/cm, 4.7 mS/cm, 6.2 mS/cm, or 8.2 mS/cm respectively The mAb02 feeds had a concentration of 13 g/L with 6% total aggregates (4% dimers) and was dialyzed into a 50 mM sodium acetate buffer at pH 4.0 having a conductivity of 2.5 mS/cm, 5.0 mS/cm, 7.0 mS/cm, or 9.0 mS/cm The conductivity of the buffers used for dialysis of the mAb02 feed were adjusted by the addition of sodium chloride The percentages included on the plots in black font indicate the cumulative monomer recovery at the last fraction before the cumulative percentage of aggregates exceeded 1% between the positively charged mAb monomer/aggregates and the negatively charged resin are strongest 3.4 Influence of solution conductivity The influence of solution conductivity on the removal of aggre® gates with Eshmuno CP-FT resin using a continuous loading process was investigated with both a mAb05 feed and a mAb02 feed A mAb05 feed containing 10% aggregates and a mAb02 feed containing 6% aggregates were dialyzed into acetate buffers that varied in conductivity We found that as the solution conductivity was decreased, more aggregates were removed from both the mAb05 feed and the mAb02 feed with a higher monomer recovery (Fig 5) However, there was a significant departure from this trend for the mAb05 feed at 2.8 mS/cm in which less aggregates were removed with a lower monomer recovery than was observed for the three other mAb05 feeds having higher solution conductivities One potential explanation for this outlier could be that the mAb05 monomer is too strongly bound to the resin at this low solution conductivity inhibiting displacement by the aggregates as is required for an efficient separation with a frontal chromatography mechanism No such exception was observed for the removal of aggregates from the mAb02 feeds where the most efficient solution condition was at the lowest conductivity of 2.5 mS/cm 3.5 Influence of residence time The influence of residence time on the removal of aggregates ® with Eshmuno CP-FT resin using a continuous loading process was investigated using a mAb05 feed containing 11% aggregates We observed that as the residence time was increased more aggregates were removed from the mAb05 feed with a higher monomer recovery (Fig 6) Longer residence times are to likely result in the more efficient removal of aggregates with a frontal chromatography mechanism because mass transfer of the aggregates into the resin limits displacement of the bound monomers However, longer residence times are not desirable as they will require longer loading times For instance, increasing the residence time from to increased loading time for the mAb05 feed from 3.3 h to 6.7 h 3.6 Influence of mAb feed concentration The influence of the mAb feed concentration on the removal of ® aggregates with Eshmuno CP-FT resin using a continuous loading process was investigated with a mAb05 feed containing 10% aggregates We observed that as the concentration of the mAb05 feed was decreased more aggregates were removed with a higher monomer recovery (Fig 7) However, lowering the concentration of the mAb feed is not desirable, because longer loading times are required to process the larger volumes of the mAb feed For instance, decreasing the concentration of the mAb05 feed from 15 g/L to g/L increased the loading time from 3.3 h to 10 h 3.7 Influence of the percentage of aggregates in the mAb feed To examine the influence of the percentage of aggregates in the mAb feed on the removal of aggregates with Eshmuno® CP-FT resin using a continuous loading process, six mAb05 feeds were prepared with varying percentages of aggregates ranging from 1.9% to 14.6% We observed for all six mAb05 feeds that a specific loading of the feed could be selected where the level of aggregates in the elution pool was reduced below 1% with a monomer recovery greater than 85% (Fig 8, top left and right) We also observed that 158 M.T Stone et al / J Chromatogr A 1599 (2019) 152–160 Fig The cumulative percentage of aggregates in the elution pool as a function of mAb05 loading (left) and the cumulative percentage of aggregates in the elution pool as a function of the cumulative mAb05 monomer recovery in the elution pool (right) The feed was processed through a 1.0 mL packed column of Eshmuno® CP-FT resin at a residence time of (180 cm/h), (90 cm/h), (60 cm/h), or (30 cm/h) The mAb05 feed had a concentration of 15 g/L with 11% total aggregates (5% dimers) and was dialyzed into a buffer composed of 100 mM sodium acetate at pH 5.0 and a conductivity of 5.0 mS/cm The percentages included on the plots in black font indicate the cumulative monomer recovery at the last fraction before the cumulative percentage of aggregates exceeded 1% Fig The cumulative percentage of aggregates in the elution pool as a function of mAb05 loading (left) and the cumulative percentage of aggregates in the elution pool as a ® function of the cumulative mAb05 monomer recovery in the elution pool (right) The mAb05 feed was processed through a 1.0 mL packed column of Eshmuno CP-FT resin at a residence time of The mAb05 feed had an initial concentration of 15 g/L with 10% total aggregates (6% dimers) and was dialyzed into a buffer composed of 100 mM sodium acetate at pH 5.0 and a conductivity of 5.0 mS/cm Portions of the mAb05 feed were diluted to g/L and 10 g/L with the dialysis buffer The percentages included on the plots in black font indicate the cumulative monomer recovery at the last fraction before the cumulative percentage of aggregates exceeded 1% Fig The cumulative percentage of aggregates in the elution pool as a function of mAb05 loading (top left) and the cumulative percentage of aggregates in the elution pool as a function of the cumulative mAb05 monomer recovery in the elution pool (top right) The cumulative percentage of aggregates in the elution pool as a function of ® the loading of mAb05 aggregates (bottom) The mAb05 feeds were processed through a 1.0 mL packed column of Eshmuno CP-FT resin at a residence time of The mAb05 feeds had a concentration of 15 g/L with 1.9%, 3.7%, 7.3%, 10.4% 12.2%, or 14.6% total aggregates and were dialyzed into a 100 mM sodium acetate buffer at pH 5.0 and a conductivity of 5.0 mS/cm The percentages included on the plots in black font indicate the cumulative monomer recovery at the last fraction before the cumulative percentage of aggregates exceeded 1% M.T Stone et al / J Chromatogr A 1599 (2019) 152–160 159 Table The effective loading range for the removal of aggregates from mAb05 feeds with varying percentages of aggregates percentage of aggregates in mAb05 feed minimum loading at which the monomer recovery in the elution pool was ≥85% (g/L) maximum loading at which the aggregates in the elution pool was ≤1% (g/L) observed effective loading range (g/L) 1.9% 3.7% 7.3% 10.4% 12.2% 14.6% 700 681 596 583 511 446 >1000 >1000 >1000 827 660 535 >300 >319 >404 245 149 89 as the percentage of aggregates in the mAb05 feed was increased, the aggregates eluted from the column at lower loadings The percentages of the aggregates in the elution pool were also plotted as a function of the loading of aggregates (Fig 8, bottom) The resulting plot shows that all six feeds had a similar shape that is consistent with a frontal chromatography mechanism where the aggregates should not begin to elute until they have exceeded the capacity of ® the Eshmuno CP-FT resin We also noted that as the percentage of aggregates in the mAb05 feed was increased, the effective loading range for the removal of aggregates became narrower (Table 2) For this experiment, we defined the effective loading range as starting when the cumulative monomer recovery in the elution pool exceeded 85% and ending when the cumulative percentage of aggregates exceeded 1% The effective loading range for the removal of aggregates was found to increase as the percentage of aggregates in the feed was decreased The effective loading ranges for the 1.9%, 3.7%, and 7.3% feeds were not fully determined, as the percentage of aggregates in the elution pool remained below 1% at a loading of 1000 g/L The effective loading range for the 1.9% and 3.7% feeds are likely to extend significantly beyond 1000 g/L as the percentage of aggregates in the elution pool was only 0.2% and 0.3% respectively when the experiment ended Discussion ® First, we sought to confirm that Eshmuno CP-FT resin was removing aggregates from a mAb05 feed according to a frontal chromatography mechanism when using a continuous loading process The composition of the mAb05 feed that eluted from the ® Eshmuno CP-FT resin as well as the composition of the mAb05 feed that was retained by the column was determined as the loading was varied (Fig 1) We observed that the mAb05 monomer eluted from the column in the earliest fractions and the dimers did not begin to elute until 600 g/L At lower loadings the composition of mAb05 feed that was retained by the column consisted primarily of monomer, but as the loading was increased the retained monomer was displaced by dimers and higher molecular weight aggregates [14,16] We also noted that dimers were the only types of aggregates observed in the elution fractions while higher molecular weight aggregates were completely retained by the column This suggests that the higher molecular weight aggregates are forming a third front that has yet to elute from the column at the end of the experiment The results of both experiments indicate the sep® aration of the mAb monomer from the aggregates with Eshmuno CP-FT resin using a continuous loading process is consistent with a frontal chromatography mechanism ® Next, we compared the removal of aggregates with Eshmuno CP-FT resin using a continuous loading process from a mAb05 feed with six commercially available strong CEX chromatography resins having sulfonate ligands (Fig 2) We chose to compare the strong CEX resins at a solution pH of 5.0 and a conductivity of mS/cm where they should have a high capacity for the mAb aggregates If a CEX resin efficiently removes aggregates from a mAb feed using a continuous loading process under these solution conditions, then very high loadings of the resin should be ® ® ® possible Eshmuno CP-FT resin, Eshmuno CPX resin, Poros XS, ® ® Poros 50 HS, and Toyopearl Gigacap S-650 M showed a gradual increase in the percentage of the aggregates in the elution pool as ® is expected with a frontal chromatography mechanism Eshmuno CP-FT resin removed significantly more aggregates with a higher monomer recovery than the other CEX chromatography resins ® Capto S ImpAct and SP SepharoseTM Fast Flow showed an immediate breakthrough of aggregates in the elution pool indicating that they are not removing aggregates according to a frontal chromatography mechanism under these solution conditions These two CEX resins are both composed of an agarose base bead and we speculate that this factor may be responsible for inhibiting a frontal chromatography mechanism under these solution conditions as all the other CEX resins are composed of polymer base beads The percentage of aggregates in the elution pool for all seven CEX resins was plotted as a function of their ionic density and their mAb05 static binding capacity (Table 1, Fig 3) A low percentage of aggregates in the elution pool was found to correlate with a low ionic density while no correlation was observed with the static binding capacities of the CEX resins for mAb05 A lower ionic density CEX resin may facilitate efficient removal of mAb aggregates by frontal chromatography because it likely has fewer electrostatic interactions with the monomer allowing it to be more easily displaced by the aggregates It is important to note that we compared the removal of aggregates with CEX resins using a continuous loading process at a single solution condition where CEX resins typically have high capacities for the mAb monomer and aggregates CEX resins with higher ionic densities may remove aggregates more efficiently at a higher solution pH and/or conductivity where the strength of the electrostatic interaction between positively charged mAb monomer/aggregates and the negatively charged resin will be weaker However, operating a CEX resin at a higher pH/conductivity will reduce its capacity for mAb aggregates and thus limit the amount of the mAb monomer that can be purified by continuous loading before elution of aggregates will occur Then we examined the influence of solution pH and conductivity ® on the removal of aggregates with Eshmuno CP-FT resin using a continuous loading process from both a mAb05 feed and a mAb02 feed We found that more aggregates were removed with higher monomer recoveries at lower solution pHs (Fig 4) and conductivities (Fig 5), which favor strong electrostatic interactions between the positively charged mAb monomer/aggregates and the negatively charged resin One exception to this trend was observed with ® mAb05 at 2.8 mS/cm, where Eshmuno CP-FT resin was significantly less efficient for the removal of aggregates than the other higher conductivities mAb05 feeds investigated One explanation is that at a solution conductivity of 2.8 mS/cm the mAb05 monomer ® is too strongly bound to the Eshmuno CP-FT resin thus preventing displacement by aggregates and inhibiting separation by a frontal chromatography mechanism [12] Liu, et al also observed that the ® removal of aggregates from a mAb feed using Poros 50 HS CEX 160 M.T Stone et al / J Chromatogr A 1599 (2019) 152–160 resin with a continuous loading process became increasingly efficient as the solution conductivity was decreased from 18 mS/cm to mS/cm, however at mS/cm the removal of aggregates was significantly worse [10] However, no such exception was observed for the mAb02 feed where the removal of aggregates was most efficient at the lowest conductivity of 2.5 mS/cm We also investigated the influence of the flow-rate and the mAb feed concentration on the removal of aggregates from a mAb05 feed ® with Eshmuno CP-FT resin using a continuous loading process The removal of aggregates was found to be most efficient at longer residence times (Fig 6) and lower mAb feed concentrations (Fig 7) ® Liu, et al also reported that the removal of aggregates with Poros 50 HS resin using a continuous loading process was most efficient at longer residence times [10] Longer residents times are likely to be advantageous for the removal of aggregates using a frontal chromatography mechanism because they give more time for mass transfer of the aggregates into the resin However, using longer residence times and lower mAb concentrations will also increase the resin loading time and thus could decrease the productivity of the resin to an unreasonably low level Finally, we investigated how the percentage of aggregates in the mAb feed influenced the removal of mAb aggregates with ® Eshmuno CP-FT resin using a continuous loading process We tested six different mAb05 feeds with levels of aggregates that varied from 1.9% to 14.6% We found that as the percentage of aggregates in the mAb05 feed was increased, the aggregates began eluting from the resin at lower loadings The level of aggregates in all six mAb05 feeds could be reduced to less than 1% with monomer recoveries greater than 85% at a particular loading (Fig 8) However, purifying mAb05 feeds containing higher percentages of aggregates using a continuous loading process is more challenging, because the effective operating range was found to decrease as the percentage of aggregates in the feed was increased (Table 2) A mAb05 feed with a higher percentage of aggregates must be processed at lower loadings and over narrower ranges to remove a sufficient amount of aggregates with a good monomer recovery While a mAb05 feed a with lower percentage of aggregates can be processed at higher loadings with a significantly wider effective loading range Conclusions ® A low ionic density CEX chromatography resin, Eshmuno CPFT resin, was investigated for the removal of aggregates from mAb feeds using a continuous loading process The removal of ® mAb aggregates with Eshmuno CP-FT resin using a continuous loading process was found to be consistent with a frontal chromatography mechanism, whereby the mAb monomers are initially retained by the column and are subsequently displaced by aggre® gates Eshmuno CP-FT resin was found to be significantly more effective for the removal of mAb aggregates using a continuous loading process compared to six commercially available strong CEX chromatography resins under solution conditions where CEX resins typically have high capacities for mAb monomer and aggregates We found that the efficient removal of aggregates using a continuous loading process correlated with CEX resins having lower ionic densities while no correlation was observed with their mAb static binding capacities Optimization studies found that the removal of ® aggregates with Eshmuno CP-FT resin using a continuous loading process was more efficient at lower solution pHs and lower solution conductivities, which favor strong electrostatic interactions between the positively charged mAb monomer/aggregates ® and the negatively charged Eshmuno CP-FT resin An important exception to this trend in the solution conditions was observed at the lowest conductivity for the mAb05 feed Optimization studies ® also found that Eshmuno CP-FT resin removes more aggregates with higher monomer recoveries at longer residence times and ® lower mAb feed concentrations Eshmuno CP-FT resin efficiently removed aggregates from mAb feeds containing between 1.9% and 14.6% aggregates using a continuous loading process, however the mAb feeds with lower percentages of aggregates had much wider effective loading ranges Conflict of interest disclosure The authors are employees of EMD Millipore Corporation which ® sells Eshmuno CP-FT resin Acknowledgements The authors thank James Hamzik, Lars Peeck, Dominic Zorn, Romas Skudas, Paul Turiano, Lloyd Gottlieb, Michael Schulte, David Beattie, and Matthias Jöhnck for their support and encouragement References [1] A.S Rosenberg, Effects of protein aggregates: an immunologic perspective, AAPS J (2006) E501–E507, http://dx.doi.org/10.1208/aapsj080359 [2] M.E.M Cromwell, E Hilario, F Jacobson, Protein aggregation and bioprocessing, AAPS J (2006) E572–E579, http://dx.doi.org/10.1208/ aapsj080366 [3] M Vazquez-Rey, D.A Lang, Aggregates in monoclonal antibody manufacturing processes, Biotechnol Bioeng 108 (2011) 1494–1508, http:// dx.doi.org/10.1002/bit.23155 [4] E.M Moussa, J.P Panchal, B.S Moorthy, J.S Blum, M.K Joubert, L.O Narhi, E.M Topp, Immunogenicity of therapeutic protein aggregates, J Pharm Sci 105 (2016) 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Rachinskii, Theory of frontal chromatography, in: The General Theory of Sorption Dynamics and Chromatography, Springer, Boston, 1965, pp 51–68, http://dx.doi.org/10.1007/978-1-4757-0061-9 [15] J.A Jonsson, Common concepts in chromatography, in: Chromatographic Theory and Basic Principles, Marcel Dekker, New York, 1987, pp 1–26 [16] D.A Hill, P Mace, D Moore, Frontal chromatographic techniques in preparative chromatography, J Chromatogr A 523 (1990) 11–21, http://dx doi.org/10.1016/0021-9673(90)85007-I [17] S Ahuja, Chromatographic methods, in: Chromatography and Separation Science, Academic Press, San Diego, 2003, pp 81–100, http://dx.doi.org/10 1016/S0149-6395(03)80024-7 [18] A.S.P Potty, A Xenopoulos, Stress-induced antibody aggregates, Bioproc Int 11 (2013) 44–52, retrieved from http://www.bioprocessintl.com  ... the cumulative percentage of aggregates exceeded 1% The effective loading range for the removal of aggregates was found to increase as the percentage of aggregates in the feed was decreased The. .. where the removal of aggregates was most efficient at the lowest conductivity of 2.5 mS/cm We also investigated the influence of the flow-rate and the mAb feed concentration on the removal of aggregates. .. 10 h 3.7 Influence of the percentage of aggregates in the mAb feed To examine the influence of the percentage of aggregates in the mAb feed on the removal of aggregates with Eshmuno® CP-FT resin