Method for Processing Solutions of Biomolecules

Abstract

Apparatus for in-line liquid exchanging a biomolecule-containing liquid is provided. The apparatus comprises a means (3) for mixing at least two liquids comprising a multiple inlet flow-controller (2), the means for mixing also comprising an outlet in fluid connection with a tangential flow filtration device (1) configured in single-pass mode.

Claims

1. Apparatus for in-line liquid exchanging a biomolecule-containing liquid comprising a means for mixing comprising a multiple inlet flow-controller further comprising two or more variable flow inlet valves for mixing at least two liquids, the means for mixing also comprising an outlet in fluid connection with a tangential flow filtration device configured in single-pass mode.

2. Apparatus according to claim 1, wherein retentate from the tangential flow filtration device is in fluid connection with a second means for mixing at least two liquids and the second means for mixing comprises an outlet in fluid connection with a second tangential flow filtration device configured in single-pass mode.

3. Apparatus according to claim 1, wherein the variable flow inlet valves are intermittent flow valves.

4. Apparatus for liquid exchanging a biomolecule-containing liquid comprising: a) a multiple inlet flow-controller comprising: i) a first inlet for a first liquid medium comprising a biomolecule; ii) at least a second inlet for a second liquid medium; iii) an outlet in fluid connection with a tangential flow filtration device; and b) a means for imparting flow of the liquids through the flow-controller and the tangential flow filtration device.

5. Apparatus according to claim 4, wherein the means for imparting flow comprises a pump located between the outlet of the multiple inlet flow-controller and the tangential flow filtration device.

6. Apparatus according to claim 5, further comprising a restrictor downstream of the tangential flow filtration device.

7. Apparatus according to claim 4, further comprising a second multiple inlet flow-controller comprising: i) a first inlet in fluid connection with the retentate from the tangential flow filtration device; ii) a second inlet for a third liquid medium; and iii) an outlet in fluid connection with a second tangential flow filtration device.

8. Apparatus according to claim 7, wherein the second multiple inlet flow-controller functions as a restrictor.

9. Apparatus according to claim 8, wherein the second multiple inlet flow-controller further comprises two or more variable flow inlet valves.

10. A method for the preparation of a biomolecule, which comprises processing a liquid medium comprising the biomolecule by liquid exchange employing an apparatus according to claim 1.

11. The method according to claim 10, wherein the variable flow inlet valves are cycled between a position achieving a first, relatively low flow rate wherein the liquid remains able to flow, or flow is prevented and at least a second, higher flow rate.

12. The method according to claim 11, wherein at least 10 cycles are employed.

13. The method according to claim 12, wherein the cycle frequency is less than 100 Hz.

14. The method according to any one of claim 10, wherein the processing comprises buffer exchange.

15. A process for the production of a biomolecule which comprises a method according to claim 10.

16. The process according to claim 15, wherein the variable flow inlet valves are cycled between a position achieving a first, relatively low flow rate wherein the liquid remains able to flow, or flow is prevented and at least a second, higher flow rate, at least 10 cycles are employed, and the cycle frequency is from 0.05 to 0.5 Hz.

Description

EXAMPLE 1

[0066] A stock of at least 400 mL of 1 mg/mL rhLactoferrin at pH 7.5 was volumetrically diluted 4-fold with buffer (A) through using a 25% gradient of rhLactoferrin on the B1 pump of a GE Healthcare ?KTA? Explorer system, whilst feeding buffer (A) through the A1 pump (75%) at a constant flow rate of 15 mL/min. The diluted rhLactoferrin was then directed in down flow mode through position 2 on the ?KTA? Explorer V2 valve into a 65 cm long, 10 kDa mPES Spectrum Labs MidiKros? hollow fibre with a surface area of 370 cm.sup.2. The hollow fibre retentate line was in turn directly connect to a downstream multiple inlet variable flow-controller. The multiple inlet variable flow-controller comprises of a custom made (Gem?) plastic two valve manifold with a single outlet having a 2 mm internal bore with a fast acting solenoid actuator under the control of a Raspberry Pi minicomputer, which controls the flow of liquid through the manifold. The manifold is configured to have the same flow path volumes from valve to the outlet. The cycle time of the multiple inlet variable flow-controller was set to 2 seconds and the retentate controlling valve was opened for 25% of the cycle to achieve the 4-fold volumetric concentration factor required to obtain the initial starting volume of the rhLactoferrin solution. The second valve position on the multiple inlet flow-controller was open for the 75% of the cycle when the first valve was closed, to allow a second 4-fold dilution of the hollow fibre retentate with buffer (B). The outlet from the multiple inlet variable flow-controller passed through a static mixer of length 10 cm and diameter 5 mm before return to valve V3, position 2 on the ?KTA? Explorer to collect conductivity, pH and 280 nm absorbance data. The F8 outlet line from the ?KTA? Explorer valve V4 was connected to the A11 feed line of the A1 pump of a second GE Healthcare ?KTA? Explorer system also running at 15 mL/min. This system was in turn connected to a second 65 cm long, 10 kDa mPES Spectrum Labs MidiKros? hollow fibre with a surface area of 370 cm.sup.2 through the ?KTA? Explorer column valve V2, again on position 2. The retentate of the hollow fibre was fed directly into a second 10 mm internal bore sized multiple inlet variable flow-controller. This valve used a cycle time of 10 seconds with retentate controlling valve being open for 4% of the cycle to obtain the 4-fold volumetric concentration factor in order to once again obtain the initial starting volume of the rhLactoferrin solution. The outlet from the multiple inlet variable flow-controller was directed through a second static mixer of length 10 cm and diameter 5 mm before returning to the ?KTA? explorer on valve V3, position 2 for collection of conductivity, pH and 280 nm absorbance data. The in-line buffer exchanged rhLactoferrin solution was collected through the outlet line F8 on the ?KTA? Explorer valve V4. The data from the first ?KTA? Explorer system demonstrated successful rapid buffer exchange using an in-line system, whilst the trace from the second ?KTA? Explorer system showed the protein concentration relative to the feed was maintained.

[0067] Absorbance, conductivity and pH traces of the in-line buffer exchanged rhLactoferrin demonstrates using two 4-fold dilutions and concentrations resulted in a ?95% exchange of buffer (A) for buffer (B). Buffer (B) conductivity 6.9 mS/cm and pH 7.47 compared well with final buffer exchanged Lactoferrin with a conductivity 7.2 mS/cm and pH 7.43. The protein concentration was maintained at around 45 mAU.

EXAMPLES 2 TO 8

[0068] The method of Example 1 was repeated, but with the conditions varied as stated in Table 1 to investigate the effect of reversing the buffer exchange or the addition of buffer components which change buffer viscosity (10% sorbitol and/or 6% propan-1,2-diol) on the operating time and different concentration/dilution ratios between the 1.sup.st and 2.sup.nd concentrators. Examples 2 and 3 used buffer (A) for the diluent, Example 4 used buffer (B), Examples 5, 6 and 7 used buffer (C) and Example 8 used buffer (D).

[0069] From the results given in Table 1, it can be seen that serial dilution and concentration achieves buffer exchanges equivalent to up to 3 diavolumes on a conventional recirculating batch TFF system. Higher buffer exchange efficiencies can be achieved by running at greater dilution rates, as seen in Examples 6 and 7.

TABLE-US-00001 TABLE 1 Combined Conductivity dilution Feed Buffer Retentate Efficiency Equivalent Feed Retentate Ex. ratio (mS/cm) (mS/cm) (mS/cm) (%) DV (mL) (mL) 1 1:16 3.57 16.49 16.14 97.9 3.9 65.7 243 2 1:16 3.64 16.49 14.48 87.8 2.1 67.5 48 3 1:16 3.64 16.49 14.76 89.5 2.3 345 550 4 1:16 14.20 7.02 7.96 86.6 2.0 350 770 5 1:16 9.34 15.15 14.70 97.0 3.5 159 220 6 1:16 9.42 15.54 14.63 94.1 2.8 154 224 1:32 14.90 95.9 3.2 7 1:12 9.42 15.54 14.36 92.4 2.6 242 251 1:16 14.64 94.2 2.8 1:24 14.88 95.8 3.2 1:32 14.99 96.5 3.4 8 1:16 14.82 11.44 12.56 126 157