HK1179272B - Process for obtaining antibodies - Google Patents

Description

Method for obtaining antibodies

The present invention relates to methods for increasing the yield of recombinant antibodies, particularly therapeutic antibodies, in the production and isolation process. These methods are particularly suitable for large scale industrial manufacture of therapeutic antibodies.

Recombinant DNA technology has rapidly developed and is particularly useful for the production of antibodies, particularly therapeutic antibodies. Systems for expressing recombinant genes are well known to those skilled in the relevant art. These systems include expression in mammalian cells, insect cells, fungal cells, bacterial cells, and transgenic animals and plants. The choice of expression system depends on the characteristics of the encoded protein, e.g., whether it has post-translational modifications. Other considerations include the time and particularly the cost to produce the required amount of material of the required quality. These latter considerations are particularly important for the production of therapeutic antibodies of the quality required for the approval of the registration and in the amounts required to treat a large number of patients.

The most widely used system for the production of recombinant proteins is based on expression in Escherichia coli (e.coli). One particular problem faced with the use of E.coli is the difficulty in producing the quantity of material of the desired quality in the quantity required for treatment. In particular, the time and cost required is too high. One particular problem of note is the loss of antibody production during the extraction of antibodies from E.coli.

Although the purification cost is proportionately a fraction of the total cost of the therapeutic antibody product, the proportion of purification cost increases further as upstream production costs become cheaper. Thus, improvements in antibody recovery and purification will drive further reductions in production costs, regardless of the mode of production (Humphreys & Glover, curr. Opin. drug Disc/' overlay & Development,2001,4: 172-. Thus, there is a need for methods that introduce time and/or cost savings into the production of therapeutic antibodies, particularly in purification, for example by increasing product recovery and/or improving the quality of the product stream.

Low product yields through fermentation or culture pathways are often a significant problem noted during the primary extraction stage; the expression of antibodies is high intracellularly but it is difficult to achieve a high percentage recovery during the primary extraction stage.

There are methods that partially solve the latter problem and enable the production of antibodies acceptable for therapeutic applications, which are described in US5,655,866. This method involves the use of heat treatment to assist in the subsequent separation of functional Fab' fragments of the antibody from non-functional antibodies, which heat treatment can be performed at any time during fermentation or culture, or at any stage during extraction and purification of the antibody. Functional antibodies are extremely stable at elevated temperatures above room temperature, while many other proteins, including host cell proteins and free light and heavy chain species, as well as non-functional fragments of antibodies, form precipitates and/or aggregates that can be easily separated from functional antibodies in primary purification steps such as filtration or centrifugation or fluidized bed chromatography. Cell extracts were prepared by incubating intact cells in Tris HCl buffer (100 mM, pH 7.4) containing 10mM EDTA, as described in U.S. Pat. No. 5,655,866.

WO2006/054063 describes that the yield of functional antibodies in the primary extraction stage is increased by introducing a non-lysing treatment in combination with a heat treatment. The essential point of this method is that after centrifugation, the cell pellet is resuspended in 1M Tris (pH 7.4) containing 100mM EDTA, followed by a non-lysing treatment and then a heat treatment.

WO2005/019466 describes the introduction of a break step under defined conditions of temperature and pH after fermentation but before downstream processing including extraction, improving the yield of recombinant protein.

Summary of The Invention

The invention described herein is based on the following surprising and unexpected observation facts: after culturing a host cell sample transformed with an expression vector encoding a recombinant antibody molecule, an increase in the pH of the sample obtained during primary recovery has a significant beneficial effect on antibody production.

The pH of the antibody, prior to processing (e.g., heating), initially ranges from 6 to 9, and unexpectedly, the pH drops even in the presence of buffering, possibly as a result of cellular metabolism. The present inventors now believe that this is extremely detrimental to yield/recovery and propose to solve this problem by adjusting the pH of the material before and/or during processing at the appropriate time to ensure that the pH is within the target range.

It has been unexpectedly found that the pH of the sample prior to the heat treatment step has a significant effect on the antibody yield in the cell sample. It has been found that adjusting the pH of the sample to 6-9 before the heat treatment step will increase the antibody yield by as much as 40%. This allows a huge beneficial saving in time and cost of producing large quantities of functional antibodies of therapeutic quality. In fact, other steps commonly used to increase yield, such as homogenization and stabilization steps, may not be required to achieve high levels of antibody yield.

In previously used methods, such as in US5,655,866, cell extracts were prepared by incubating intact cells in a buffer at pH 7.4. It has been found that the pH of a cell sample actually decreases gradually over time despite the addition of a buffer that is expected to maintain the pH of the sample at a constant level. Under certain circumstances, such as a long time after addition of the buffer, the pH of the sample was found to be as low as pH5.5 prior to the heat treatment step. It has been found that detecting and optionally adjusting the pH prior to heat treatment to ensure that the pH of the sample is between 6 and 9 results in a surprising increase in antibody yield.

Without wishing to be bound by theory, it is believed to be important to maintain the pH in the range of 6-9 during processing steps such as heat treatment. Adjusting the pH prior to processing (e.g., a heat treatment step) helps maintain the pH within an appropriate range. Thus, in one aspect, an antibody extraction step is provided wherein the pH is substantially maintained in the range of 6-9 for substantially the entire duration of substantially the entire process.

Without being bound by theory, it is believed that the method provided by the present invention enables recovery of recombinant proteins from the periplasm in a primary separation process that are not released under standard extraction conditions.

Accordingly, in a first aspect of the invention, there is provided a method for producing a recombinant antibody molecule comprising culturing a sample of host cells transformed with an expression vector encoding a recombinant antibody molecule; adding an extraction buffer to the sample; and carrying out a heat treatment step on the sample; wherein the pH of the sample is detected after addition of the extraction buffer and optionally adjusted to ensure that the pH of the sample is 6-9 prior to the heat treatment step.

Monitoring of pH at this stage is critical to establishing pH control.

In an alternative aspect, a method is provided for extracting a recombinant antibody molecule from a sample of host cells transformed with an expression vector encoding the recombinant antibody molecule; which comprises the following steps:

adjusting the pH of the composition of cells to a range of 6-9 such that the pH is maintained within this range during the subsequent extraction step,

the cells are subjected to an extraction step, such as a heat treatment step,

wherein the pH is monitored at least one point in time before and/or during the extraction step.

It has also been found that an increase in the pH of the extraction buffer results in a surprising increase in the antibody yield of the sample after the heat treatment step.

Accordingly, a second aspect of the invention provides a method for the manufacture of a recombinant antibody molecule comprising culturing a sample of host cells transformed with an expression vector encoding a recombinant antibody molecule; adding an extraction buffer to the sample at a pH of 7.5-9.0; and performing a heat treatment step on the sample.

Detailed Description

An antibody molecule as used herein is intended to refer to a complete antibody or binding fragment thereof, in particular a complete antibody or a Fab fragment.

In a first aspect of the invention, the sample has or is adjusted to a pH of 6 to 9 prior to the heat treatment step.

In a preferred embodiment, the sample before the heat treatment step has a pH of 6.5-8.5, a pH of 6.5-8.0, a pH of 7.0-9.0, a pH of 7.0-8.5, a pH of 7.0-8.0, a pH of 7.1-8.0, a pH of 7.5-8.0, a pH of 7.0-7.8, a pH of 7.1-7.7, a pH of 7.2-7.6, a pH of 7.3-7.5, a pH of 7.1, a pH of 7.2, a pH of 7.3, a pH of 7.4, a pH of 7.5, a pH of 7.6, a pH of 7.7, a pH of 7.8 or a pH of 7.9, such as a pH of 7.4, in particular a pH of 6.8.

The pH measurements referred to herein are typically standardized to 20 ℃.

The heat treatment step in the method of the present invention is a step of maintaining the temperature of the sample at a desired high temperature after the desired high temperature is reached during the heating period. Suitable temperature ranges for the heat treatment step include 30-70 ℃.

In the context of the present invention, the expression "before the heat treatment step" refers to and includes the point in time before the sample reaches the desired high temperature and the heat treatment step (maintained at the high temperature) begins. To reach the high temperature required for the heat treatment step, the sample may undergo a "heating period" in which the temperature of the sample is raised to the required high temperature. In one embodiment, the method according to the invention comprises subjecting the sample to a heating period and a heat treatment step.

In the method of the invention, the sample has a pH of 6-9, for example pH6.8, prior to the heat treatment step. In this context, "before the heat treatment step" means that the pH of the sample is at the desired level before or at the point in time when the sample reaches the high temperature required for the heat treatment step. In embodiments in which the method includes subjecting the sample to a heating period and a heat treatment step, the sample may be at a desired pH level prior to the beginning of the heating period and/or at a desired pH level during the heating period.

In a preferred embodiment, the sample is at the desired pH level, pH6-9, before the heating period begins.

In one embodiment, the present invention provides a method for producing a recombinant antibody molecule comprising culturing a sample of host cells transformed with an expression vector encoding a recombinant antibody molecule; adding an extraction buffer to the sample; and subjecting the sample to a heating period and a heat treatment step; wherein the pH of the sample is measured after addition of the extraction buffer and optionally adjusted to ensure that the pH of the sample is between pH6 and 9, for example between pH7 and 9, such as between pH7 and 8, prior to the heating period.

In an alternative embodiment, the present invention provides a method for producing a recombinant antibody molecule comprising culturing a sample of host cells transformed with an expression vector encoding a recombinant antibody molecule; adding an extraction buffer to the sample; and subjecting the sample to a heating period and a heat treatment step; wherein the pH of the sample is detected and optionally adjusted after addition of the extraction buffer to ensure that the pH of the sample is 6-9, preferably 6-8, more preferably 6-7 during the heating period.

In one embodiment, the pH of the sample is detected and optionally adjusted to ensure that the pH of the sample is at a first pH prior to the heating period and at a second pH during the heating period. Preferably, the first and second pH levels are preferably not the same. Preferably, the second pH is lower than the first pH. Accordingly, the present invention provides a method for producing a recombinant antibody molecule comprising culturing a sample of host cells transformed with an expression vector encoding a recombinant antibody molecule; adding an extraction buffer to the sample; and subjecting the sample to a heating period and a heat treatment step; wherein the pH of the sample is measured and optionally adjusted after addition of the extraction buffer to ensure that the pH of the sample is between 7 and 9, preferably between 7 and 8, prior to the heating period and to ensure that the pH of the sample is between 6 and 8, preferably between 6 and 7, during the heating period. In this embodiment, the sample pH can be measured and optionally adjusted prior to the heating period, and optionally adjusted during the heating period.

In a preferred embodiment, the sample shortly before the heating period has a pH of 6-9, preferably 7-9, more preferably 7-8. Additionally or alternatively, shortly before the heat treatment step in the heating phase (optionally including the point in time when the sample reaches the desired elevated temperature and the heat treatment step begins), the sample has a pH of 6-9, preferably a pH of 6-8, more preferably a pH of 6-7. It has been found that the pH of the sample shortly before the heating period or shortly before the heat treatment step has a significant effect on the yield of recombinant antibody.

In the context of the present invention, the term "shortly before … …" preferably means within 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, 4 minutes or less, 3 minutes or less, 2 minutes or less, 1 minute or less, 30 seconds or less, 10 seconds or less, 5 seconds or less, 1 second or less, before the heating period or heat treatment step. The term "shortly before … …" may also include a solution pH of 6-9 at the beginning of the heating period or the beginning of the heat treatment step.

The pH of the sample was checked after addition of extraction buffer. The pH of the sample can be detected using any suitable pH measuring device known in the art. The pH of the sample can be measured at one or more separate time points in the method, such as at the time point of addition of the extraction buffer, shortly after addition of the extraction buffer, shortly before the start of the heating period, at the start of the heating period, during the heating period (including the time point at which the sample reaches the elevated temperature required for the heat treatment step), during the heat treatment step, and after the heat treatment step. Alternatively, the pH of the sample is detected by continuous monitoring. In embodiments wherein the pH of the sample is continuously monitored, the pH of the sample is preferably continuously monitored after the step of culturing the cells, preferably from after the centrifugation step after culturing until the heat treatment step begins. In a preferred embodiment, the pH of the sample is continuously monitored from the point in time when the extraction buffer is added until the heat treatment step begins. However, the pH may also be monitored during the culturing step and/or during the heat treatment step.

Thus, in one embodiment, the pH profile of the heating step is controlled.

In a preferred embodiment, the pH of the sample is detected and optionally adjusted, and then the heating period is started (preferably automatically) when the sample reaches the desired pH.

The extraction buffer is added after the step of culturing the cell sample. If the method comprises a centrifugation step after the incubation step, an extraction buffer may be added before and/or during and/or after the centrifugation step. Preferably, an extraction buffer is added after the centrifugation step to resuspend the cell pellet formed by centrifugation.

In one embodiment of the invention, the extraction buffer has a suitable pH to ensure that the pH of the sample before the heat treatment step is in the range of pH6-9, for example in the range of pH 6-8. In embodiments wherein the extraction buffer has a suitable pH to ensure that the pH of the sample prior to the heat treatment step is between pH6 and 9, e.g., between pH6 and 8, the extraction buffer has, e.g., a pH of between pH7.5 and 9.0, between pH7.5 and 8.8, between pH7.5 and 8.5, between pH 8.0 and 9.0, between pH 8.5 and 9.0, between pH 8.6 and 8.9, between pH 8.0, between pH 8.1, between pH 8.2, between pH 8.3, between pH 8.4, between pH 8.5, between pH 8.6, between pH 8.7, between pH 8.8, between pH 8.9 or between pH 9.0. Preferably, the heating period and the heat treatment step are performed shortly after, preferably immediately after, the addition of the extraction buffer, thus ensuring that the sample has the desired pH before the heat treatment step. For example, the heating period or heat treatment step can be performed within 4 hours or less, 3 hours or less, 2 hours or less, 1 hour or less, 30 minutes or less, 10 minutes or less, or 5 minutes or less after addition of the extraction buffer.

Thus, the method of the invention may not require a step of pH adjustment of the sample. The pH of the sample can be measured after addition of the extraction buffer, maintained within the desired pH range of 6-9. This may for example be the case where the pH of the extraction buffer is suitable for adjusting the pH of the sample to 6-9 as described above, e.g. where the extraction buffer has a pH of 7.5-9.0 and the heat treatment step is performed shortly thereafter.

However, generally due to the time interval between the addition of extraction buffer and the heat treatment step, the method according to the invention requires, in addition to any pH adjustment that may be caused by the addition of extraction buffer, a step of detecting and adjusting the pH of the sample to ensure that the pH of the sample is between 6 and 9 prior to the heat treatment step.

In this embodiment, where the method includes pH detection and adjustment, the pH of the extraction buffer may be below pH8, such as pH7.4 or lower, for example, pH 6.0-7.4, pH 6.5-7.4, or pH 7.0-7.4, such as pH 6.8.

Alternatively, in a preferred embodiment, the extraction buffer is at pH7.5-9.0, pH 7.5-8.8, pH 7.5-8.5, pH 8.0-9.0, pH 8.5-9.0, pH 8.6-8.9, pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4, pH 8.5, pH 8.6, pH 8.7, pH 8.8, pH, 8.9, pH 9.0, most preferably pH 8.0.

In this embodiment, the sample pH may be adjusted by any suitable means at any suitable time in the process. The pH of the sample may be adjusted before and/or after the addition of the extraction buffer.

In one embodiment, the pH of the sample is adjusted prior to adding the extraction buffer. In this embodiment, if the method comprises a centrifugation step after the culturing step, a pH adjustment step may be performed before and/or after the centrifugation step. After culturing the cells, optionally after centrifugation, the pH of the sample is generally lower. For example, the sample may have a pH of about pH 5.5. Thus, the pH of the sample may be adjusted after culturing the cells and optionally after one or more additional steps, such as centrifugation. For example, the pH of the sample may be adjusted to pH 6.5-8.0, preferably pH 7.0-8.0, pH 6.5-7.5, pH 6.6-7.4, pH 6.7-7.3, pH 6.8-7.2, pH 6.9-7.1, most preferably pH6.9 prior to addition of the extraction buffer.

In one embodiment, where the sample pH before the heat treatment step is less than pH7 (e.g., pH 6.9) as required for the sample pH before the extraction buffer is added, the sample pH needs to be further increased by the addition of extraction buffer and/or by other pH adjustments after the addition of extraction buffer such that the sample pH is 7-9 before the heat treatment step.

In a preferred embodiment of the invention, the sample pH is adjusted to pH6-9 after addition of the extraction buffer and before the heat treatment step. At this stage, the sample is preferably adjusted to pH 7.0-9.0, pH 7.0-8.5, pH 7.0-8.0, pH 7.1-8.0, pH 7.5-8.0, pH 7.0-7.8, pH 7.1-7.7, pH 7.2-7.6, pH 7.3-7.5, pH 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 7.9, most preferably pH 7.4. In one embodiment, the pH of the sample is adjusted prior to the heating period. Preferably, the sample pH is adjusted to pH 7-9, preferably pH 7-8, prior to the heating period. Alternatively or additionally, the pH adjustment of the sample is adjusted during the heating period. Preferably, the sample pH is adjusted to pH 6-8, preferably pH6-7, during the heating period.

In a preferred embodiment of the invention, an extraction buffer having a pH of 7.4 or pH8 is added to the sample and the sample pH is measured and subsequently adjusted to pH7.4 before the heat treatment step, preferably before the heating period, more preferably shortly before the heating period.

In embodiments where the pH of the sample is detected after addition of the extraction buffer and adjusted prior to the heat treatment step, the pH of the sample may be detected and adjusted prior to the beginning of the heating period. Additionally or alternatively, the sample pH may be detected and adjusted during the heating period.

In a preferred embodiment, the pH of the sample is detected and adjusted immediately prior to the heating period. Additionally or alternatively, the pH of the sample is detected and adjusted immediately prior to the heat treatment step of the heating period (optionally including the point in time at which the sample reaches the desired elevated temperature and the heat treatment step begins).

In the context of the present invention, the term "shortly before … …" preferably means that the pH of the sample is detected and adjusted to pH6-9 in a time of 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, 4 minutes or less, 3 minutes or less, 2 minutes or less, 1 minute or less, 30 seconds or less, 10 seconds or less, 5 seconds or less, 1 second or less before the heating period or before the heat treatment step. The term "shortly before … …" may also include the pH of the solution being detected and adjusted at the beginning of the heating period or the beginning of the heat treatment step. In a preferred embodiment, the heating period and/or the heat treatment step is (preferably automatically) initiated upon detection of a pH of the sample of 6-9.

The pH of the sample may be detected and adjusted by any single or multiple pH adjustment steps as described above. Thus, the pH can be adjusted in the following stages:

just before addition of extraction buffer;

only after addition of extraction buffer but before the heat treatment step; or

Before addition of the extraction buffer and after addition of the extraction buffer but before the heat treatment step.

The pH can be adjusted multiple times, e.g., 1, 2, 3, 4 or more times, after addition of the extraction buffer.

In one embodiment, the pH of the sample is continuously adjusted, preferably between the addition of the extraction buffer and the heat treatment step.

In one embodiment, the pH of the sample may additionally be detected and optionally adjusted during the heat treatment step. Therefore, the method according to the present invention may further comprise the step of adjusting the pH of the sample during the heat treatment step. Preferably, the pH of the sample is adjusted to pH 6.0-9.0, pH6.5-8.5, pH 6.5-8.0, pH 7.0-9.0, pH 7.0-8.5, pH 7.0-8.0, pH 7.1-8.0, pH 7.5-8.0, pH 7.0-7.8, pH 7.1-7.7, pH 7.2-7.6, pH 7.3-7.5, pH 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 7.9 in the heat treatment step.

In accordance with a second aspect of the present invention, there is provided a method for the manufacture of a recombinant antibody molecule comprising culturing a sample of host cells transformed with an expression vector encoding a recombinant antibody molecule; adding an extraction buffer having a pH of 7.5-9.0 to the sample; and performing a heat treatment step on the sample.

As described above in the first aspect of the text, the extraction buffer preferably has a pH of 7.5-9.0, a pH of 7.5-8.8, a pH of 7.5-8.5, a pH of 8.0-9.0, a pH of 8.5-9.0, a pH of 8.6-8.9, a pH of 8.0, a pH of 8.1, a pH of 8.2, a pH of 8.3, a pH of 8.4, a pH of 8.5, a pH of 8.6, a pH of 8.7, a pH of 8.8, a pH of 8.9 or a pH of 9.0. In this aspect of the invention, it is less important to detect the pH of the sample. The method according to the second aspect of the invention may comprise detecting the pH of the sample and adjusting the pH of the sample as described in the first aspect of the invention. However, the steps involved in detecting the pH or adjusting the pH are not critical. In one embodiment, the method according to the second aspect of the invention does not comprise a step of detecting the pH of the sample or a step of adjusting the pH.

The following detailed description of the invention applies to embodiments of the first and second aspects of the invention.

pH adjusting reagent and extraction buffer

The pH must be adjusted so that the pH during the heat treatment step is maintained/maintained within the desired range of pH 6-9.

In one embodiment, the pH is adjusted with a base, such as an inorganic base, e.g. sodium hydroxide, or an organic base, such as triethylamine or trimethylamine.

Any suitable reagent may be used to adjust the pH of the sample. The reagent may be an extraction buffer or may be added before and/or after the extraction buffer. Typical agents useful for adjusting pH comprise or consist of one or more of the following: NaOH, NH₄OH, sulfuric acid, EDTA and Tris buffer solution. Preferably, the pH of the sample is determined using a base (e.g., hydrogen and oxygen)Sodium hydroxide or ammonium hydroxide).

In one embodiment, the extraction buffer is a Tris (hydroxymethyl) methylamine/anhydrous ethylenediaminetetraacetic acid disodium salt (Tris/EDTA) buffer, which is typically adjusted to the desired pH by the addition of HCl. Without being bound by any theory, it is believed that Tris and EDTA act synergistically to release Lipopolysaccharide (LPS) from the outer membrane of E.coli. EDTA removes divalent cations of LPS in the stabilizing outer membrane. Tris is believed to bind to LPS and replace Ca²⁺And Mg²⁺. This results in a decrease in the interaction between LPS molecules and therefore in an Increase in Permeability of the Outer membrane (Vaara, M.1992.Agents That Increate the Permeability of the Outer membrane. American Society for Microbiology56: 395-411).

Heat treatment step

Preferably, the heat treatment step in the process of the present invention is as described in detail in US5,665,866 (the content of which is incorporated herein by reference). The heat treatment step makes it possible to obtain soluble, correctly folded and assembled antibody samples by facilitating the removal of other antibody-related materials. An antibody that is "properly folded and assembled" appears as a single band on a non-reducing SDS PAGE corresponding to the expected molecular weight of the assembled heavy and light chains. Other antibody-related materials are typically free heavy and light chains or portions thereof, as well as partially degraded fragments of properly folded and assembled antibodies.

The heat treatment step is carried out by bringing the sample to the desired high temperature. Most preferably, the heat treatment step is performed in the range of 30 ℃ to 70 ℃. The temperature may be selected as desired or according to the stability of the antibody being purified. In another embodiment, the temperature is in the range of 40 ℃ to 65 ℃, or preferably in the range of 40 ℃ to 60 ℃, more preferably in the range of 45 ℃ to 60 ℃, even more preferably in the range of 50 ℃ to 60 ℃, most preferably in the range of 55 ℃ to 60 ℃,58 ℃ to 60 ℃ or 59 ℃. Thus, the minimum temperature is 30 ℃, 35 ℃ or 40 ℃ and the maximum temperature is 60 ℃,65 ℃ or 70 ℃.

Preferably, the heat treatment step will last for an extended period of time. The length of time of the heat treatment is preferably between 1 and 24 hours, more preferably between 4 and 18 hours, even more preferably between 6 and 16 hours, most preferably between 10 and 14 hours or 10 and 12 hours, for example 12 hours. Therefore, the heat treatment is carried out for a minimum time of 1, 2 or 3 hours and a maximum time of 20, 22 or 24 hours.

In a particular embodiment, the heat treatment is carried out at 50 ℃ to 60 ℃ for 10 to 16 hours, more preferably at 59 ℃ for 10 to 12 hours. One skilled in the art will appreciate that the temperature and time may be selected to accommodate the characteristics of the sample to be processed and the antibody being produced.

In one embodiment, the method according to the present disclosure does not include a pre-treatment step to maintain the cells under controlled conditions prior to performing the heat treatment step.

In a preferred embodiment, the present invention provides a method for the manufacture of a recombinant antibody molecule comprising culturing a sample of host cells transformed with an expression vector encoding a recombinant antibody molecule and adding an extraction buffer to the sample, and subjecting the sample to a heat treatment step at a temperature in the range of 40 ℃ to 70 ℃ (preferably 59 ℃) for up to 15 hours (preferably 10 to 12 hours); wherein the pH of the sample is monitored and adjusted prior to the heat treatment step, preferably shortly before the heating period, such that the pH of the sample is between pH7 and 8, preferably pH 7.4. Preferably, an extraction buffer having a pH of 7.4 or 8.0 is added to the sample.

Preferably, the heat treatment step is carried out on a shaker set at a suitable RPM, such as 200 RPM. However, the appropriate RPM will vary depending on the scale of the process.

Fermentation of

The step of culturing the host cell sample may comprise fermentation of any desired scale. In the methods of the invention, the sample may be a fermentation product including bacteria, particularly gram negative bacteria, or yeast, cell culture, such as but not limited to mammalian or insect cell culture. Most preferably, the sample is a fermentation product, including E.coli expressing recombinant antibodies, wherein the antibodies produced may be a mixture of functional and non-functional antibodies. If desired, the host cells can be collected from the fermentation medium, for example, the host cells can be collected from the sample by centrifugation, filtration, or by concentration. In particular, the method of the invention is suitable for large scale industrial manufacture of antibodies of therapeutic quality.

Other steps

The method according to the invention may comprise one or more further steps.

In one embodiment, the method according to the invention comprises a centrifugation step after the culturing step, followed by resuspension of the cells by addition of an extraction buffer.

The process may additionally include primary purification methods such as filtration and/or centrifugation. Fluidized bed chromatography is also included. Preferred downstream purification methods include ion exchange chromatography, microfiltration, ultrafiltration, diafiltration, and fixed bed capture and expanded bed capture, and any combination of these.

Non-lysing treatment step

The method may further comprise subjecting the sample to a non-lysing treatment step prior to subjecting the sample to the heat treatment step. This step may also be referred to as a homogenization step (homogenization step). Non-lysing treatment steps can further increase the yield of functional antibodies isolated or obtained and simplify large scale sample processing. Lysis results in an increase in viscosity, which can create problems in downstream processing and purification of functional antibodies. In particular, lysis of the host cells results in the release of host cell proteins, making purification more time and money consuming, as more purification steps may be required and/or larger amounts of chromatographic material are required to achieve the desired purity. Almost complete release of host cell DNA increases the viscosity of the sample, causing difficulties in filtration and centrifugation, which are major causes of protein loss during clarification. Lysed samples (i.e., samples containing host cell proteins and DNA) can also cause clogging of chromatographic materials. Preferably, the non-lysing treatment step is carried out as described in WO2006/054063 (the contents of which are incorporated herein by reference). As described in WO2006/054063, the non-lysing treatment step includes any treatment that does not produce lysis of a substantial proportion of bacteria, mammalian cells, yeast, insect cells or other organisms used for recombinant antibody expression (e.g. e. In a most preferred embodiment, the non-lysing treatment comprises pressure treatment. Alternatively, the non-lysing treatment comprises a pre-conditioning step such as shaking or stirring. "substantial proportion" includes that 80% or more of the organisms in the fermentation or culture are present in intact form, more preferably more than 85%, even more preferably more than 90%, most preferably 95% or more are in intact form.

Lysis may be judged in any manner known in the art, including: by microscopic observation, Fluorescence Activated Cell Sorting (FACS) analysis and determination of total protein compared to protein in supernatant and/or organism (cell) pellet. In one embodiment, the presence or absence of lysis can be judged after non-lytic treatment by comparing the total protein in the sample before and after treatment. If the treatment causes lysis, the amount of total protein present in the supernatant of the treated sample will increase compared to the amount of total protein present in the untreated sample, as measured, for example, using the Bradford assay. In a preferred embodiment, FACS analysis is performed wherein the sample is labeled with a fluorescent dye followed by non-lysing treatment and FACS analysis. Most preferably, FACS analysis is performed prior to treatment to give a baseline value for comparison.

Thus, non-lysing treatment may include preconditioning, for example in buffer, by gently resuspending over a period of time, for example by shaking or stirring, or by manually resuspending, for example by pipetting through a pipette tip. In one embodiment, preconditioning is performed for 1 to 24 hours, more preferably 1 to 20 hours, more preferably 2 to 18 hours, 4 to 16 hours, 6 to 16 hours, and most preferably 12, 14, or 16 hours. Thus, the minimum time for preconditioning is 1, 2 or 4 hours and the maximum time is 16, 18 or 24 hours. The preconditioning may be carried out by spinning at 50-250rpm, preferably at 60-100 rpm, and most preferably for 14 or 16 hours. During preconditioning, the cells are maintained at a temperature in the range of 4 ℃ to 30 ℃, more preferably between 4 ℃ and 20 ℃, and most preferably at room temperature.

In one embodiment, the preconditioning step does not include a machined portion.

In a preferred embodiment, the non-lysing treatment comprises subjecting the host cells to high pressure, for example using french press or nitrogen reduced pressure. In one specific example, the sample is an escherichia coli fermentation product that expresses recombinant antibodies that are subjected to pressure treatment in a french press. The pressure may range from 750psi or thereabouts to 5000psi or thereabouts. In one embodiment, the pressure treatment is performed at 1000psi or 1250psi, 1500psi, 1750psi, 2000psi, 2250psi, 2500psi, 2750psi, 3000psi, 3250psi, 3500psi, 4000psi, 4250psi, 4500psi, or 4750 psi. More preferably, the pressure treatment is carried out at 1000psi to 3000psi, most preferably at 2000 psi. Pressure treatment is almost non-lysing (i.e., less than 20% lysis), and can be determined by simple experimentation, depending on the buffer and the cell type containing the sample, and the pressure.

In one embodiment of the invention, the method does not include a non-lysing treatment step as described above, such as subjecting the host cells to increased pressure or preconditioning by gentle resuspension over a period of time. It is known that the inclusion of such a non-lysing treatment step can improve the yield of recombinant protein (WO 2006/054063). However, we have surprisingly found that improved antibody production is achieved by the methods of the invention which may or may not include such a non-lysing treatment step. Thus, in embodiments in which the method does not include a non-lysing treatment step, the present invention provides a more simplified and economical way of making recombinant antibodies.

Stabilization step

In one embodiment, the method according to the invention comprises the step of interrupting said method between the step of culturing the host sample and the addition of the extraction buffer. In the interruption step, the sample is stabilized at a suitable temperature. Preferably, this step of interrupting the process is carried out as described in WO 2005/019466. This step may also be referred to as the cytosol stabilization step (CSH). Preferably, the process is interrupted for at least one hour, 1 hour to 72 hours, 12 hours to 48 hours, 12 hours, 24 hours, 33 hours or 48 hour period.

Preferably, the sample is stabilized at a suitable temperature, such as 18 ℃, during the interruption of the method.

In one embodiment of the invention, the method does not comprise an interruption step after the step of culturing the host cell sample, as described in WO 2005/019466. It is known that the inclusion of such interruptions increases the yield of recombinant proteins (WO 2005/019466). We have surprisingly found that a similar increase in antibody yield is achieved by the method of the invention, with or without the inclusion of such a disruption step, i.e. no further increase in yield is observed when a disruption step is included in the method. Thus, in embodiments in which the method does not comprise an interruption step, the present invention provides a more simplified and economical way of making recombinant antibodies. Thus, in one embodiment, the time period between the step of culturing the host cell sample and the step of adding the extraction buffer is less than 12 hours, preferably 10 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, 2 hours or less, 1 hour or less than 1 hour.

Antibodies

As used herein, "functional antibodies" include antibody molecules that retain the ability to specifically recognize or bind to the cognate antigen against which they are raised. The production of functional antibodies appears as a single band on non-reducing SDS-PAGE corresponding to the expected molecular weight of the antibody or is determined directly by using BIAcore or other methods known to those skilled in the art, such as but not limited to ELISA. Non-functional antibodies include fragments that do not recognize their cognate antigen, and include incorrectly folded or incorrectly assembled antibodies, free heavy and light chains, and fragments thereof, including partially degraded fragments of an antibody that do not recognize or bind to their cognate antigen.

In a preferred embodiment, the recombinant antibody molecules are at least part of an antibody light chain and at least part of an antibody heavy chain, such that at least some of the expressed light and heavy chain antibody molecules are capable of combining to form a functional antibody.

As used herein, "antibody" includes antibodies having full-length heavy and light chains; a functionally active fragment, derivative or analogue thereof, and may be, but is not limited to, VH, VL, VHH, Fab, modified Fab, altered hinge region Fab, Fab ', F (ab')₂Or an Fv fragment; light or heavy chain monomers or dimers; single chain antibodies such as single chain Fv in which the heavy and light chain variable domains are connected by a peptide linker, or bispecific antibodies such as Fab-dabs, as described in PCT/GB 2008/003331.

The antibody may be a polyclonal, monoclonal, bivalent, trivalent or tetravalent antibody, humanized or chimeric antibody. These antibodies and fragments thereof may be naturally occurring, humanized, chimeric or CDR-grafted antibodies, and amino acids or domains may be modified, added or deleted, if desired, using standard molecular biology techniques. Humanized antibodies are antibodies from non-human species that have one or more Complementarity Determining Regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule (see, e.g., US5,585,089). Antibody molecules purified using the methods of the invention can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass of immunoglobulin molecule.

Methods for producing these antibody molecules are known in the art (see, e.g., Shrader et al, WO 92/02551; Ward et al, 1989, Nature,341:544; Orlandi et al, 1989, Proc. Natl.Acad.Sci.USA,86:3833; Riechmann et al,1988, Nature,322:323; Bird et al,1988, Science,242:423; Queen et al, U.S. Pat. No. 5,585,089; Adair, WO91/09967; Moutain and Adair,1992, Biotechnol. Genet. Eng.Rev,10:1-142; Verma et al, 1998, Journal Immunological Methods,216: 165-.

Monoclonal Antibodies can be prepared by any method known in the art, such as hybridoma technology (Kohler & Milstein,1975, Nature,256: 495-.

Chimeric antibodies are those antibodies encoded by immunoglobulin genes that have been genetically engineered so that the light and heavy chain genes are composed of immunoglobulin gene segments belonging to different species. These chimeric antibodies may be less antigenic. Bivalent antibodies can be generated by methods known in the art (Milstein et al, 1983, Nature 305:537-539; WO93/08829, Traunecker et al, 1991, EMBO J.10: 3655-3659). Bivalent, trivalent, and tetravalent antibodies may include multiple specificities, or be monospecific (see, e.g., WO 92/22853).

Antibody sequences can also be generated using a single lymphocyte antibody method based on the molecular cloning and expression of immunoglobulin variable region cDNAs generated from a single lymphocyte selected for the production of a specific antibody, as described in Babcook, J.et al, 1996, Proc.Natl.Acad.Sci.USA 93(15): 7843-. The latter method relies on the isolation of individual antibody-producing cells, which are then clonally expanded, followed by selection of clones producing antibodies recognizing their cognate antigen, and, if desired, followed by their variable heavy chain (V)_H) And light chain (V)_L) The sequence of the gene is identified. Alternatively, cells producing antibodies that recognize their cognate antigen can be cultured together after screening.

The antibodies produced using the methods of the invention are most preferably humanized antibodies, which can then be linked to toxins, drugs, cytotoxic compounds or multimers or other compounds that can prolong the half-life of the antibody after administration to a patient.

The antibody may be specific for any target antigen. The antigen may be a cell-bound protein, for example a cell surface protein or a soluble protein on a cell, such as a bacterial cell, a yeast cell, a T-cell, an endothelial cell or a tumor cell. The antigen of interest may also be any medically relevant protein, such as a protein that is upregulated in disease or infection, e.g., a receptor and/or its corresponding ligand. Specific examples of cell surface proteins include adhesion molecules, e.g. integrins such as β 1 integrins, e.g. VLA-4, E-selectin, P-selectin or L-selectin, CD2, CD3, CD4, CD5, CD7, CD8, CD11a, CD11b, CD18, CD19, CD20, CD23, CD25, CD33, CD38, CD40, CD40 40, CD134(OX40), ICOS, BCMP 40, CD137, CD27 40, CDCP 40, CSF 40 or CSF 40-receptor, DPCR 40, dulindu 40, FLJ 84, FLJ 78407, HEK 40, KIAA0634, KIAA0659, KIAA1246, mrkiaa 5, mr3672, MHC 20572, MHC 2053672, MHC 40, nk3672, MHC 40, tnf 40, protein receptor types including optionally human milk fat receptor antigens, MHC receptor, protein, ttc 40, nkt 40, MHC receptor, MHC 40, MHC receptor types including ttc 40, MHC receptor d40, MHC receptor, MHC 40, MHC receptor types including ttc 40, MHC receptor types (E-40, ttc 36.

Soluble antigens include interleukins such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-12, IL-13, IL-14, IL-16 or IL-17 such as IL17A and/or IL17F, viral antigens such as respiratory syncytial virus or cytomegalovirus antigens, immunoglobulins such as IgE, interferons such as alpha, beta or gamma interferon, tumor necrosis factor TNF (previously referred to as tumor necrosis factor-alpha), tumor necrosis factor-beta, colony stimulating factors such as G-CSF or GM-CSF, and platelet-derived growth factors such as PDGF-alpha and PDGF-beta, and where appropriate receptors therefor. Other antigens include bacterial cell surface antigens, bacterial toxins, viruses such as influenza, EBV, HepA, B and C, bioterrorism agents, radionuclides and heavy metals, and snake and spider venoms and toxins.

In one embodiment, the antibody can be used to functionally alter the activity of an antigen of interest. For example, an antibody may directly or indirectly neutralize, antagonize, or agonize the activity of the antigen.

In a preferred embodiment, the antibody is an anti-TNF antibody, more preferably an anti-TNF Fab', as described in WO01/094585 (the contents of which are incorporated herein by reference).

Methods for expressing recombinant proteins are known in the art. Suitable examples of host cells for expression of recombinant antibody molecules include bacteria such as gram-positive or gram-negative bacteria, e.g. e.coli, or yeast cells, e.g. brewer's yeast (s.cerevisiae) or mammalian cells, e.g. CHO cells and myeloma or hybridoma cell lines, e.g. NSO cells. Most preferably, in the methods of the invention, recombinant antibodies are produced in bacteria such as E.coli (see Verma et al,1988, J.Immunol. methods 216:165-181; Simmons et al, 2002, J.Immunol. methods 263: 133-147).

Cells

The term "sample" as used herein refers to a population of cells transformed with an expression vector encoding a recombinant antibody molecule. The sample may be of any suitable scale, from small-scale antibody production to large-scale antibody manufacture for commercial purposes.

The cells used in the present invention may be, for example, but not limited to, bacteria (particularly gram-negative bacteria), yeast, mammals or insects. Most preferably, the cell is E.coli. The cell may be a wild-type cell or a genetically engineered recombinant cell. The E.coli host cell may be a naturally occurring E.coli strain or a mutant strain capable of producing a recombinant protein. Examples of specific host E.coli strains include MC4100, TG1, TG2, DHB4, DH5 α, DH1, BL21, K12, XL1Blue and JM 109. An example is E.coli W3110(ATCC 27,325), a commonly used host strain for recombinant protein fermentation. Examples also include modified E.coli strains, such as metabolic mutants and protease deficient strains.

Recombinant antibodies produced using the methods of the invention are typically expressed in the periplasm of E.coli host cells or in the host cell culture supernatant, depending on the nature of the protein and the scale of production. Methods for targeting proteins to these compartments are known in the art and are reviewed in Makrides, Microbiological Reviews,1996,60, 512-. Examples of suitable signal sequences for introducing proteins into the periplasm of E.coli cells include E.coli PhoA, OmpA, OmpT, LamB and OmpF signal sequences. Proteins can be targeted to the supernatant by causing protein secretion either by relying on the natural secretory pathway or by inducing restricted leakage of the outer membrane, examples being the use of pelB leader, protein a leader, co-expression with a bacterin release protein, expression of mitomycin-induced bacterin release protein and addition of glycine to the culture medium, and co-expression of the kil gene associated with membrane permeability. Most preferably, in the method of the invention, the recombinant protein is expressed in the periplasm of E.coli cells.

Expression of the recombinant protein in the E.coli host cell may also be controlled by an inducible system, and thus expression of the recombinant protein in the E.coli host cell may also be controlled by an inducible promoter. A number of inducible promoters which can be used in E.coli are known in the art, and depending on the nature of the promoter, expression of the recombinant protein can be induced by different factors, such as temperature or the concentration of particular substances in the growth medium (Baneyx, Current Opinion in Biotechnology,1999,10: 411. sup. 421; Goldstein and Doi,1995, Biotechnology. Annu. Rev, 105. sup. 128). Examples of inducible promoters include the E.coli lac, tac and trc promoters, which are induced by lactose or non-hydrolysable lactose analogues, isopropyl-beta-D-1-thiogalactoside (IPTG), and the phoA, trp and araBAD promoters, which are induced by phosphate, tryptophan and L-arabinose, respectively. Expression can be induced, for example, by the addition of an inducer or by a change in temperature (when induction is temperature dependent). In the case where induction of recombinant protein expression is achieved by adding an inducer to the culture, the inducer may be added by any suitable method, depending on the fermentation system and the inducer, for example, by single or multiple additions or by feeding stepwise additions of the inducer. It will be appreciated that there may be a delay between the addition of the inducer and the actual induction of protein expression, for example where the inducer is lactose, a delay may occur before induction of protein expression when any existing carbon source is used before lactose.

The E.coli host cell culture (fermentate) may be cultured in any medium that supports E.coli growth and expression of the recombinant protein. The medium may be any chemically defined medium, such as those provided in Principles of Microbe and Cell culture, Blackwell Scientific Publications, Pirt S.J (1975), which may be modified as appropriate to control growth rate, as described herein. An example of a suitable medium is "SM 6E", as described by Humphreys et al, 2002, Protein Expression and purification,26: 309-.

The cultivation of the E.coli host cells may be carried out in any suitable vessel, such as a shake flask or a fermenter, depending on the scale of production desired. There are a variety of large scale fermenters available, having a volume of over 1,000 liters up to about 100,000 liters. Preferably, a1,000-50,000 liter fermentor is used, more preferably 1,000-10,000 or 12,000 liters. Smaller scale fermenters, with a volume of 0.5-1,000 liters, may also be used.

The fermentation of E.coli may be carried out in any suitable system, for example in a continuous, batch or batch fermentation system (Thiry & Cinglani, 2002, Trends in Biotechnology,20: 103-105), depending on the desired protein and yield. When batch fermentation is used, nutrients or elicitors can be added at one time when needed. Alternatively, a batch culture may be used, the culture grown in a batch fermentation prior to induction has the maximum specific growth rate, and the culture growth may be maintained until fermentation is complete using the nutrients initially present in the fermentor and one or more feeding rules for controlling the growth rate. Batch fermentation can also be used prior to induction to control the metabolism of E.coli host cells and to achieve higher cell densities (Lee, 1996, Tibtech,14: 98-105).

Preferred features of each embodiment of the present invention are the result of modifications, as necessary, made to other embodiments. All publications, including but not limited to patents and patent applications cited in this specification are herein incorporated by reference, and for each individual publication specifically and individually indicated to be incorporated by reference, although the entirety of each individual publication has been specifically and individually indicated to be incorporated by reference herein as if fully set forth herein.

In one aspect, antibodies obtained or obtainable from the methods are provided.

In one aspect, there is provided the use of pH control means, such as a buffer, for improving the extraction, e.g. primary extraction, of antibodies, particularly wherein said control ensures that the pH in an extraction step, such as a thermal extraction step, is maintained within a range of pH 6-9.

The pH control means as employed herein are buffers, bases and/or acids.

The invention will now be described with reference to the following examples, which are illustrative only and are not to be construed as limiting the scope of the invention in any way.

FIG. 1 is a graph showing the pH of cells resuspended in Tris/EDTA extraction buffer at pH7.4 and pH8 over a period of time.

FIG. 2a is a histogram showing the effect of pH of the extraction buffer on antibody A production.

FIG. 2b is a histogram showing the pH of the cell sample (resuspended cell paste) immediately after addition of the extraction buffer with pH 7.4-9.0 and the pH of the cell sample 1 hour after addition of the buffer but before the heating period.

FIG. 3a is a histogram showing the effect of adjusting the pH of the sample before the heat treatment step on antibody A production. The numbers above each bar represent the percent increase in yield compared to the control without the pH adjustment step.

Figure 3b is a graph showing the pH change of the sample at various stages of the process: cell paste (after culture and centrifugation); after addition of buffer (immediately after addition of extraction buffer); before pH adjustment; after pH adjustment, before the heating period; and after the heat treatment step.

FIG. 4 shows SDS-PAGE analysis of antibody A samples extracted from cells after heat treatment. Lane 1 is the molecular weight marker, lane 2 is the antibody a sample, lane 3 is the sample without pH adjustment, and lanes 4-8 show the samples after pH adjustment to 7.0,7.2,7.4,7.6, and 7.8, respectively, prior to the heat treatment step.

FIG. 5 is a histogram showing the effect on antibody A production using an extraction buffer of pH8 and adjusting the sample pH to pH7.4 prior to the heat treatment step. Figure 5 also shows the effect of including a homogenization step or a cell paste stabilization step. The numbers above each bar represent the percent increase in yield compared to the control with homogenization but without the pH adjustment step.

FIG. 6 shows SDS-PAGE analysis of antibody A samples extracted from cells after heat treatment.

Lane 1 is molecular weight markers;

lane 2 is antibody a sample;

lane 3 is a sample after the homogenization step but without pH adjustment and without cytoplasm stabilization;

lane 4 is a sample that was treated with extraction buffer pH8 and adjusted to pH7.4 before heat treatment, and that was subjected to a homogenization step without cytoplasm stabilization;

lane 5 is a sample without pH adjustment, without homogenization, and without cytosol stabilization;

lane 6 is a sample that was adjusted to pH7.4 after treatment with extraction buffer pH8 and before heat treatment, and that was not homogenized and cell paste stabilized;

lane 7 is a sample after the cytosol stabilization step but without pH adjustment, without homogenization;

lane 8 is a sample that was adjusted to pH7.4 after treatment with extraction buffer pH8 and before heat treatment, and that was cytosolically stabilized without homogenization.

FIG. 7 is a graph showing the pH of samples over time, respectively: a control sample without pH adjustment, a sample treated with an extraction buffer of pH8, a sample treated with an extraction buffer of pH7.4 and pH adjusted to pH7.4 prior to the heat treatment step, and a sample treated with an extraction buffer of pH8 and pH adjusted to pH7.4 prior to the heat treatment step. The first peak indicates the point of addition of extraction buffer and the second peak indicates the point at which the pH of both samples was adjusted prior to the heat treatment step.

FIG. 8 is a histogram showing the effect on antibody A production on control samples without pH adjustment, samples treated with extraction buffer pH7.4 and the pH of the samples adjusted to pH7.4 prior to the heat treatment step, samples treated with extraction buffer pH8, and samples treated with extraction buffer pH8 and the pH of the samples adjusted to pH7.4 prior to the heat treatment step. The numbers above each bar represent the percent increase in yield compared to the control without pH adjustment.

Figure 9 is a histogram showing the effect on antibody a production on control samples without pH adjustment, samples treated with extraction buffer pH7.4 and adjusting the sample pH to pH7.4 in the heating phase prior to the heat treatment step, and samples treated with extraction buffer pH8 and adjusting the sample pH to pH7.4 in the heating phase prior to the heat treatment step. The numbers above each bar represent the percent increase in yield compared to the control without pH adjustment.

Figure 10 is a histogram showing the effect of pH adjustment to 6.6, 7.0, 7.4 and 7.8 units on Fab' titer compared to non-pH adjusted controls. The experiment was repeated with three different pretreatment steps (no pretreatment, cytosol stabilization and homogenization, respectively) before extraction.

FIG. 11 is a histogram showing the mean Fab' titers under the following conditions: no pH adjustment, no pretreatment and pH adjustment (to a range of 6.6-7.8 units), homogenization and pH adjustment (to a range of 6.6-7.8 units) and all pH adjustment conditions (homogenization and no pretreatment). Error bars show the standard deviation of the mean.

General procedure

In the following examples, unless otherwise indicated, the process was carried out as follows:

cell culture step and centrifugation:

antibody A (Fab') was expressed in E.coli W3110 cells using vector pTT0D with DNA insert encoding antibody A. Fermentation was carried out for 30 hours at 25 ℃ after induction with lactose for harvest. 50ml or 1L of the harvested culture samples were centrifuged at 4200RPM for 1 hour at 4 ℃.

The supernatant was discarded and to simulate the production scale clarification process, a small portion of the supernatant was added to the cells such that the resulting cells would make up 35% of the harvested weight of the sample.

Cell paste stabilization step (CSH):

in some experiments a cell plasma stabilization step was performed, in which the samples were stabilized at 18 ℃ and 200RPM for 33 hours before addition of extraction buffer.

Addition of extraction buffer:

stocks of 300mM Tris and 30mM EDTA were prepared to final concentrations of 100mM Tris and 10mM EDTA, adjusted to pH7.4 with hydrochloric acid, and used to resuspend the resulting plasma samples (hereinafter referred to as samples). In the experiments described below, the pH of the extraction buffer was adjusted from the pH of the control, 7.4, to a higher pH level, between pH7.4 and 9.0.

Homogenization step (homogenization):

in some experiments, the homogenization step was performed by passing the sample a single pass at 1500psi after addition of the extraction buffer.

pH adjustment before heating period:

in some experiments, the pH of the samples was adjusted to the desired level, between 7.0 and 7.8, with 5M NaOH before the heating period began.

Heating period:

the sample is subjected to a heating period in which the temperature of the sample is raised from 18 ℃ to the desired elevated temperature of 59 ℃, at which temperature the heat treatment step is initiated.

pH adjustment during heating period:

in some experiments, the pH of the sample was adjusted to the desired level, 7.4 ± 0.02, with 5M NaOH during the heating period until the desired high temperature of 59 ℃ was reached.

A heat treatment step:

the sample was stabilized at 59 ℃ at 200RPM for 10-12 hours.

After heat treatment, the resuspended cell pellet was clarified by centrifugation at 4200rpm for 1 hour at 4 ℃. Fab' in the supernatant containing functional antibody a was determined using protein G HPLC analysis in 20mM phosphate buffer. Antibody A was eluted using a pH gradient solution where pH 7.0 was reduced to pH2.5 at the time of injection.

The reduced extract was analyzed on a Tris-glycine SDS-PAGE gel at a loading concentration of about 1. mu.g.

Example 1 Effect on sample pH after addition of extraction buffer

The cell culture step and the step of adding an extraction buffer, wherein the buffer has a pH of 8.0 or a pH of 7.4, are performed as described in the general methods section. The pH of the sample was measured from the time of addition of the extraction buffer. Figure 1 shows that the sample pH drops rapidly after addition of buffer and that the pH drops rapidly below pH7, especially when the buffer has a pH of 7.4.

Example 2 Effect of extraction buffer pH on antibody production

The cell culture step, the addition of extraction buffer step, the heating phase and the heat treatment step are performed as described in the general methods section.

The cell paste stabilization step, homogenization step and pH adjustment step were not performed before or during heating.

The pH of the extraction buffer varied as follows: 7.4, 8.0.8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 and 9.0. The results are shown in FIG. 2a, showing the Fab' concentration after heat extraction. It can be seen that raising the pH of the extraction buffer above 7.4 results in a significant increase in Fab' recovery, up to pH 8.8 can lead to increased yields. Above 8.8 the Fab' concentration begins to decrease.

TABLE 1

Table 1 and fig. 2b show the pH of the cell sample (resuspended cell paste) immediately after addition of the extraction buffer with pH 7.4-9.0 and the pH of the cell sample 1h after addition of the extraction buffer and before the heating period.

Example 3 Effect of pH before heating phase on antibody production

The cell culture step, the extraction buffer addition step, the homogenization step, the pH adjustment step before heating, the heating period and the heat treatment step were performed as described in the general methods section. The control did not undergo a pH adjustment step.

In the control experiment, the extraction buffer was pH7.4, in other experiments the pH was adjusted before the heating period, and the extraction buffer was pH 8.0.

The cell paste stabilization step and the pH adjustment step in heating were not performed.

In the control, no pH adjustment was performed prior to the heating period. In other experiments, the pH of the samples was adjusted to pH 7.0,7.2,7.4,7.6 and 7.8 prior to the heating period.

The results are shown in figure 3a, which shows that this pH adjustment step results in improved Fab' recovery. Figure 3a shows how a set point in the pH range of 7.0 to 7.8 increases product recovery by 26-40% compared to a control sample without pH adjustment.

TABLE 2

In the method, the pH of the sample is measured at different time points. Table 2 and figure 3b above show the pH change of the samples at different stages of the process: cell paste (after culture and centrifugation); after addition of buffer (immediately after addition of extraction buffer); before pH adjustment; after pH adjustment but before the heating period; and after the heat treatment step.

The SDS-PAGE gel in FIG. 4 shows the protein profile of the sample after extraction.

Lane 1 is the molecular weight marker, lane 2 is the sample of antibody a, lane 3 is the sample without pH adjustment, and lanes 4-8 show samples with pH adjusted to 7.0,7.2,7.4,7.6 and 7.8, respectively, prior to the heat treatment step.

The sample loading was normalized to 1. mu.g of Fab'. No significant difference in protein profile was observed between the control and the pH adjusted samples prior to heat treatment.

Example 4 Effect of extraction buffer pH and pH adjustment on antibody production with or without the cell paste stabilization and homogenization steps

The following experiments were performed as described in the general methods section.

Control (homogenized): a cell culture step, a step of adding an extraction buffer (pH 7.4), a homogenization step, a heating period, and a heat treatment step;

buffer pH8 and adjusted to pH7.4 before heating (homogenized): a cell culture step, a step of adding an extraction buffer (pH 8), a homogenization step, a step of adjusting pH to pH7.4 before heating, a heating period, and a heat treatment step;

control (no homogenization or CSH): a cell culture step, wherein an extraction buffer solution is added (pH 7.4), a heating period and a heat treatment step are performed;

buffer pH8 and adjusted to pH7.4 before heating (not homogenized or CSH): a cell culture step of adding an extraction buffer (pH 8), adjusting the pH to pH7.4 before heating, a heating period, and a heat treatment step;

control (over CSH): a cell culture step, a cell plasma stabilization step, an extraction buffer adding step (pH 7.4), a heating period and a heat treatment step; and

buffer pH8 and adjusted to pH7.4 before heating (over CSH): a cell culture step, a cell plasma stabilization step, an extraction buffer addition step (pH 8), a step of adjusting pH to pH7.4 before heating, a heating period, and a heat treatment step.

Fig. 5 shows the results of the above experiment. It can be seen that the addition of a pH adjustment step prior to heating resulted in a-34% increase in extraction titer compared to the control. It can also be seen that the inclusion of the homogenization step has no effect on the yield compared to the process with a pH adjustment step. Cytosolic stabilization provided increased yield when compared to the control extract, but not when compared to the extract with the pH adjustment step.

The SDS-PAGE gel of FIG. 6 shows the protein profile of the samples after extraction.

Lane 1 is molecular weight markers;

lane 2 is antibody a sample;

lane 3 is a sample after the homogenization step but without pH adjustment and without cytoplasm stabilization;

lane 4 is a sample that was treated with extraction buffer pH8 and adjusted to pH7.4 before heat treatment, and that was subjected to a homogenization step without cytoplasm stabilization;

lane 5 is a sample without pH adjustment, without homogenization, and without cytosol stabilization;

lane 6 is a sample that was adjusted to pH7.4 after treatment with extraction buffer pH8 and before heat treatment, and that was not homogenized and cell paste stabilized;

lane 7 is a sample after stabilization of the cytoplasm but without pH adjustment, without homogenization;

lane 8 is a sample that was adjusted to pH7.4 after treatment with extraction buffer pH8 and before heat treatment, and that was cytosolically stabilized without a homogenization step.

The protein profile of the pH adjusted extract was similar to the results of the cytosol stabilized control.

Example 5 Effect of the extraction buffer pH and/or pH adjustment step before heating on sample pH and Fab' production.

The cell culture step, the addition of extraction buffer step, the heating phase and the heat treatment step are performed as described in the general methods section. Two experiments included pH adjustment prior to the heating step, and two did not.

The cell paste stabilization step, homogenization step and pH adjustment step in heating were not performed.

Four different pH control protocols were performed:

1. comparison: the pH of the extraction buffer solution is 7.4, and the pH is not adjusted before heating;

2. the extraction buffer pH was 7.4, the pH was adjusted to pH7.4 before heating;

a buffer solution with pH8, wherein the pH of the extraction buffer solution is 8.0, and the pH is not adjusted before heating; and

4. the extraction buffer pH was 8.0 and the pH was adjusted to pH7.4 before heating.

The pH was monitored throughout the primary recovery, starting from the cytosol until addition of buffer (first peak), pH adjustment before heating (second peak) and heat treatment (pH drop). Figure 7 shows the pH profile in these methods.

The effect on Fab ' yield is shown in FIG. 8, where it can be seen that all pH-increasing protocols (1-3) resulted in increased Fab ' yield, while protocol 4 used a high buffer, combined with pH adjustment prior to heating, resulting in the highest Fab ' recovery.

Example 6 Effect of pH of extraction buffer and/or pH adjustment step during heating on sample pH and Fab' production.

The cell culture step, the addition of extraction buffer step, the heating phase and the heat treatment step are performed as described in the general methods section. Two experiments included pH adjustment during the heating step, while the control did not.

The cell paste stabilization step, homogenization step and pH adjustment step before heating were not performed.

Different pH control protocols were implemented:

1. comparison: extraction buffer pH7.4 and no pH adjustment during heating;

extraction buffer pH7.4 and adjusting pH to 7.4 while heating;

extraction buffer pH 8.0 and adjusting the pH to 7.4 during heating.

The effect on Fab ' yield is shown in figure 9 and it can be seen that all pH-raising protocols (2 and 3) resulted in increased Fab ' yield, whereas protocol 4 used high buffer, combined with pH adjustment during heating, resulting in the highest Fab ' recovery.

Example 7

The experiment was carried out as follows: most of the spent medium is removed by removing the fermentation broth and centrifuging and thereby producing a cell paste. In the case of cytosolic stabilization, this cytosol was stabilized for 33 hours. Cells were resuspended in extraction buffer with homogenization, without pretreatment, and either homogenized or heat extracted without any pretreatment. After the cell paste was stabilized, the cells were resuspended in extraction buffer. Under all conditions, once resuspended in extraction buffer, the pH was adjusted to the desired set point (shown in figure 12) and the thermal extraction process was initiated (10 hours at 59 ℃). The extract was clarified by centrifugation after hot extraction to determine Fab' titers in the liquid phase.

The following data is also shown in figure 12.

No pretreatment, no pH

Situation of adjustment

Pretreatment of	pH adjustment	After extraction (g/L)	Increase of control (%)
				CSH	na	0.419	19.27
CSH	n/a	0.402	14.52
				CSH	7	0.412	17.40
CSH	7.4	0.408	16.29
				CSH	7.8	0.435	23.86
Is free of	na	0.351	0.00
				Is free of	6.6	0.421	19.89
Is free of	7	0.412	17.40
				Is free of	7.4	0.399	13.63
Is free of	7.8	0.403	14.72
				Homogenization	na	0.359	2.29
Homogenization	6.6	0.392	11.79
				Homogenization	7	0.394	12.16
Homogenization	7.4	0.403	14.80
				Homogenization	7.8	0.420	19.60

The control was a control without pretreatment and without pH adjustment

Claims (10)

1. A method for making a recombinant antibody molecule comprising

a) Culturing a sample of host cells transformed with an expression vector encoding a recombinant antibody molecule;

b) adding an extraction buffer to the sample; and

c) carrying out a heat treatment step on the sample;

wherein the pH of the host cell sample is detected after addition of the extraction buffer and optionally adjusted to ensure that the pH of the sample is 6-9 prior to the heat treatment step.

2. The method according to claim 1, wherein the heat treatment step is performed in the range of 30 ℃ to 70 ℃.

3. The method according to claim 1, wherein the extraction buffer has a pH of 7.5 to 9.

4. The method according to claim 1, wherein the extraction buffer is selected from NaOH, NH₄OH, sulfuric acid, EDTA, Tris buffer, and combinations thereof.

5. The method according to claim 1, wherein the antibody is selective for tumor necrosis factor or CD 40L.

6. The method according to claim 1, wherein the antibody molecule is selected from the group consisting of: VH, VL, VHH, Fab, modified Fab, altered hinge region Fab, Fab ', F (ab') 2 or Fv fragments; light or heavy chain monomers or dimers; single chain antibodies in which the heavy and light chain variable domains are linked by a peptide linker, and bispecific antibodies.

7. The method according to claim 6, wherein the single chain antibody is a single chain Fv.

8. A method according to claim 6 wherein the bispecific antibody is a Fab-dAb.

9. The method according to claim 1, wherein the pH is adjusted to ensure that the pH of the sample is 6-9 before the heat treatment step.

10. Use of a buffer to improve antibody extraction, wherein the buffer maintains the pH shortly before the heat extraction step in the range of pH 6-9.