5 Monoclonal Antibody Capture Case Study
Monoclonal antibody (IgG1 with 1.2 mg/mL titre) was captured from clarified cell culture harvest using the CaptureSMB process [1]. The process was run on Contichrom® equipment from ChromaCon, using the ChromIQ®
Lab-10 operating software.
Two columns of 0.5 cm inner diameter and 10 cm length were packed with AmsphereTM JWT-203 protein A (JSR Life Sciences), 1 column volume (CV) was 2.0 mL. One UV detector was mounted at the outlet of each column.
With respect to other impurities like Host Cell Proteins (HCP) or DNA, the purity is expressed in terms of amount impurity per amount of product, e.g. [ng HCP/ mg mAb] or [ppm HCP] and is typically determined by ELISA or other fluorescent assays.
The process yield Y is defined as the ratio of the product mass recovered in the product pool mpool and the product mass supplied through the feed mfeed, within one cycle. In CaptureSMB chromatography, the yield is measured after the startup cycle, i.e. when the process has started up and the UV profiles do not change anymore from cycle to cycle (after the startup cycle):
The recovery and regeneration protocol had been determined previously through single column batch capture runs and is reported in Table 1. Buffer A was 20 mM Phos, 150 mM NaCl, pH 7.5; buffer B was 20 mM Phos, 1M NaCl, pH 7.5; buffer C was 50 mM Na-Cit, pH 3.2; and buffer D was 0.1 M NaOH. The flow rate was 1 mL/min (300 cm/h) except for the CIP step were it was 0.33 mL/min (100 cm/h).
Table 1. Recovery and regeneration protocol of batch chromatography Step
Wash 1 Wash 2 Wash 3 Elution CIP
The mass balance closure mb is defined as the ratio of the product mass leaving the system mout including all outlet streams and the product mass entering the system via the feed within the same time period (mfeed). In batch and CaptureSMB chromatography (after the startup cycle) the mass balance closure is typically referred to one cycle:
Equilibrate 1 Equilibrate 2
Buffer A B
A C D C A
Number of column volumes [CV] 2 5 5 5
7.5 (100 cm/h) 2 3
The product pool concentration was determined using analytical Protein A chromatography (Poros A20 column, 2.1 mm x 30 mm, Life technologies), aggregate content was measured by size exclusion chromatography (TSK-Gel G3000SWXL, 7.8 mm x 300 mm, Tosoh), and HCP values were determined using a CHO-HCP ELISA kit (# F550, Cygnustechnologies), and DNA content was quantified by fluorescence (Quant-iTTM PicoGreen®
The CaptureSMB operating parameters were determined from a breakthrough curve of clarified harvest with a single column (10 cm length, feed flow rate of 300 cm/h). The ChromIQ®
operating software of the Contichrom®
The load L is defined as the ratio between product mass in the feed and the total bed volume Vcol (in CaptureSMB including all columns), measured within the same time period (typically one cycle):
equipment platform allows for fully automated determination of CaptureSMB operating parameters.
Essentially, the calculations include the determination of the amount of mAb that is contained in the upstream and downstream columns, respectively, in dependence of the amount loaded on the two interconnected columns. In the presented case, the columns were loaded until 70% of the feed concentration was reached at the column outlet of the upstream column (determined by offline fraction analysis of the breakthrough curve). Since the time required for B is fixed by the recovery and regeneration protocol, and the feed flow rate of IC is known (maximum desired feed flow rate should be chosen, in this case 300 cm/h), the remaining parameters QB (feed flow rate of batch phase) and tIC (duration of interconnected phase) can be determined. QB was multiplied with a safety factor of 80% to account for resin degradation over time.
The productivity Prod is defined as the ratio of the product mass contained in the product pool, mpool, of one cycle, the cycle duration tcycle and the total stationary phase volume Vcol:
dsDNA kit, Life technologies).
The buffer consumption BC is defined as the ratio of the buffer volume consumed Vbuff and the mass obtained in the product pool mpool, measured within the same time period (typically one cycle):
The capacity utilisation CU is defined as the ratio of the load L and the static capacity Qsat, which corresponds to the maximum binding capacity.
Figure 3. Chromatograms of a batch (top) and CaptureSMB cycle (bottom). The markers indicate the beginning of load, wash, elution and CIP phases, respectively. In the first interconnected (IC) phase of the process column 1 (red UV profile) is upstream of column 2 (blue UV profile) and in the second IC phase vice versa.
WWW.LABMATE-ONLINE.COM
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76 |
Page 77 |
Page 78 |
Page 79 |
Page 80 |
Page 81 |
Page 82 |
Page 83 |
Page 84 |
Page 85 |
Page 86 |
Page 87 |
Page 88 |
Page 89 |
Page 90 |
Page 91 |
Page 92 |
Page 93 |
Page 94 |
Page 95 |
Page 96
