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Efficient capture of monoclonal antibody from cell culture supernatant using protein A media contained in a cuboid packed-bed device
Journal of Chromatography B 1134–1135 (2019) 121853
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/jchromb
Efficient capture of monoclonal antibody from cell culture supernatant using protein A media contained in a cuboid packed-bed device
T
Guoqiang Chena, Alisha Gerriora, Yves Durocherb,c, Raja Ghosha,
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a
Department of Chemical Engineering, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S 4L7, Canada National Research Council of Canada, Montreal, QC H4P 2R2, Canada c Département de biochimie et médecine moléculaire, Faculté de médecine, Université de Montréal, Montreal, QC H3C 3J7, Canada b
ARTICLE INFO
ABSTRACT
Keywords: Column Cuboid packed-bed Chromatography Capture Monoclonal antibody Protein A Affinity chromatography
A box-shaped or cuboid packed-bed device was used for monoclonal antibody (mAb) separation using protein A affinity chromatography. The separation efficiency of the device was compared with an equivalent column i.e. packed with same resin, and having identical bed height and bed volume. The protein A media packed cuboid device had a larger number of theoretical plates than its equivalent column, e.g. 8750/m as opposed to about 4700/m at a flow rate of 0.5 mL/min. In mAb purification experiments, the impurity flow-through and eluted mAb peaks were shaper with the cuboid device. This implied that the effective separation time and buffer consumption with this device was lower, the purified mAb pooled volume was smaller, and the mAb concentration in the pooled volume was greater. Equivalent separation efficiency could be obtained with the cuboid device using higher flow rates than that used with the column. For instance, elution peaks equivalent to those obtainable by the column could be obtained at a 5 times greater flow rate using the cuboid device. The results discussed in this paper clearly demonstrate the potential for improving the efficiency of protein A affinity chromatography based mAb purification by using a cuboid packed-bed device.
1. Introduction Monoclonal antibodies (mAbs) are widely used to diagnose and treat cancer and infectious diseases, as well as to modulate immune responses [1,2]. Most mAbs used as biopharmaceuticals belong to the IgG1 subclass [3,4]. Compared to other biopharmaceutical proteins or even small molecule drugs, the therapeutic mAbs are administered in quite large doses [5]. Mammalian cells such as Chinese Hamster Ovary (CHO) cells and NS0 (mouse myeloma) cells are generally used to make mAbs [6]. Spectacular developments in upstream processing over the past decades have resulted in increase in mAb titers from less than 0.5 g/L to 10 g/L [7,8]. The challenge in mAb production has therefore shifted from upstream to downstream processing, primarily due to high purification cost [9,10]. The biopharmaceutical industry employs a platform downstream process for mAbs which consists of protein A affinity chromatography, low pH virus inactivation, polishing steps, virus filtration, and ultrafiltration/diafiltration for formulation [10–13]. Although the specific sequence and type of polishing and further processing techniques used vary from manufacturer to manufacturer, protein A chromatography is nearly always employed as the primary mAb capture step [10] due to its high selectivity [11].
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However, protein A affinity media is very expensive and this has been flagged as a major limitation [11,14]. The motivation to reduce purification cost has led to search for alternatives to protein A affinity chromatography. Techniques that have been considered include protein A mimetic chromatography [15], hydrophobic charge induction chromatography [15], cation exchange chromatography [16], mixed mode chromatography [17], as well as non-chromatographic methods such as precipitation [18], aqueous twophase system [19], and protein A affinity membrane chromatography [20]. However, most of these techniques give considerably lower selectivity and several of these need to be used in combination. Therefore, the advantage of the lower media cost is often offset by the elaborate and complex nature of these alternative separation processes as well as additional time required for development and retrofitting of existing platform processes. A more practical approach would therefore be to use the protein A affinity media more efficiently. For instance, a continuous multi-column based protein A chromatography could improve the mAb binding capacity utilization [21]. The problem of flow maldistribution in columns could become a severe limiting factor when designing and developing such advanced separation systems. More generally, better devices and processes could improve the efficiency of
Corresponding author. E-mail address: [email protected] (R. Ghosh).
https://doi.org/10.1016/j.jchromb.2019.121853 Received 20 June 2019; Received in revised form 27 September 2019; Accepted 28 October 2019 Available online 12 November 2019 1570-0232/ Crown Copyright © 2019 Published by Elsevier B.V. All rights reserved.
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Fig. 1. The cuboid packed-bed device used in this study (A) photograph (left) and schematic diagram (right), and (B) plan view of components.
asymmetry factor were systematically compared. These results clearly demonstrated the advantages of using the protein A cuboid packed-bed device over its equivalent column.
protein A affinity chromatography and bring down the manufacturing cost. The importance of chromatography device is gradually being recognized by the more and more researchers. The term chromatography immediately conjures up the image of a resin-packed column. However, such traditional columns are not always best suited for efficient separation. Non-uniform packing and column flow maldistribution frequently lead to low efficiency and poorly resolved peaks [22–29]. The problem of flow maldistribution is generally addressed through header modification [22,23], or improvement in flow distributor/collector/frit design [24–26]. Alternative column types such as radial flow [27], parallel segmented flow [28] and curtain flow [29] columns have also been proposed. Our research group has been actively involved with the design and development of cuboid packed-bed devices which are suitable for efficient chromatographic separations [30–34]. In such a device, a box-shaped resin-bed is fed from an upper lateral channel and is drained using a complimentary lower channel (see Fig. 1A). Such fluidic arrangement results in a narrow residence time distribution (RTD) within the device, and thereby efficient chromatographic separation [30,31]. The effects of the device design specifics such as the cuboid length to width ratio [32] and process parameters [33] on separation efficiency have been examined. The use of cuboid packed-bed devices for high-resolution separation of protein mixtures using cation and anion exchange resins have been reported [30,31,34]. In the current, we examine if a cuboid packed-bed device could be used to increase the efficiency of protein A affinity chromatography. Protein A affinity resin containing cuboid packed-bed device (Fig. 1A) and its equivalent column (i.e. having similar bed-height and bed-volume) were first used to separate the mAb from model impurity bovine serum albumin (BSA) for preliminary comparison. These devices were then used to capture the mAb from cell culture supernatant. Process efficiency metrics such as the height and shape of flow through impurity and eluted mAb peaks, mAb recovery and concentration in pooled fractions were compared. Effects of process parameters such as flow rate and loading volume were examined. Chromatographic separation attributes such as the number of theoretical plates and
2. Materials and methods Sodium phosphate monobasic (S0751), sodium phosphate dibasic (S0876), citric acid (C0759), sodium citrate tribasic (S4641), and bovine serum albumin (BSA, A7906) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium chloride (SOD002.205) was purchased from Bioshop (Burlington, ON, Canada). Monoclonal antibody hIgG1CD4 (Batch 12, stock concentration: 10 mg in 2.1 mL) was kindly donated by the Therapeutic Antibody Center, University of Oxford, Oxfordshire, UK. This monoclonal antibody will be referred to as mAb-1 in the paper. Monoclonal antibody Trastuzumab containing cell culture supernatant (lot: 20170920BC), produced using Chinese Hamster Ovary (CHO) cell line was kindly donated by the National Research Council Canada, Montreal, QC, Canada. This monoclonal antibody will be referred to as mAb-2 in this paper. Both mAb-1 and mAb-2 were IgG1 antibodies. Ultrapure water was obtained from a Diamond NANOpure water purification unit (Barnstead, IA, USA). Millipore Vantage® L Laboratory Column VL 16 × 250 (product number 96160250) was kindly provided by PlantForm Corporation, Guelph, ON, Canada. Protein A affinity MabSelect SuRe resin (17-5438-03) was procured from GE Healthcare Biosciences, QC, Canada. The number of theoretical plates in the cuboid packed-bed device and its equivalent column was determined using 0.4 M NaCl solution as the mobile phase and 0.8 M NaCl solution as the tracer. The buffers utilized for the affinity chromatography experiments were 20 mM phosphate buffer, 150 mM NaCl, pH 7.2 (binding buffer) and 0.1 M citrate buffer, pH 3.0 (eluting buffer) respectively. The NaCl solutions and buffers were filtered and degassed before use. Protein samples (BSA, mAb-1 solutions and their mixture) was prepared in the binding buffer. The CHO cell culture supernatant used in the mAb-2 purification experiment was directly loaded on the chromatography device without 2
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modification. The dimensions of the packed-bed within the cuboid device used in this study were 20 mm (length) × 10 mm (width) × 25 mm (height). The drawings of the device is shown in Fig. 1B. The design features of the device were similar to those used in our previous studies [30,31,33,34], except that the tapered portions on the vent sides of the upper and lower channels were removed to reduce the dead volume. The external length and width of the three components comprising the cuboid device, i.e. the top plate (acrylic), the central frame (polyvinyl chloride, PVC) and bottom plate (acrylic) were 80 mm and 30 mm respectively. The top and bottom plates were provided with pillared lateral channels to facilitate flow distribution. The chromatographic media was retained within the central frame using stainless steel mesh on both sides (0.0017 in. opening, 9319T188, McMaster Carr, USA) on each side. MabSelect SuRe resins were packed in the cuboid device and the equivalent column to obtain identical bed height (25 mm) and bed volume (5 mL). During the chromatography experiments, the cuboid device or the column was connected to an AKTA prime liquid chromatography system (GE Healthcare Biosciences, QC, Canada) using the same set of connecting tubings to keep the extra-device volume the same. The UV absorbance was monitored using a UV detector having 2 mm optical path length. The number of theoretical plates in the chromatography devices was determined by injecting 100 μL (i.e. 2% of bed volume) tracer solution. The retention volume (VR) and peak width at 50% height (w0.5) for a given operating condition was determined and the number of theoretical plates (N) was calculated as follows:
N = 5.545 ×
VR W0.5
Table 1 Comparison of salt peak attributes obtained with protein A containing cuboid packed-bed device and its equivalent column (bed volume: 5 mL, media: MabSelect SuRe resin, mobile phase: 0.4 M NaCl solution, tracer: 0.8 M NaCl solution, loop: 0.1 mL).
Column Cuboid Column Cuboid
R W0.1 L W0.1
Pab × 100% Pt
w0.5 (mL)
N¯ (/m)
10 10 0.5 0.5
7.36 6.82 6.58 6.28
2.07 1.6 1.45 1
2810 4027 4692 8750
± ± ± ±
AF 253 108 1078 252
1.24 0.95 1.25 1.06
± ± ± ±
0.11 0.07 0.17 0.14
2
(1)
Fig. 2. Single BSA flow through peaks at the flow rate of 10 mL/min using the protein A cuboid packed-bed device and its equivalent column. (packed-bed: 5 mL MabSelect SuRe resin, loop: 0.5 mL, sample: 2 mg/mL BSA, running buffer: 20 mM phosphate buffer, 150 mM NaCl, pH 7.2).
(2)
The recovery of mAb in the pooled fractions was determined by peak integration. The zero baseline during mAb elution was determined and the total peak area was considered to be 100%. Based, on this, the % recovery (R) of mAb in a given pool was determined by:
R=
VR (mL)
Standard deviation: Each device was repacked three times using the same protocol, and each incarnation was assessed at the two flow rates in triplicate.
The asymmetry factor (AF) was calculated based on the left and right half peak widths at 10% peak height (i.e. w0.1L and w0.1R):
AF =
Flow rate (mL/min)
(3)
where Pab is the area under the curve from the starting point a to the ending point b, and Pt is the total eluted peak area. The mAb concentration in a given pool (Cp) could be obtained as follows:
CP =
C0 Vo R VP
(4)
where C0 is the mAb concentration in the injected feed material, V0 is the volume of the feed material injected, and Vp is the pool volume. 3. Results Table 1 summarizes the attributes of the salt tracer peaks obtained with the protein A media containing cuboid packed-bed device and its equivalent column at two different flow rates, i.e. 10 and 0.5 mL/min. The data includes the retention volume (VR), peak width at half height (w0.5), the number of theoretical plates per meter bed height (N¯ ) and the asymmetry factor (AF). The reproducibility of protein A packing for the cuboid device and the column was verified by repacking each device three times, followed by efficiency testing of these in terms of the number of theoretical plates. The salt peaks obtained with the cuboid packed-bed device were narrower, higher and more symmetric and the (N¯ ) value was higher at both flow rates examined. For both devices, as
Fig. 3. Single BSA flow through peaks at the flow rate of 0.5 mL/min using the protein A cuboid packed-bed device and its equivalent column. (packed-bed: 5 mL MabSelect SuRe resin, loop: 0.5 mL, sample: 2 mg/mL BSA, running buffer: 20 mM phosphate buffer, 150 mM NaCl, pH 7.2).
the flow rate decreased from 10 mL/min to 0.5 mL/min, the (N¯ ) value increased. Figs. 2 and 3 show single BSA flow through peaks obtained with the protein A media containing cuboid packed-bed device and its equivalent column at flow rates of 10 and 0.5 mL/min respectively. In these 3
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Fig. 5. Separation of mAb-2 from impurities in cell culture supernatant (low load) using the protein A cuboid packed-bed device and its equivalent column. (packed-bed: 5 mL MabSelect SuRe resin, 10 mL/min for the flow through phase and 0.5 mL/min for the eluting phase, loop: 5 mL, mAb concentration in the sample: 1.035 mg/mL, binding buffer: 20 mM phosphate buffer, 150 mM NaCl, pH 7.2, eluting buffer: 0.1 M citrate buffer, pH 3.0, step change elution started 22 mL after injection).
Fig. 4. Separation of mAb-1 from BSA using the protein A cuboid packed-bed device and its equivalent column. (packed-bed: 5 mL MabSelect SuRe resin, 10 mL/min for the flow through phase and 0.5 mL/min for the eluting phase, loop: 0.5 mL, sample: 2 mg/mL BSA + 0.5 mg/mL mAb-1, binding buffer: 20 mM phosphate buffer, 150 mM NaCl, pH 7.2, eluting buffer: 0.1 M citrate buffer, pH 3.0, gradient started 19 mL after injection).
experiments, 0.5 mL of 2 mg/mL BSA solution was injected to obtain the simulated “impurity flow through” peak. At both flow rates, the BSA peak obtained with the cuboid packed-bed device was narrower and higher than that obtained with the column. Fig. 4 shows the chromatograms obtained during separation of mAb-1 from BSA using the protein A containing cuboid packed-bed device and its equivalent column. In these experiments, 0.5 mL of simulated feed solution containing 2 mg/mL BSA and 0.5 mg/mL mAb-1 was injected. These separation experiments were carried out using a combination of two different flow rates. A flow rate of 10 mL/min was used for equilibration, sample loading and chase. A chase volume of 19 mL was imposed between sample loading and initiation of mAb-1 elution to ensure that BSA was completely removed from the devices. During elution, which was carried out by step change to eluting buffer, the flow rate was decreased to 0.5 mL/min. The flow rate was reduced in order to obtain sharper eluted peaks to ensure that the dilution of mAb in the eluate was minimized. The eluted peak obtained with the cuboid packed-bed device was sharper and narrower, indicating the potential for obtaining a higher concentration of eluted mAb-1 in pooled fraction. Fig. 5 shows chromatograms obtained during protein A based affinity separation of mAb-2 from impurities present in CHO cell culture supernatant using the cuboid packed-bed device and its equivalent column. The flow rate combination strategy used in the previously reported experiments, i.e. 10 mL/min for equilibration, sample loading and chase, and 0.5 mL/min for elution was also employed in these experiments. The volume of cell culture media loaded was 5 mL and it had a mAb-2 concentration of 1.035 mg/mL. The high flow rate used during loading could potentially reduce the binding capacity of the protein A media to some extent. Therefore, a slightly lower protein loading was used. Due to the larger loading volume (i.e. 5 mL), a longer chase volume between loading and elution (i.e. 22 mL) was imposed to ensure that impurities such as host cell proteins, endotoxins and DNA present in the cell culture supernatant could be better removed before initiation of step change elution. As with mAb-1, the eluted mAb-2 peak obtained with the cuboid packed-bed device was sharper and higher, indicating a higher concentration of the eluted mAb in the pooled fraction. Table 2 summarizes the % recovery, volume and concentration of eluted mAb-2 peaks obtained from the experiments shown in Fig. 5. For peak cutting, the initial value for UV absorbance (at 280 nm) for the peak collection start point was arbitrarily set as 10 mAU and the final
Table 2 Recovery, pool volume and concentration of eluted mAb obtained from protein A packed cuboid packed-bed device and its equivalent column during purification of mAb-2 (bed volume: 5 mL, media: MabSelect SuRe resin, flow rate during impurity flow through: 10 mL/min, loop size: 5 mL, mAb concentration in the sample: 1.035 mg/mL, binding buffer: 20 mM phosphate buffer, 150 mM NaCl, pH 7.2, eluting buffer: 0.1 M citrate buffer, pH 3.0, gradient: Step-change initiated 22 mL after injection).
Column Cuboid
Flow rate during elution (mL/min)
Recovery (%)
Pool volume (mL)
Column volume (CV)
Concentration (mg/mL)
0.5 0.5 1 1.5 2 2.5
94.09% 94.73% 96.80% 96.43% 95.41% 95.05%
3.87 2.61 3.12 3.46 3.7 3.85
0.774 0.522 0.624 0.692 0.74 0.77
1.26 1.88 1.61 1.44 1.33 1.28
Peak collection start point: 10 mAU, peak collection end point: 50 mAU.
value for peak collection end point was set at 50 mAU. When elution was carried out at 0.5 mL/min flow rate, the recovery obtained from both devices were comparable. However, mAb-2 was eluted in a significantly smaller volume from the cuboid packed-bed device (i.e. 2.61 mL, i.e. 0.522 column volumes, as opposed to 3.87 mL, i.e. 0.774 column volumes). Therefore, mAb-2 concentration in the eluted peak obtained from the cuboid packed-bed device was correspondingly greater. More mAb-2 purification experiments were carried out with the cuboid packed bed device at different higher elution flow rates (i.e. 1.0, 1.5, 2.0 and 2.5 mL/min), by loading the same amount of CHO cell culture supernatant, i.e. 5 mL. The % recovery, volume and concentration of eluted mAb-2 data obtained from these experiments are also shown in Table 2. Fig. 6 shows an analysis of the effect of the cumulative volume of the pooled material collected from the eluted peaks shown in Fig. 5 on the % recovery of mAb-2. The start point for pool collection was arbitrarily set at 10 mAU. For similar volume of cumulatively pooled material, the % recovery was significantly greater with the cuboid packedbed device. Fig. 7 shows an analysis of the effect of the cumulative volume of the pooled material collected from the eluted peaks shown in Fig. 5 on the concentration of mAb-2. For similar volume of 4
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Fig. 8. Separation of mAb-2 from impurities in cell culture supernatant (high load) using the protein A cuboid packed-bed device and its equivalent column. (packed-bed: 5 mL MabSelect SuRe resin, 2 mL/min for loading and 5 mL/min for wash and elution, binding buffer: 20 mM phosphate buffer, 150 mM NaCl, pH 7.2, eluting buffer: 0.1 M citrate buffer, pH 3.0, loading: 80 mL 1.035 mg/ mL IgG1 containing CHO supernatant, wash: 100 mL using binding buffer, elution: 30 mL by step change to 100% eluting buffer).
Fig. 6. Recovery analysis of the eluted peaks from the cuboid and the column. (Peak collection start point: 10 mAU).
4. Discussion The data shown in Table 1 presented preliminary evidence on the superiority of the cuboid packed-bed device over its equivalent column. In recent studies [30–34], we have shown that a cuboid packed-bed device has several advantages in term of fluidics, such as greater uniformity in flow path length and residence time, and, superior flow distribution and collection. The sharper, narrower and more symmetric peaks, and the greater number of theoretical plates obtained with the cuboid packed-bed device could be attributed to its superior fluidic attributes which resulted in a narrower solute residence time distribution (RTD) than in a column [30–34]. In our earlier studies, the narrower RTD resulted in better resolution of multiple eluted peaks [30,31,34]. In the current study, the protein A based affinity purification process involved could be described as binary separation, i.e. the material present in the feed is segregated into two fractions, the flow through consisting of the impurities and the eluate consisting of the purified mAb. Therefore, the impact of the superior fluidics would be in terms of narrower and sharper flow through and eluted peaks. A sharper flow through peak would make it possible to initiate the elution process earlier, thereby making the overall separation faster. An earlier elution also implies that the mAb remains bound to the column for a smaller duration, thereby lowering the chances of on-column degradation. In the experimental results shown in Figs. 2–4, BSA represents a model impurity. These experiments were carried out to compare the cuboid packed-bed device and its equivalent column in terms of a simplified simulated monoclonal antibody purification process. The sharper BSA flow through peaks obtained with the cuboid device (see Figs. 2 and 3) was consistent with the effect of process parameters on flow through peaks discussed in one of our previous papers [33], where it was reported that the difference in efficiency between the cuboid device and the column was substantial when the loaded sample volume was lower than 20% of the device bed volume. At both flow rates examined, i.e. 10 and 0.5 mL/min, it took longer with the column for the UV absorbance to reach the baseline, the difference being greater at the lower flow rate on account of the greater difference in efficiency (see data shown in Table 1). However, carrying out the feed loading at 0.5 mL/min would tremendously slow down the overall purification process. Therefore, BSA/mAb-1 separation was carried out by loading
Fig. 7. Concentration analysis of the eluted peaks from the cuboid and the column. (Peak collection start point: 10 mAU).
cumulatively pooled material, the mAb-2 concentration was significantly greater with the cuboid packed-bed device. To further demonstrate the advantages of using the cuboid packedbed device, mAb-2 purification experiments were carried out by loading 80 mL of CHO cell culture supernatant. The concentration of mAb-2 in the feed was 1.035 mg/mL, and the total amount of the mAb loaded corresponded to about 80% of the 10% dynamic binding capacity (DBC) of the protein A media. The DBC value was determined from a break through curve of purified mAb-2 from a 1 mL column using the same buffer and at the same residence time (data not shown). The flow rate used during mAb-2 loading was 2 mL/min, while chase (with approximately 100 mL binding buffer) and elution (in step mode) was carried out at a flow rate of 5 mL/min. The chromatograms from these highload purification experiments are shown in Fig. 8. Following sample loading, the UV absorbance took considerably longer to reach the baseline with the column. Consistent with earlier results, the eluted mAb-2 peak was sharper and higher with the cuboid packed-bed device. Also, its area under the curve was greater, indicating higher mAb binding or recovery. In addition, the increase in UV absorbance at the end of the loading plateau obtained with the column presumably indicated mAb-2 loss in the flow through. 5
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the feed solution at 10 mL/min, the flow rate being decreased to 0.5 mL/min during elution (see Fig. 4). The lower flow rate during elution enabled collection of mAb-1 as a smaller and more concentrated fraction. This would make it easier to further process the monoclonal antibody obtained by protein A chromatography by different combinations of ion-exchange and hydrophobic interaction chromatography for polishing, i.e. removing trace impurities such as endotoxins, leached protein A and aggregates. Fig. 5 shows that sharper impurity flow through and eluted mAb-2 peaks could be obtained with the cuboid packed-bed device using the separation strategy used for BSA/mAb-1 separation, i.e. by loading and washing at 10 mL/min flow rate and eluting at 0.5 mL/min flow rate. The respective differences in peak attributes between the cuboid device and the column were even more significant than those observed in BSA/ mAb-1 separation. The impact of the difference in eluted peak attributes on the difference in separation efficiency of the two devices is clearly reflected in the data shown in Table 2. Complete collection of eluted mAb-2 (arbitrarily defined by 10 mAU collection start and 50 mAU collection end) could be achieved in a significantly lower volume with the cuboid packed-bed device. As explained in the previous paragraph, this would make it easier to further process the monoclonal antibody. The above mentioned advantages, i.e. smaller eluate volume and higher mAb-2 concentration in eluate are also evident from the data shown in Figs. 6-7. For instance, if the % recovery were to be set at 90%, the pooled eluate volume for the column would be more than 50% greater than that with the cuboid packed-bed device, and the mAb-2 concentration in the column eluate could be correspondingly lower. The chromatograms obtained from the high-load protein A affinity purification experiments shown in Fig. 8 highlight two very important factors. Firstly, the UV absorbance during the chase reached the baseline considerably faster with the cuboid packed-bed device, the difference in volume being more than 80 mL. This implied that the elution process could have been initiated much earlier with the cuboid packedbed device, leading to a significantly faster separation, less mAb-2 oncolumn residence, and significant savings in terms of buffer usage. The chromatograms shown in Fig. 8 also indicate that the effective mAb binding capacity was higher with the cuboid packed-bed device. When 80 mL of CHO cell culture supernatant was loaded on the column, some of the mAb-2 was lost in the flow through as indicated by the increase in UV absorbance from the flow through plateau at around 80 mL effluent volume. For the same volume of CHO cell culture supernatant loaded, there was no corresponding mAb-2 loss with the cuboid packed-bed device. The greater mAb-2 capture with the cuboid packed-bed is also evident from the greater area under the curve of the eluted peak. It should be noted that a flow rate of 2 mL/min (2.5 min residence time) during sample loading was used to speed up the process. This resulted in a slightly lower dynamic binding capacity (i.e. around 21 mg/mL resin) than that reported by the manufacturer for a typical 20 cm bed height column operated at a residence time of greater than 6 min (i.e. 30 mg/mL resin for IgG1). The above discussion establishes that the use of a cuboid packedbed device could significantly increase the efficiency of protein A affinity chromatography based monoclonal antibody purification. This device consistently outperformed its equivalent column in terms of all the separation efficiency metrics examined. Depending on how the protein A affinity purification process is designed and operated, the higher separation efficiency of the cuboid packed-bed device would lead to the following potential advantages: higher capture efficiency, faster separation, higher productivity, higher recovery, smaller pool volume, higher eluate concentration, lower buffer consumption, and shorter exposure to low pH environment and the consequent lower risk of mAb aggregation. Moreover, the high-efficiency separation capability at low bed height obtainable with the cuboid packed-bed device could potentially be useful for developing continuous multi-column purification processes for biologics purification. However, adoption of the cuboid device for industrial scale processes will only take place after
more thorough studies on process engineering parameters have been conducted. Such studies would typically involve investigation of alternative cuboid design variants, alternative flow distribution arrangements, and the investigation of pressure distribution, wall effect, and impact of dead corners on column sanitization in process-scale chromatography. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements We thank the Natural Science and Engineering Research Council (NSERC) of Canada for funding this research work. We thank Paul Gatt for fabricating the cuboid packed-bed device based on design provided by R.G. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jchromb.2019.121853. References [1] T. Waldmann, Monoclonal antibodies in diagnosis and therapy, Science 252 (1991) 1657–1662. [2] H.M. Shepard, G.L. Phillips, C.D. Thanos, M. Feldmann, Developments in therapy with monoclonal antibodies and related proteins, Clin. Med. 17 (2017) 220–232. [3] G. Dekkers, A.E.H. Bentlage, T.C. Stegmann, H.L. Howie, S. Lissenberg-Thunnissen, J. Zimring, T. Rispens, G. Vidarsson, Affinity of human IgG subclasses to mouse Fc gamma receptors, mAbs 9 (2017) 767–773. [4] N.A. Buss, S.J. Henderson, M. McFarlane, J.M. Shenton, L. de Haan, Monoclonal antibody therapeutics: history and future, Curr. Opin. Pharmacol. 12 (2012) 615–622. [5] S. Aldington, J. Bonnerjea, Scale-up of monoclonal antibody purification processes, J. Chromatogr. B. 848 (2007) 64–78. [6] A.D. Bandaranayake, S.C. Almo, Recent advances in mammalian protein production, FEBS Lett. 588 (2014) 253–260. [7] F. Li, N. Vijayasankaran, A. Shen, R. Kiss, A. Amanullah, Cell culture processes for monoclonal antibody production, mAbs 2 (2010) 466–477. [8] R.A. Rader, E.S. Langer, 30 years of upstream productivity improvements, BioProcess Int. 13 (2015). [9] P. Gronemeyer, R. Ditz, J. Strube, Trends in upstream and downstream process development for antibody manufacturing, Bioengineering 1 (2014) 188–212. [10] A.A. Shukla, L.S. Wolfe, S.S. Mostafa, C. Norman, Evolving trends in mAb production processes, Bioeng. Transl. Med. 2 (2017) 58–69. [11] A.A. Shukla, B. Hubbard, T. Tressel, S. Guhan, D. Low, Downstream processing of monoclonal antibodies—application of platform approaches, J. Chromatogr. B. 848 (2007) 28–39. [12] H.F. Liu, J. Ma, C. Winter, R. Bayer, Recovery and purification process development for monoclonal antibody production, mAbs 2 (2010) 480–499. [13] B. Kelley, Industrialization of mAb production technology: The bioprocessing industry at a crossroads, mAbs 1 (2009) 443–452. [14] A.L. Grilo, M. Mateus, M.R. Aires-Barros, A.M. Azevedo, Monoclonal antibodies production platforms: an opportunity study of a non-protein-A chromatographic platform based on process economics, Biotech. J. 12 (2017) Special Issue: AFOB Special Issue on Stem Cells in Tissue Engineering and Regenerative Medicine. [15] S. Ghose, B. Hubbard, S.M. Cramer, Evaluation and comparison of alternatives to Protein A chromatography: mimetic and hydrophobic charge induction chromatographic stationary phases, J. Chromatogr. A 1122 (2006) 144–152. [16] Y. Tao, A. Ibraheem, L. Conley, D. Cecchini, S. Ghose, Evaluation of high-capacity cation exchange chromatography for direct capture of monoclonal antibodies from high-titer cell culture processes, Biotechnol. Bioeng. 111 (2014) 1354–1364. [17] J. Pezzini, G. Joucla, R. Gantier, M. Toueille, A.-M. Lomenech, C. Le Sénéchal, B. Garbay, X. Santarelli, C. Cabanne, Antibody capture by mixed-mode chromatography: a comprehensive study from determination of optimal purification conditions to identification of contaminating host cell proteins, J. Chromatogr. A 1218 (2011) 8197–8208. [18] J. Ma, H. Hoang, T. Myint, T. Peram, R. Fahrner, J.H. Chou, Using precipitation by polyamines as an alternative to chromatographic separation in antibody purification processes, J. Chromatogr. B 878 (2010) 798–806. [19] A.M. Azevedo, P.A.J. Rosa, I.F. Ferreira, J. de Vries, T.J. Visser, M.R. Aires-Barros, Downstream processing of human antibodies integrating an extraction capture step and cation exchange chromatography, J. Chromatogr. B 877 (2009) 50–58.
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G. Chen, et al. [20] C. Boi, S. Dimartino, G.C. Sarti, Performance of a new protein A affinity membrane for the primary recovery of antibodies, Biotechnol. Progr. 24 (2008) 640–647. [21] D. Baur, M. Angarita, T. Müller-Späth, F. Steinebach, M. Morbidelli, Comparison of batch and continuous multi-column protein A capture processes by optimal design, Biotech. J. 11 (2016) 920–931. [22] Q.S. Yuan, A. Rosenfeld, T.W. Root, D.J. Klingenberg, E.N. Lightfoot, Flow distribution in chromatographic columns1, J. Chromatogr. A 831 (1999) 149–165. [23] C. Johnson, V. Natarajan, C. Antoniou, Evaluating two process scale chromatography column header designs using CFD, Biotechnol. Progr. 30 (2014) 837–844. [24] G.-H. Klepp, S. Böcker, J. Strube, H. Kansy, H. Schmale, Liquid distributor and liquid collector for chromatography columns, U.S. Patent 9383344 B2, 2016. [25] M. LePlang, D. Chabrol, Fluid distributor and device for treating a fluid such as a chromatograph equipped with said distributor, U.S. Patent 5141635 A, 1992. [26] W. Sun, D. Hu, X. Mei, X. Zhou, Influence of ring frit structure on column efficiency, Chromatographia 78 (2015) 467–472. [27] T. Besselink, A. van der Padt, A.E.M. Janssen, R.M. Boom, Are axial and radial flow chromatography different? J. Chromatogr. A 1271 (2013) 105–114. [28] M. Camenzuli, H.J. Ritchie, J.R. Ladine, R.A. Shalliker, Enhanced separation performance using a new column technology: parallel segmented outlet flow, J.
Chromatogr. A 1232 (2012) 47–51. [29] D. Foley, L. Pereira, M. Camenzuli, T. Edge, H. Ritchie, R.A. Shalliker, Curtain flow chromatography (‘the infinite diameter column’) with automated injection and high sample through-put: The results of an inter-laboratory study, Microchem. J. 110 (2013) 127–132. [30] R. Ghosh, Using a box instead of a column for process chromatography, J. Chromatogr. A 1468 (2016) 164–172. [31] R. Ghosh, G. Chen, Mathematical modelling and evaluation of performance of cuboid packed-bed devices for chromatographic separations, J. Chromatogr. A 1515 (2017) 138–145. [32] G. Chen, R. Ghosh, Effect of the length-to-width aspect ratio of a cuboid packed-bed device on efficiency of chromatographic separation, Processes 6 (2018) 160. [33] G. Chen, R. Ghosh, Effects of process parameters on the efficiency of chromatographic separations using a cuboid packed-bed device, J. Chromatogr. B 1086 (2018) 23–28. [34] G. Chen, A. Gerrior, R. Ghosh, Feasibility study for high-resolution multi-component separation of protein mixture using a cation-exchange cuboid packed-bed device, J. Chromatogr. A 1549 (2018) 25–30.
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/jchromb
Efficient capture of monoclonal antibody from cell culture supernatant using protein A media contained in a cuboid packed-bed device
T
Guoqiang Chena, Alisha Gerriora, Yves Durocherb,c, Raja Ghosha,
⁎
a
Department of Chemical Engineering, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S 4L7, Canada National Research Council of Canada, Montreal, QC H4P 2R2, Canada c Département de biochimie et médecine moléculaire, Faculté de médecine, Université de Montréal, Montreal, QC H3C 3J7, Canada b
ARTICLE INFO
ABSTRACT
Keywords: Column Cuboid packed-bed Chromatography Capture Monoclonal antibody Protein A Affinity chromatography
A box-shaped or cuboid packed-bed device was used for monoclonal antibody (mAb) separation using protein A affinity chromatography. The separation efficiency of the device was compared with an equivalent column i.e. packed with same resin, and having identical bed height and bed volume. The protein A media packed cuboid device had a larger number of theoretical plates than its equivalent column, e.g. 8750/m as opposed to about 4700/m at a flow rate of 0.5 mL/min. In mAb purification experiments, the impurity flow-through and eluted mAb peaks were shaper with the cuboid device. This implied that the effective separation time and buffer consumption with this device was lower, the purified mAb pooled volume was smaller, and the mAb concentration in the pooled volume was greater. Equivalent separation efficiency could be obtained with the cuboid device using higher flow rates than that used with the column. For instance, elution peaks equivalent to those obtainable by the column could be obtained at a 5 times greater flow rate using the cuboid device. The results discussed in this paper clearly demonstrate the potential for improving the efficiency of protein A affinity chromatography based mAb purification by using a cuboid packed-bed device.
1. Introduction Monoclonal antibodies (mAbs) are widely used to diagnose and treat cancer and infectious diseases, as well as to modulate immune responses [1,2]. Most mAbs used as biopharmaceuticals belong to the IgG1 subclass [3,4]. Compared to other biopharmaceutical proteins or even small molecule drugs, the therapeutic mAbs are administered in quite large doses [5]. Mammalian cells such as Chinese Hamster Ovary (CHO) cells and NS0 (mouse myeloma) cells are generally used to make mAbs [6]. Spectacular developments in upstream processing over the past decades have resulted in increase in mAb titers from less than 0.5 g/L to 10 g/L [7,8]. The challenge in mAb production has therefore shifted from upstream to downstream processing, primarily due to high purification cost [9,10]. The biopharmaceutical industry employs a platform downstream process for mAbs which consists of protein A affinity chromatography, low pH virus inactivation, polishing steps, virus filtration, and ultrafiltration/diafiltration for formulation [10–13]. Although the specific sequence and type of polishing and further processing techniques used vary from manufacturer to manufacturer, protein A chromatography is nearly always employed as the primary mAb capture step [10] due to its high selectivity [11].
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However, protein A affinity media is very expensive and this has been flagged as a major limitation [11,14]. The motivation to reduce purification cost has led to search for alternatives to protein A affinity chromatography. Techniques that have been considered include protein A mimetic chromatography [15], hydrophobic charge induction chromatography [15], cation exchange chromatography [16], mixed mode chromatography [17], as well as non-chromatographic methods such as precipitation [18], aqueous twophase system [19], and protein A affinity membrane chromatography [20]. However, most of these techniques give considerably lower selectivity and several of these need to be used in combination. Therefore, the advantage of the lower media cost is often offset by the elaborate and complex nature of these alternative separation processes as well as additional time required for development and retrofitting of existing platform processes. A more practical approach would therefore be to use the protein A affinity media more efficiently. For instance, a continuous multi-column based protein A chromatography could improve the mAb binding capacity utilization [21]. The problem of flow maldistribution in columns could become a severe limiting factor when designing and developing such advanced separation systems. More generally, better devices and processes could improve the efficiency of
Corresponding author. E-mail address: [email protected] (R. Ghosh).
https://doi.org/10.1016/j.jchromb.2019.121853 Received 20 June 2019; Received in revised form 27 September 2019; Accepted 28 October 2019 Available online 12 November 2019 1570-0232/ Crown Copyright © 2019 Published by Elsevier B.V. All rights reserved.
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Fig. 1. The cuboid packed-bed device used in this study (A) photograph (left) and schematic diagram (right), and (B) plan view of components.
asymmetry factor were systematically compared. These results clearly demonstrated the advantages of using the protein A cuboid packed-bed device over its equivalent column.
protein A affinity chromatography and bring down the manufacturing cost. The importance of chromatography device is gradually being recognized by the more and more researchers. The term chromatography immediately conjures up the image of a resin-packed column. However, such traditional columns are not always best suited for efficient separation. Non-uniform packing and column flow maldistribution frequently lead to low efficiency and poorly resolved peaks [22–29]. The problem of flow maldistribution is generally addressed through header modification [22,23], or improvement in flow distributor/collector/frit design [24–26]. Alternative column types such as radial flow [27], parallel segmented flow [28] and curtain flow [29] columns have also been proposed. Our research group has been actively involved with the design and development of cuboid packed-bed devices which are suitable for efficient chromatographic separations [30–34]. In such a device, a box-shaped resin-bed is fed from an upper lateral channel and is drained using a complimentary lower channel (see Fig. 1A). Such fluidic arrangement results in a narrow residence time distribution (RTD) within the device, and thereby efficient chromatographic separation [30,31]. The effects of the device design specifics such as the cuboid length to width ratio [32] and process parameters [33] on separation efficiency have been examined. The use of cuboid packed-bed devices for high-resolution separation of protein mixtures using cation and anion exchange resins have been reported [30,31,34]. In the current, we examine if a cuboid packed-bed device could be used to increase the efficiency of protein A affinity chromatography. Protein A affinity resin containing cuboid packed-bed device (Fig. 1A) and its equivalent column (i.e. having similar bed-height and bed-volume) were first used to separate the mAb from model impurity bovine serum albumin (BSA) for preliminary comparison. These devices were then used to capture the mAb from cell culture supernatant. Process efficiency metrics such as the height and shape of flow through impurity and eluted mAb peaks, mAb recovery and concentration in pooled fractions were compared. Effects of process parameters such as flow rate and loading volume were examined. Chromatographic separation attributes such as the number of theoretical plates and
2. Materials and methods Sodium phosphate monobasic (S0751), sodium phosphate dibasic (S0876), citric acid (C0759), sodium citrate tribasic (S4641), and bovine serum albumin (BSA, A7906) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium chloride (SOD002.205) was purchased from Bioshop (Burlington, ON, Canada). Monoclonal antibody hIgG1CD4 (Batch 12, stock concentration: 10 mg in 2.1 mL) was kindly donated by the Therapeutic Antibody Center, University of Oxford, Oxfordshire, UK. This monoclonal antibody will be referred to as mAb-1 in the paper. Monoclonal antibody Trastuzumab containing cell culture supernatant (lot: 20170920BC), produced using Chinese Hamster Ovary (CHO) cell line was kindly donated by the National Research Council Canada, Montreal, QC, Canada. This monoclonal antibody will be referred to as mAb-2 in this paper. Both mAb-1 and mAb-2 were IgG1 antibodies. Ultrapure water was obtained from a Diamond NANOpure water purification unit (Barnstead, IA, USA). Millipore Vantage® L Laboratory Column VL 16 × 250 (product number 96160250) was kindly provided by PlantForm Corporation, Guelph, ON, Canada. Protein A affinity MabSelect SuRe resin (17-5438-03) was procured from GE Healthcare Biosciences, QC, Canada. The number of theoretical plates in the cuboid packed-bed device and its equivalent column was determined using 0.4 M NaCl solution as the mobile phase and 0.8 M NaCl solution as the tracer. The buffers utilized for the affinity chromatography experiments were 20 mM phosphate buffer, 150 mM NaCl, pH 7.2 (binding buffer) and 0.1 M citrate buffer, pH 3.0 (eluting buffer) respectively. The NaCl solutions and buffers were filtered and degassed before use. Protein samples (BSA, mAb-1 solutions and their mixture) was prepared in the binding buffer. The CHO cell culture supernatant used in the mAb-2 purification experiment was directly loaded on the chromatography device without 2
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modification. The dimensions of the packed-bed within the cuboid device used in this study were 20 mm (length) × 10 mm (width) × 25 mm (height). The drawings of the device is shown in Fig. 1B. The design features of the device were similar to those used in our previous studies [30,31,33,34], except that the tapered portions on the vent sides of the upper and lower channels were removed to reduce the dead volume. The external length and width of the three components comprising the cuboid device, i.e. the top plate (acrylic), the central frame (polyvinyl chloride, PVC) and bottom plate (acrylic) were 80 mm and 30 mm respectively. The top and bottom plates were provided with pillared lateral channels to facilitate flow distribution. The chromatographic media was retained within the central frame using stainless steel mesh on both sides (0.0017 in. opening, 9319T188, McMaster Carr, USA) on each side. MabSelect SuRe resins were packed in the cuboid device and the equivalent column to obtain identical bed height (25 mm) and bed volume (5 mL). During the chromatography experiments, the cuboid device or the column was connected to an AKTA prime liquid chromatography system (GE Healthcare Biosciences, QC, Canada) using the same set of connecting tubings to keep the extra-device volume the same. The UV absorbance was monitored using a UV detector having 2 mm optical path length. The number of theoretical plates in the chromatography devices was determined by injecting 100 μL (i.e. 2% of bed volume) tracer solution. The retention volume (VR) and peak width at 50% height (w0.5) for a given operating condition was determined and the number of theoretical plates (N) was calculated as follows:
N = 5.545 ×
VR W0.5
Table 1 Comparison of salt peak attributes obtained with protein A containing cuboid packed-bed device and its equivalent column (bed volume: 5 mL, media: MabSelect SuRe resin, mobile phase: 0.4 M NaCl solution, tracer: 0.8 M NaCl solution, loop: 0.1 mL).
Column Cuboid Column Cuboid
R W0.1 L W0.1
Pab × 100% Pt
w0.5 (mL)
N¯ (/m)
10 10 0.5 0.5
7.36 6.82 6.58 6.28
2.07 1.6 1.45 1
2810 4027 4692 8750
± ± ± ±
AF 253 108 1078 252
1.24 0.95 1.25 1.06
± ± ± ±
0.11 0.07 0.17 0.14
2
(1)
Fig. 2. Single BSA flow through peaks at the flow rate of 10 mL/min using the protein A cuboid packed-bed device and its equivalent column. (packed-bed: 5 mL MabSelect SuRe resin, loop: 0.5 mL, sample: 2 mg/mL BSA, running buffer: 20 mM phosphate buffer, 150 mM NaCl, pH 7.2).
(2)
The recovery of mAb in the pooled fractions was determined by peak integration. The zero baseline during mAb elution was determined and the total peak area was considered to be 100%. Based, on this, the % recovery (R) of mAb in a given pool was determined by:
R=
VR (mL)
Standard deviation: Each device was repacked three times using the same protocol, and each incarnation was assessed at the two flow rates in triplicate.
The asymmetry factor (AF) was calculated based on the left and right half peak widths at 10% peak height (i.e. w0.1L and w0.1R):
AF =
Flow rate (mL/min)
(3)
where Pab is the area under the curve from the starting point a to the ending point b, and Pt is the total eluted peak area. The mAb concentration in a given pool (Cp) could be obtained as follows:
CP =
C0 Vo R VP
(4)
where C0 is the mAb concentration in the injected feed material, V0 is the volume of the feed material injected, and Vp is the pool volume. 3. Results Table 1 summarizes the attributes of the salt tracer peaks obtained with the protein A media containing cuboid packed-bed device and its equivalent column at two different flow rates, i.e. 10 and 0.5 mL/min. The data includes the retention volume (VR), peak width at half height (w0.5), the number of theoretical plates per meter bed height (N¯ ) and the asymmetry factor (AF). The reproducibility of protein A packing for the cuboid device and the column was verified by repacking each device three times, followed by efficiency testing of these in terms of the number of theoretical plates. The salt peaks obtained with the cuboid packed-bed device were narrower, higher and more symmetric and the (N¯ ) value was higher at both flow rates examined. For both devices, as
Fig. 3. Single BSA flow through peaks at the flow rate of 0.5 mL/min using the protein A cuboid packed-bed device and its equivalent column. (packed-bed: 5 mL MabSelect SuRe resin, loop: 0.5 mL, sample: 2 mg/mL BSA, running buffer: 20 mM phosphate buffer, 150 mM NaCl, pH 7.2).
the flow rate decreased from 10 mL/min to 0.5 mL/min, the (N¯ ) value increased. Figs. 2 and 3 show single BSA flow through peaks obtained with the protein A media containing cuboid packed-bed device and its equivalent column at flow rates of 10 and 0.5 mL/min respectively. In these 3
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Fig. 5. Separation of mAb-2 from impurities in cell culture supernatant (low load) using the protein A cuboid packed-bed device and its equivalent column. (packed-bed: 5 mL MabSelect SuRe resin, 10 mL/min for the flow through phase and 0.5 mL/min for the eluting phase, loop: 5 mL, mAb concentration in the sample: 1.035 mg/mL, binding buffer: 20 mM phosphate buffer, 150 mM NaCl, pH 7.2, eluting buffer: 0.1 M citrate buffer, pH 3.0, step change elution started 22 mL after injection).
Fig. 4. Separation of mAb-1 from BSA using the protein A cuboid packed-bed device and its equivalent column. (packed-bed: 5 mL MabSelect SuRe resin, 10 mL/min for the flow through phase and 0.5 mL/min for the eluting phase, loop: 0.5 mL, sample: 2 mg/mL BSA + 0.5 mg/mL mAb-1, binding buffer: 20 mM phosphate buffer, 150 mM NaCl, pH 7.2, eluting buffer: 0.1 M citrate buffer, pH 3.0, gradient started 19 mL after injection).
experiments, 0.5 mL of 2 mg/mL BSA solution was injected to obtain the simulated “impurity flow through” peak. At both flow rates, the BSA peak obtained with the cuboid packed-bed device was narrower and higher than that obtained with the column. Fig. 4 shows the chromatograms obtained during separation of mAb-1 from BSA using the protein A containing cuboid packed-bed device and its equivalent column. In these experiments, 0.5 mL of simulated feed solution containing 2 mg/mL BSA and 0.5 mg/mL mAb-1 was injected. These separation experiments were carried out using a combination of two different flow rates. A flow rate of 10 mL/min was used for equilibration, sample loading and chase. A chase volume of 19 mL was imposed between sample loading and initiation of mAb-1 elution to ensure that BSA was completely removed from the devices. During elution, which was carried out by step change to eluting buffer, the flow rate was decreased to 0.5 mL/min. The flow rate was reduced in order to obtain sharper eluted peaks to ensure that the dilution of mAb in the eluate was minimized. The eluted peak obtained with the cuboid packed-bed device was sharper and narrower, indicating the potential for obtaining a higher concentration of eluted mAb-1 in pooled fraction. Fig. 5 shows chromatograms obtained during protein A based affinity separation of mAb-2 from impurities present in CHO cell culture supernatant using the cuboid packed-bed device and its equivalent column. The flow rate combination strategy used in the previously reported experiments, i.e. 10 mL/min for equilibration, sample loading and chase, and 0.5 mL/min for elution was also employed in these experiments. The volume of cell culture media loaded was 5 mL and it had a mAb-2 concentration of 1.035 mg/mL. The high flow rate used during loading could potentially reduce the binding capacity of the protein A media to some extent. Therefore, a slightly lower protein loading was used. Due to the larger loading volume (i.e. 5 mL), a longer chase volume between loading and elution (i.e. 22 mL) was imposed to ensure that impurities such as host cell proteins, endotoxins and DNA present in the cell culture supernatant could be better removed before initiation of step change elution. As with mAb-1, the eluted mAb-2 peak obtained with the cuboid packed-bed device was sharper and higher, indicating a higher concentration of the eluted mAb in the pooled fraction. Table 2 summarizes the % recovery, volume and concentration of eluted mAb-2 peaks obtained from the experiments shown in Fig. 5. For peak cutting, the initial value for UV absorbance (at 280 nm) for the peak collection start point was arbitrarily set as 10 mAU and the final
Table 2 Recovery, pool volume and concentration of eluted mAb obtained from protein A packed cuboid packed-bed device and its equivalent column during purification of mAb-2 (bed volume: 5 mL, media: MabSelect SuRe resin, flow rate during impurity flow through: 10 mL/min, loop size: 5 mL, mAb concentration in the sample: 1.035 mg/mL, binding buffer: 20 mM phosphate buffer, 150 mM NaCl, pH 7.2, eluting buffer: 0.1 M citrate buffer, pH 3.0, gradient: Step-change initiated 22 mL after injection).
Column Cuboid
Flow rate during elution (mL/min)
Recovery (%)
Pool volume (mL)
Column volume (CV)
Concentration (mg/mL)
0.5 0.5 1 1.5 2 2.5
94.09% 94.73% 96.80% 96.43% 95.41% 95.05%
3.87 2.61 3.12 3.46 3.7 3.85
0.774 0.522 0.624 0.692 0.74 0.77
1.26 1.88 1.61 1.44 1.33 1.28
Peak collection start point: 10 mAU, peak collection end point: 50 mAU.
value for peak collection end point was set at 50 mAU. When elution was carried out at 0.5 mL/min flow rate, the recovery obtained from both devices were comparable. However, mAb-2 was eluted in a significantly smaller volume from the cuboid packed-bed device (i.e. 2.61 mL, i.e. 0.522 column volumes, as opposed to 3.87 mL, i.e. 0.774 column volumes). Therefore, mAb-2 concentration in the eluted peak obtained from the cuboid packed-bed device was correspondingly greater. More mAb-2 purification experiments were carried out with the cuboid packed bed device at different higher elution flow rates (i.e. 1.0, 1.5, 2.0 and 2.5 mL/min), by loading the same amount of CHO cell culture supernatant, i.e. 5 mL. The % recovery, volume and concentration of eluted mAb-2 data obtained from these experiments are also shown in Table 2. Fig. 6 shows an analysis of the effect of the cumulative volume of the pooled material collected from the eluted peaks shown in Fig. 5 on the % recovery of mAb-2. The start point for pool collection was arbitrarily set at 10 mAU. For similar volume of cumulatively pooled material, the % recovery was significantly greater with the cuboid packedbed device. Fig. 7 shows an analysis of the effect of the cumulative volume of the pooled material collected from the eluted peaks shown in Fig. 5 on the concentration of mAb-2. For similar volume of 4
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Fig. 8. Separation of mAb-2 from impurities in cell culture supernatant (high load) using the protein A cuboid packed-bed device and its equivalent column. (packed-bed: 5 mL MabSelect SuRe resin, 2 mL/min for loading and 5 mL/min for wash and elution, binding buffer: 20 mM phosphate buffer, 150 mM NaCl, pH 7.2, eluting buffer: 0.1 M citrate buffer, pH 3.0, loading: 80 mL 1.035 mg/ mL IgG1 containing CHO supernatant, wash: 100 mL using binding buffer, elution: 30 mL by step change to 100% eluting buffer).
Fig. 6. Recovery analysis of the eluted peaks from the cuboid and the column. (Peak collection start point: 10 mAU).
4. Discussion The data shown in Table 1 presented preliminary evidence on the superiority of the cuboid packed-bed device over its equivalent column. In recent studies [30–34], we have shown that a cuboid packed-bed device has several advantages in term of fluidics, such as greater uniformity in flow path length and residence time, and, superior flow distribution and collection. The sharper, narrower and more symmetric peaks, and the greater number of theoretical plates obtained with the cuboid packed-bed device could be attributed to its superior fluidic attributes which resulted in a narrower solute residence time distribution (RTD) than in a column [30–34]. In our earlier studies, the narrower RTD resulted in better resolution of multiple eluted peaks [30,31,34]. In the current study, the protein A based affinity purification process involved could be described as binary separation, i.e. the material present in the feed is segregated into two fractions, the flow through consisting of the impurities and the eluate consisting of the purified mAb. Therefore, the impact of the superior fluidics would be in terms of narrower and sharper flow through and eluted peaks. A sharper flow through peak would make it possible to initiate the elution process earlier, thereby making the overall separation faster. An earlier elution also implies that the mAb remains bound to the column for a smaller duration, thereby lowering the chances of on-column degradation. In the experimental results shown in Figs. 2–4, BSA represents a model impurity. These experiments were carried out to compare the cuboid packed-bed device and its equivalent column in terms of a simplified simulated monoclonal antibody purification process. The sharper BSA flow through peaks obtained with the cuboid device (see Figs. 2 and 3) was consistent with the effect of process parameters on flow through peaks discussed in one of our previous papers [33], where it was reported that the difference in efficiency between the cuboid device and the column was substantial when the loaded sample volume was lower than 20% of the device bed volume. At both flow rates examined, i.e. 10 and 0.5 mL/min, it took longer with the column for the UV absorbance to reach the baseline, the difference being greater at the lower flow rate on account of the greater difference in efficiency (see data shown in Table 1). However, carrying out the feed loading at 0.5 mL/min would tremendously slow down the overall purification process. Therefore, BSA/mAb-1 separation was carried out by loading
Fig. 7. Concentration analysis of the eluted peaks from the cuboid and the column. (Peak collection start point: 10 mAU).
cumulatively pooled material, the mAb-2 concentration was significantly greater with the cuboid packed-bed device. To further demonstrate the advantages of using the cuboid packedbed device, mAb-2 purification experiments were carried out by loading 80 mL of CHO cell culture supernatant. The concentration of mAb-2 in the feed was 1.035 mg/mL, and the total amount of the mAb loaded corresponded to about 80% of the 10% dynamic binding capacity (DBC) of the protein A media. The DBC value was determined from a break through curve of purified mAb-2 from a 1 mL column using the same buffer and at the same residence time (data not shown). The flow rate used during mAb-2 loading was 2 mL/min, while chase (with approximately 100 mL binding buffer) and elution (in step mode) was carried out at a flow rate of 5 mL/min. The chromatograms from these highload purification experiments are shown in Fig. 8. Following sample loading, the UV absorbance took considerably longer to reach the baseline with the column. Consistent with earlier results, the eluted mAb-2 peak was sharper and higher with the cuboid packed-bed device. Also, its area under the curve was greater, indicating higher mAb binding or recovery. In addition, the increase in UV absorbance at the end of the loading plateau obtained with the column presumably indicated mAb-2 loss in the flow through. 5
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the feed solution at 10 mL/min, the flow rate being decreased to 0.5 mL/min during elution (see Fig. 4). The lower flow rate during elution enabled collection of mAb-1 as a smaller and more concentrated fraction. This would make it easier to further process the monoclonal antibody obtained by protein A chromatography by different combinations of ion-exchange and hydrophobic interaction chromatography for polishing, i.e. removing trace impurities such as endotoxins, leached protein A and aggregates. Fig. 5 shows that sharper impurity flow through and eluted mAb-2 peaks could be obtained with the cuboid packed-bed device using the separation strategy used for BSA/mAb-1 separation, i.e. by loading and washing at 10 mL/min flow rate and eluting at 0.5 mL/min flow rate. The respective differences in peak attributes between the cuboid device and the column were even more significant than those observed in BSA/ mAb-1 separation. The impact of the difference in eluted peak attributes on the difference in separation efficiency of the two devices is clearly reflected in the data shown in Table 2. Complete collection of eluted mAb-2 (arbitrarily defined by 10 mAU collection start and 50 mAU collection end) could be achieved in a significantly lower volume with the cuboid packed-bed device. As explained in the previous paragraph, this would make it easier to further process the monoclonal antibody. The above mentioned advantages, i.e. smaller eluate volume and higher mAb-2 concentration in eluate are also evident from the data shown in Figs. 6-7. For instance, if the % recovery were to be set at 90%, the pooled eluate volume for the column would be more than 50% greater than that with the cuboid packed-bed device, and the mAb-2 concentration in the column eluate could be correspondingly lower. The chromatograms obtained from the high-load protein A affinity purification experiments shown in Fig. 8 highlight two very important factors. Firstly, the UV absorbance during the chase reached the baseline considerably faster with the cuboid packed-bed device, the difference in volume being more than 80 mL. This implied that the elution process could have been initiated much earlier with the cuboid packedbed device, leading to a significantly faster separation, less mAb-2 oncolumn residence, and significant savings in terms of buffer usage. The chromatograms shown in Fig. 8 also indicate that the effective mAb binding capacity was higher with the cuboid packed-bed device. When 80 mL of CHO cell culture supernatant was loaded on the column, some of the mAb-2 was lost in the flow through as indicated by the increase in UV absorbance from the flow through plateau at around 80 mL effluent volume. For the same volume of CHO cell culture supernatant loaded, there was no corresponding mAb-2 loss with the cuboid packed-bed device. The greater mAb-2 capture with the cuboid packed-bed is also evident from the greater area under the curve of the eluted peak. It should be noted that a flow rate of 2 mL/min (2.5 min residence time) during sample loading was used to speed up the process. This resulted in a slightly lower dynamic binding capacity (i.e. around 21 mg/mL resin) than that reported by the manufacturer for a typical 20 cm bed height column operated at a residence time of greater than 6 min (i.e. 30 mg/mL resin for IgG1). The above discussion establishes that the use of a cuboid packedbed device could significantly increase the efficiency of protein A affinity chromatography based monoclonal antibody purification. This device consistently outperformed its equivalent column in terms of all the separation efficiency metrics examined. Depending on how the protein A affinity purification process is designed and operated, the higher separation efficiency of the cuboid packed-bed device would lead to the following potential advantages: higher capture efficiency, faster separation, higher productivity, higher recovery, smaller pool volume, higher eluate concentration, lower buffer consumption, and shorter exposure to low pH environment and the consequent lower risk of mAb aggregation. Moreover, the high-efficiency separation capability at low bed height obtainable with the cuboid packed-bed device could potentially be useful for developing continuous multi-column purification processes for biologics purification. However, adoption of the cuboid device for industrial scale processes will only take place after
more thorough studies on process engineering parameters have been conducted. Such studies would typically involve investigation of alternative cuboid design variants, alternative flow distribution arrangements, and the investigation of pressure distribution, wall effect, and impact of dead corners on column sanitization in process-scale chromatography. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements We thank the Natural Science and Engineering Research Council (NSERC) of Canada for funding this research work. We thank Paul Gatt for fabricating the cuboid packed-bed device based on design provided by R.G. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jchromb.2019.121853. References [1] T. Waldmann, Monoclonal antibodies in diagnosis and therapy, Science 252 (1991) 1657–1662. [2] H.M. Shepard, G.L. Phillips, C.D. Thanos, M. Feldmann, Developments in therapy with monoclonal antibodies and related proteins, Clin. Med. 17 (2017) 220–232. [3] G. Dekkers, A.E.H. Bentlage, T.C. Stegmann, H.L. Howie, S. Lissenberg-Thunnissen, J. Zimring, T. Rispens, G. Vidarsson, Affinity of human IgG subclasses to mouse Fc gamma receptors, mAbs 9 (2017) 767–773. [4] N.A. Buss, S.J. Henderson, M. McFarlane, J.M. Shenton, L. de Haan, Monoclonal antibody therapeutics: history and future, Curr. Opin. Pharmacol. 12 (2012) 615–622. [5] S. Aldington, J. Bonnerjea, Scale-up of monoclonal antibody purification processes, J. Chromatogr. B. 848 (2007) 64–78. [6] A.D. Bandaranayake, S.C. Almo, Recent advances in mammalian protein production, FEBS Lett. 588 (2014) 253–260. [7] F. Li, N. Vijayasankaran, A. Shen, R. Kiss, A. Amanullah, Cell culture processes for monoclonal antibody production, mAbs 2 (2010) 466–477. [8] R.A. Rader, E.S. Langer, 30 years of upstream productivity improvements, BioProcess Int. 13 (2015). [9] P. Gronemeyer, R. Ditz, J. Strube, Trends in upstream and downstream process development for antibody manufacturing, Bioengineering 1 (2014) 188–212. [10] A.A. Shukla, L.S. Wolfe, S.S. Mostafa, C. Norman, Evolving trends in mAb production processes, Bioeng. Transl. Med. 2 (2017) 58–69. [11] A.A. Shukla, B. Hubbard, T. Tressel, S. Guhan, D. Low, Downstream processing of monoclonal antibodies—application of platform approaches, J. Chromatogr. B. 848 (2007) 28–39. [12] H.F. Liu, J. Ma, C. Winter, R. Bayer, Recovery and purification process development for monoclonal antibody production, mAbs 2 (2010) 480–499. [13] B. Kelley, Industrialization of mAb production technology: The bioprocessing industry at a crossroads, mAbs 1 (2009) 443–452. [14] A.L. Grilo, M. Mateus, M.R. Aires-Barros, A.M. Azevedo, Monoclonal antibodies production platforms: an opportunity study of a non-protein-A chromatographic platform based on process economics, Biotech. J. 12 (2017) Special Issue: AFOB Special Issue on Stem Cells in Tissue Engineering and Regenerative Medicine. [15] S. Ghose, B. Hubbard, S.M. Cramer, Evaluation and comparison of alternatives to Protein A chromatography: mimetic and hydrophobic charge induction chromatographic stationary phases, J. Chromatogr. A 1122 (2006) 144–152. [16] Y. Tao, A. Ibraheem, L. Conley, D. Cecchini, S. Ghose, Evaluation of high-capacity cation exchange chromatography for direct capture of monoclonal antibodies from high-titer cell culture processes, Biotechnol. Bioeng. 111 (2014) 1354–1364. [17] J. Pezzini, G. Joucla, R. Gantier, M. Toueille, A.-M. Lomenech, C. Le Sénéchal, B. Garbay, X. Santarelli, C. Cabanne, Antibody capture by mixed-mode chromatography: a comprehensive study from determination of optimal purification conditions to identification of contaminating host cell proteins, J. Chromatogr. A 1218 (2011) 8197–8208. [18] J. Ma, H. Hoang, T. Myint, T. Peram, R. Fahrner, J.H. Chou, Using precipitation by polyamines as an alternative to chromatographic separation in antibody purification processes, J. Chromatogr. B 878 (2010) 798–806. [19] A.M. Azevedo, P.A.J. Rosa, I.F. Ferreira, J. de Vries, T.J. Visser, M.R. Aires-Barros, Downstream processing of human antibodies integrating an extraction capture step and cation exchange chromatography, J. Chromatogr. B 877 (2009) 50–58.
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