DESC:FIELD OF INVENTION
The present invention relates to the field of preparative chromatography. The invention provides a rapid method to assess the chromatographic column lifetime during a preparative scale chromatography.
BACKGROUND OF THE INVENTION 5
Preparative chromatography is an essential method for production of therapeutic proteins and is mostly performed in commercial scale using packed bed chromatographic columns. Accordingly, a successful preparative chromatography rests on the chromatographic column performance. Monitoring of column performance is one of the best possible method to ensure the product quality and 10 quantity (yield) in any chromatography, particularly in a preparative scale chromatography. Accordingly, a change in column performance is largely used as a tool to indicate the lifetime / shelf-life of a chromatographic column.
Prior art discloses multiple methods to monitor and/or evaluate column performance such as, pulse test, change in peak width, peak asymmetry factor and 15 transition analysis. Pulse test and transition analysis of data, despite being established tools to monitor column integrity and process performance, suffer from certain disadvantages. On-line pulse tests are more likely to result in "false positives" and do not provide a sufficiently sensitive and encompassing means for detecting changes or degradation in column performance. On the other hand, 20 transition analysis requires analysis of multivariate data, together with process data, generated over extended periods of time to evaluate column performance. Moreover, these methods are subjective as the evaluation requires visual
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monitoring of chromatographic peaks and the conclusions may be based on the analyst subjective experience. In addition, these test methods are time consuming and hence find limited use.
With increasing number of protein therapeutics demanding production using highly efficient process, there is a marked need for more objective methods to 5 assess column performance and its re-usability.
The objective of the present invention was thus to develop an evaluation method relying on quantifiable chromatographic parameter/s to assess column integrity and performance, and consequently its re-usability or lifetime.
SUMMARY OF THE INVENTION 10
The present invention describes a method to assess the resin integrity, and consequently the lifetime of a chromatographic column during preparative scale chromatography.
The instant invention discloses a method wherein, two alternating flow rates viz., high and low, were employed during the cleaning procedure of a chromatographic 15 run/batch, and the back pressures exerted by the column at the two alternating flow rates were measured. The difference in the measured back pressures (i.e. delta pressure), was then used to assess the integrity of the chromatographic column resin/matrix and, consequently its performance and re-usability for the next batch/run. When first flow rate is higher than second flow rate, a positive delta 20 pressure value denotes re-usability of the chromatography column for batches to
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follow, while a negative delta pressure indicates a loss of resin/matrix integrity, and thus non-reusability of the column for the following batch.
The invention is advantageous, as the use of the delta pressure value in determining the integrity of the column is simple, quick and serve as an objective tool of assessment (as then does not depend on the analyst subjective experience). Further, 5 the steps of alternating flow rates can be incorporated as a standard operating procedure [SOP] during the cleaning step of a chromatographic process.
Cleaning of chromatographic column is a routinely performed step ‘before’ and ‘after’ any chromatographic run. The incorporation of the current method, in a ‘cleaning-in-place’ step of SOP, evaluates the chromatographic column integrity 10 and its re-usability even before loading of a protein onto the column, thus preventing the loss of a vital therapeutic protein.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. An overlay chromatogram of five different batches (these batches are not consecutive batches) where CM Ceramic HyperD resin is used in flow through 15 mode for protein purification. Batch 5 is an example of chromatogram that indicates loss of resin integrity.
Figure 2. Column back pressure of CM Ceramic HyperD resin, at high and low flow rate, during the cleaning process (performed post a chromatographic batch/run) for the five batches represented in Figure 1. 20
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DETAILED DESCRIPTION OF THE INVENTION
Preparative chromatographic columns for purification of therapeutic proteins are expensive and thus its maximum utilization is expected. However multiple use of the column in preparative scale chromatography would affect it’s mid to long term performance, indicated by a peak shift in the chromatogram (Figure 1). Though a 5 peak shift indicates a change in column performance, it is not an indication of column integrity and/or its re-usability.
In other words, despite a shift in chromatographic peak, a column can, and often is reused in commercial scale process development as long as the change in column performance does not always affect the purity or quality of the protein product. 10 However, it has also been observed during chromatography that even when the purity & yield level of a protein are within acceptable range in one batch, there is a sudden loss of column integrity in the following batch. The sudden “crash” of the column, particularly during the purification process, leads to significant loss of the product, and significantly impacts the re-usability of a chromatographic column 15 and the development cost associated with them. The impact becomes substantial in a preparative scale chromatography.
The present invention describe a method to assess or predict in advance viz., before loading a protein, the potential loss of integrity, and hence the possibilities of “crashing” of a chromatographic matrix/resin in a column. 20
A chromatographic column, post installation and equilibration with mobile phase, exhibits certain pressure which is considered as normal ‘back pressure’ of the
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column. Major factors affecting the back pressure include column length, particle size of the matrix, viscosity of the solvent and the flow rate. In particular, there exists a linear correlation between the column back pressure and the flow rate viz., an increase in column flow rate results in an increase in back pressure and a decrease in flow-rate decreases the column back pressure. This positive correlation 5 between the flow rate and column back pressure, and the flexibility in modulating the flow rate, makes it an important factor in controlling the column back pressure.
The invention accordingly utilizes this correlation and employs altering flow rates to monitor for any deviance/alteration in the pressure chromatogram. Specifically, the altering of flow rates were incorporated during the cleaning step of the 10 chromatographic procedure, and deviations in pressure measured to assess the loss of column integrity. Thus, the integrity of the column and hence its re-usability was assessed for the subsequent chromatographic run.
The present invention discloses a method wherein, two alternating flow rates viz., a high and low flow rates were employed during the cleaning procedures and the 15 back pressures exerted by the column at the two alternating flow were measured. The difference in back pressure (delta pressure), is then used to assess the integrity of the column resin /matrix and consequently its performance and re-usability for the next batch/run
In one embodiment, the invention discloses a method to assess the 20 chromatographic column integrity and its re-usability during a chromatographic process, comprising steps of;
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a) Washing the column at an initial first flow rate, during cleaning step of the said chromatographic process
b) Measuring the column back pressure, p1, in step (a)
c) Washing the column with a second flow rate
d) Measuring the column back pressure, p2, in step (c) 5
e) Obtaining a delta pressure value from the values of p1 and p2, viz., p1- p2 and
f) Using the said value from step e) to assess the loss of column integrity,
wherein,
a negative delta pressure value indicates a loss of column integrity and thus 10 non-usability of the column for a next run, and a positive delta pressure value indicates integrity of the column and its potential re-usability, when first flow rate is greater than second flow rate,
or
a positive delta pressure value indicates a loss of column integrity and thus non-15 usability of the column for a next run, and a negative delta pressure value indicates integrity of the column and its potential re-usability, when second flow rate is greater than first flow rate.
In another embodiment, the invention discloses a method to assess the chromatographic column integrity and its re-usability during a chromatographic 20 process, comprising steps of;
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a) Washing the column at an initial first flow rate, during cleaning step of the said chromatographic process
b) Measuring the column back pressure, p1, in step (a)
c) Washing the column with a second flow rate
d) Measuring the column back pressure, p2, in step (c) 5
e) Obtaining a delta pressure value from the values of p1 and p2, viz., p1- p2 and
f) Using the said value from step e) to assess the loss of column integrity,
wherein, a negative delta pressure value indicates a loss of column integrity and thus non-usability of the column for a next run, and a positive delta 10 pressure value indicates integrity of the column and its potential re-usability, when first flow rate is greater than second flow rate.
In another embodiment, the invention discloses a method to assess the chromatographic column integrity and its re-usability during a chromatographic process, comprising steps of; 15
a) Washing the column at an initial first flow rate, during cleaning step of the said chromatographic process
b) Measuring the column back pressure, p1, in step (a)
c) Washing the column with a second flow rate
d) Measuring the column back pressure, p2, in step (c) 20
e) Obtaining a delta pressure value from the values of p1 and p2, viz., p1- p2 and
f) Using the said value from step e) to assess the loss of column integrity,
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wherein, a positive delta pressure value indicates a loss of column integrity and thus non-usability of the column for a next run, and a negative delta pressure value indicates integrity of the column and its potential re-usability, when second flow rate is greater than first flow
In another embodiment, the invention discloses a method wherein the said second 5 flow rate is kept about 20 litre/hour higher or lower than the first flow rate.
In another embodiment, the invention discloses a method wherein the said second flow rate is kept about 50 litre/hour higher lower than the first flow rate.
In any of the embodiments mentioned above, the said chromatographic column is an ion exchange column. 10
In another embodiment, the said ion exchange column is CM Ceramic HyperD.
In another embodiment, the said CM Ceramic HyperD column bed height and diameter is about 30 cm and 20 cm respectively.
In any of the embodiments mentioned above, the said delta pressure value is at least about ±0.10 bar. 15
The invention is effected using a composite resin, CM ceramic HyperD, that is a mixture of rigid (non-compressible) and soft (compressible) gel matrix. Hence, the invention can be readily employed to predict the integrity of both rigid and soft column chromatographic matrix type.
The method according to the present invention can also be alternatively employed 20 for any chromatographic column chromatography such as protein-A affinity
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chromatography or a protein-G affinity chromatography or cation exchange chromatography or a hydrophobic charge induction chromatography (HCIC), or a hydrophobic interaction chromatography (HIC) or a mixed mode chromatography.
The invention is more fully understood by reference to the following examples. These examples should not, however, be construed as limiting the scope of the 5 invention.
Example 1
Cation exchange chromatography in Flow through mode
This chromatography is performed in an isocratic mode. The product is loaded onto a CM Ceramic HyperD® (Pall Corporation, U.S.A.) resin and eluted using 10 a constant mobile phase. The bed height of the column was 30cm. The pH and conductivity of the load and equilibration buffer are maintained throughout the operation. The flow-through purification process using CM-Hyper D resin has been shown in Figure 1 for five different batches. Batch 5 is an example of chromatogram where resin integrity has been lost. 15
Example 2
Column cleaning, regeneration and storage
CM-Hyper D used in example 1 was subjected to cleaning cycle for reuse. The column was cleaned using 2 CV of 25mM Tris+ 500mM of NaCl (pH-7.1, flow rate 125.6 liter/hour) followed by 5 CV of 25mM Tris+ 2M of NaCl (pH-7.1, flow 20 rate 125.6 litre/hour). This high salt cleaning was followed by low salt cleaning
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using 3 CV of 500mM NaCl (flow rate 125.6 litre/hour) and by passing 6 CV of 0.5N NaOH (flow rate 70.69 litre/hour) and incubating for 60-90 minutes. After the incubation in 0.5 N NaOH, the column was washed by passing 5 CV each of 25mM Tris+ 500 mM of NaCl (pH-7.1, flow rate 70 liter/hour) and 20 CV of 50mM NaCl (flow rate 70 litre/hour). The column was stored in 3 CV of 1M NaCl 5 in 20% ethanol (v/v). The column back pressure was monitored throughout the washing. The detail of column back pressure for different batches during cleaning has been shown in Figure 2. The difference between the column back pressure (delta pressure) at highest flow rate and lowest flow rate, during the cleaning-in-place step has been furnished in Table 1. 10
Table 1: Difference of column back pressure (delta pressure) at highest flow rate and lowest flow rate for different chromatographic batches
,CLAIMS:We claim:
1. A method to assess the chromatographic column integrity and its re-usability during a chromatographic process, comprising steps of;
a) Washing the column at an initial first flow rate, during cleaning step of the said chromatographic process
b) Measuring the column back pressure, p1, in step (a)
c) Washing the column with a second flow rate
d) Measuring the column back pressure, p2, in step (c)
e) Obtaining a delta pressure value from the values of p1 and p2, viz., p1- p2 and
f) Using the said value from step e) to assess the loss of column integrity,
wherein, a negative delta pressure value indicates a loss of column integrity and thus non-usability of the column for a next run, and a positive delta pressure value indicates integrity of the column and its potential re-usability, when first flow rate is greater than second flow rate.
2. A method to assess the chromatographic column integrity and its re-usability during a chromatographic process, comprising steps of;
a) Washing the column at an initial first flow rate, during cleaning step of the said chromatographic process
b) Measuring the column back pressure, p1, in step (a)
c) Washing the column with a second flow rate
d) Measuring the column back pressure, p2, in step (c)
e) Obtaining a delta pressure value from the values of p1 and p2, viz., p1- p2 and
f) Using the said value from step e) to assess the loss of column integrity,
wherein, a positive delta pressure value indicates a loss of column integrity and thus non-usability of the column for a next run, and a negative delta pressure
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value indicates integrity of the column and its potential re-usability, when second flow rate is greater than first flow rate.
3. A method according to claim 1 or claim 2, wherein the difference between the first and the second flow rate is kept at least at 20 litres per hour.
4. A method according to claim 1 or claim 2, wherein the delta pressure is at least about ±0.10 bar.
5. A method according to claim 1 or claim 2, wherein the resin is CM Ceramic HyperD®.