EA042536B1 - GETTING PROTEIN - Google Patents

Description

The field of technology to which the invention belongs

The present invention relates to a method for obtaining a protein expressed in a host cell; more specifically, the invention relates to a method for producing a protein that results in a protein having a reduced content of related impurities.

The state of the art to which the invention relates

In the field of therapeutics, the use of proteins and antibodies, and in particular of molecules derived from antibodies, has always been and has been of importance, and therefore, at the same time, the need for a controlled method for their production has developed. The commercialization of therapeutic proteins requires their production in large quantities. To this end, the protein is often expressed in a host cell, followed by reduction and purification prior to being converted into a usable form.

Depending on the protein to be expressed, the host cell of choice may be a mammalian host cell, often a CHO (Chinese Hamster Ovary) cell or a bacterial host cell. In the first case, the protein is usually extracted into the cell culture supernatant, which is reconstituted and the solution is further subjected to a protein purification procedure.

In the case where the host cell is a gram-negative prokaryotic cell, the often preferred expression system includes the newly synthesized protein accumulated therein and extracted from the periplasmic space. In this case, after reaching the desired level of protein expression, it is the cells that are harvested and processed. The protein is further recovered by subjecting the harvested cells to a protein extraction process, which involves releasing the protein from the periplasm into solution, followed by removal of cellular debris and other impurities. These steps from cell harvest to protein release are usually included in what is referred to as primary recovery. The resulting protein containing solution is then subjected to a protein purification procedure. Preferred Gram-negative prokaryotic cells used for periplasmic expression are in general strains of Escherichia coli or Pseudomonas fluorescens cells.

Recovery of a heterologous protein expressed in a prokaryotic host cell during primary extraction from the periplasmic space is often a problem. In this direction, various methods are described in the prior art. For example, US Pat. No. 5,655,866 describes the use of heat treatment to facilitate subsequent extraction of functional Fab' antibody fragments. WO 2006/054063 describes the inclusion of a non-lyzing treatment in combination with heat treatment in the primary extraction step. Application 2005/019466 describes the inclusion of an interruption step after the fermentation step but before extraction. US 2013/0060009 describes adjusting the pH of samples prior to their heat treatment during the extraction step.

Despite this, another significant problem is the high concentration of impurities present after the primary extraction, which increases the burden on subsequent purification steps and the overall efficiency of the entire purification process. These impurities may be in the form of cellular debris, host cell proteins (HCPs), DNA, or related impurities such as processed forms of expressed products, aggregates, or other modified forms such as deamidated, isomerized, oxidized, and other conjugated forms. Among them, recombinant protein degradation products, also called related impurities, are often the most difficult to remove during the heat extraction and primary reduction process because they have similar physicochemical properties to the target protein, such as melting point (Tm).

Although the final protein solution obtained after extraction will then be subjected to the protein purification process, the purity of this solution affects the overall efficiency and costs associated with the purification steps and thus the production of the therapeutic protein as a whole. Specifically related impurities affect the quality profile of the final product, the control of which is particularly important for consistency across batches.

For this reason, there remains a need in the art for methods that improve the removal of contaminants during protein extraction from cell culture.

Brief description of the drawings

In FIG. 1 shows the redox potential and temperature profiles of the extracts. As can be seen, maintaining the nitrogen layer during the cooking step maintains low redox values throughout, where the minimum is approximately -435 mV and is typically -375 mV while maintaining a temperature of 59°C. A similar low redox potential is observed with nitrogen bubbling. When the buffer containing the host cells is supplied with air instead of nitrogen, it can be seen that the redox potential values rise significantly during extraction during the heating step and become positive (typically approximately +45 mV) while maintaining the temperature at 59°C. A similar high redox potential is observed with air bubbling. The gas bubbling is converted to a bed in the course of the extraction due to the observed high levels of foaming.

In FIG. 2 shows sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of samples obtained after extraction. Lane M represents standard molecular weight markers (Mark 12, Invitrogen). Lane 1 is the reference standard for anti-TNF (TNF) alpha Fab'. Lane 2 is the DsbC standard (protein disulfide isomerase). Line 3 is a post-extraction sample where a nitrogen blanket is applied during extraction. Line 4 represents the sample after extraction, where air is applied during extraction. Line 5 represents the sample after extraction, where nitrogen is bubbled through the sample during extraction. Line 6 represents the sample after extraction, where air is bubbled through the sample during extraction. The figure demonstrates that in the presence of nitrogen, the reducing conditions during the heat extraction process are sufficient to provide a significant reduction in the concentration of related impurities compared to extractions carried out in the presence of air.

In FIG. 3 shows the total number of Fab' fragments, expressed as % of all related compounds, as measured by cation exchange (CEX) (high performance liquid chromatography (HPLC, HPLC) after purification of protein L. One can observe the obvious effect that the supply of a layer of nitrogen on purification from related impurities.Extract #8, where the nitrogen bed is not applied, has a higher concentration of related impurities.On the other hand, the figure also suggests that the speed of the nitrogen bed can affect the clearance of these impurities when comparing extracts 1 and 2 as well as 7 and 8.

In FIG. 4 shows the total number of Fab' fragments, expressed as % of all related compounds, as measured by cation exchange HPLC after purification of protein L. The results again demonstrate the advantage of the presence of nitrogen, which leads to a significant reduction in the concentrations of related impurities remaining after extraction by heating. An additional beneficial effect is obtained by adjusting the pH before the extraction by heating, since it appears that the concentrations of related impurities are further reduced compared to the previous examples.

In FIG. 5 shows the end point of the measurement of the redox potential as a function of the nominal volume of the shake flask. These results demonstrate that shake flasks that are made in the presence of a nitrogen blanket maintain a more reducing extract environment compared to flasks that are made in the presence of air. The figure also shows that the larger nominal volume flasks are more reducing than the lower nominal volume flasks, most likely due to the slower rate of extract oxidation due to the smaller surface area, with no correlation with the nominal volume.

In FIG. 6 shows the total number of Fab' fragments, expressed as % of all related compounds, as measured by cation exchange HPLC after purification of protein L. These results demonstrate that shake flasks, which are made in the presence of nitrogen, have significantly lower concentrations of related impurities after extraction. . All flasks prepared in the presence of nitrogen (ROP endpoint range of -10 to -116 mV) show significantly lower concentrations of related impurities compared to all flasks prepared in the presence of air (ROP endpoint range of +79 up to +97 mV).

In FIG. 7 shows the effect of pH and reducing medium on the Tm of purified Fab' fragments. The figure shows the results of determining the thermal stability for the two main compounds of related impurities. A correlation has been demonstrated between the redox potential and thermal stability (melting point); the lower the redox potential, the lower the melting point of the protein. A similar effect can be described for pH as it becomes more neutral. Both indicators provide a greater propensity for related impurities to degrade, thus facilitating their removal.

Detailed description of the invention

The present invention addresses the need outlined above for providing a new method for producing a protein, wherein the protein is expressed in a host cell. In particular, the present invention provides a method suitable for industrial production.

In a first embodiment, the invention provides a method for producing a protein of interest, wherein said protein is expressed in a host cell, comprising: culturing said host cells under conditions that cause them to express said protein;

collecting host cells from the culture fluid;

adding buffer to host cells; and subjecting the host cells to the heat treatment buffer, wherein the redox potential of the buffer is maintained below 0 mV during said heat treatment step.

In a specific embodiment, the host cells are prokaryotic cells, preferably Gram-negative bacteria. In another embodiment, host cells

- 2 042536 are E. coli cells or P. fluorescens cells. Prokaryotic host cells for protein expression are well known in the art (Terpe, K. (2006), Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems, Appl. Microbiol. Biotechnol., 72, 211 -222). Host cells according to the invention include recombinant cells that have been genetically engineered to produce a protein of interest, such as an antibody fragment. Recombinant E. coli host cells can be obtained from any suitable E. coli strain, including MC4100, TG1, TG2, DHB4, DH5a, DH1, BL21, K12, XLlBlue, and JM109. One example is E. coli strain W3110 (ATCC 27.325), a host strain often used for recombinant protein fermentation. Antibody fragments can also be obtained by culturing modified E. coli strains, such as metabolic mutants or E. coli strains lacking a protease, such as those disclosed in WO 2011/086138, WO 2011/086139, WO 2011/086136 and WO 2013/007388 which are incorporated herein in their entirety.

The protein of interest that can be purified according to the methods of the present invention is typically extracted from the periplasm of the E. coli host cell. Methods for targeting proteins to this compartment are well known in the art (Makrides, S.C. (1996), Strategies for achieving high-level expression of genes in Escherichia coli, Microbiol. Rev., 60, 512-538). Examples of suitable signal sequences for directing proteins to the E. coli periplasm include the E. coli PhoA, OmpA, OmpT, LamB and OmpF signal sequences.

Expression of the recombinant protein in E. coli host cells can also be under the control of an inducible system, whereby expression of the recombinant antibody in E. coli is under the control of an inducible promoter. A variety of inducible promoters suitable for use in E. coli are known in the art and, depending on the expression of the recombinant protein promoter, can be induced by various factors such as temperature or the concentration of a particular substance in the growth medium. Examples of inducible promoters include the E. coli lac, tac, and trc promoters, which are induced by lactose or the non-hydrolyzable lactose analog, isopropyl-B-D-1-thiogalactopyranoside (IPTG, IPTG), and the phoA, trp, and araBAD promoters, which are induced by phosphate. , tryptophan and L-arabinose, respectively. Expression can be induced by, for example, adding an inducer or changing temperature, in case the induction is temperature dependent. In the case where induction of recombinant protein expression is achieved by adding the inducer to the culture, the inducer can be added by any suitable method depending on the fermentation system and the inducer, for example, by single or multiple injections, or by gradually adding the inducer through the supply system. It will be appreciated by those skilled in the art that there may be a delay between the addition of the inducer and the direct induction of protein expression, for example where the inducer is lactose there may be a delay before induction of protein expression as any already existing carbon source is utilized to lactose.

Alternatively, a protein of interest that can be purified according to the methods of the present invention is extracted from the periplasm of P. fluorescens. Suitable sequences that target proteins to the periplasmic space of P. fluorescens include sec-type secretion leader proteins as described in the art (D.M. Retallack et al., Transport of heterologous proteins to the periplasmic space of Pseudomonas fluorescens using a variety of native signal sequences, Biotechnol Lett., 29 (2007), pp. 1483-1491). Expression of a protein of interest may be under the control of an inducible system, where expression is under the control of an inducible promoter, as described in the previous paragraph. In this case, well-known inducers such as IPTH can be used, and alternatively inducers such as anthranilate benzoate, which induces the Pben509 and Pben278 promoters, respectively, as described in P. fluorescens expression systems.

Prokaryotic host cell cultures (fermentations) can be grown in any medium that supports host cell growth and recombinant protein expression. The medium may be any chemically defined medium such as, for example, described for E. coli in Durany O. et al. (2004), Studies on the expression of recombinant fuculose-1-phosphate aldolase in Escherichia coli, Process. Biochem., 39, 1677-1684.

Fermentation of the prokaryotic host cells can take place in any suitable system, eg, continuous, batch or fed-batch mode, depending on the protein to be produced. Intermittent mode can be used with injections of nutrients or inducers as needed. Alternatively, a fed-batch culture and pre-induction batch cultures can be used at a maximum specific growth rate that can be maintained with nutrients initially present in the fermenter and one or more nutrient feed regimes applied to control the growth rate. until fermentation is complete. A fed-batch regimen can also be used prior to induction to control the metabolism of prokaryotic host cells and to ensure that a higher

- 3 042536 cell density.

The host cells are then harvested from the culture medium, eg host cells are harvested from the sample by centrifugation, filtration or concentration.

In one embodiment of the method according to the present invention, the step of harvesting the cells includes the step of centrifuging and recovering the cells. In a particular preferred embodiment, said centrifugation step is a continuous centrifugation in which the cell suspension is reconstituted.

For culture processes of a prokaryotic host cell (eg, E. coli), where the protein of interest, such as an antibody fragment, is found in the periplasmic space of the host cell, release of the protein from the host cell is necessary. Release can be achieved by any suitable method or combination of methods, such as cell lysis by mechanical or pressure treatment, freeze-thaw cycles, osmotic shock, extraction agents, or heat treatment. Such extraction methods for protein release are well known in the art. In the process of the present invention, protein extraction is achieved by a heat treatment step, i.e. maintaining the sample at an elevated temperature for a specified period of time.

In accordance with the method of the present invention, the buffer is added to the cells collected from the culture fluid prior to the heat treatment step. In a specific embodiment of the process of the invention, said buffer is 100 mmol Tris and 10 mmol ethylenediaminetetraacetic acid (EDTA) at pH 7.4; in a further embodiment, said extraction buffer is 75 mM Tris and 7.5 mM EDTA at pH 7.4, 200 mM Tris and 20 mM EDTA at pH 7.4, or 300 mM Tris and 30 mM EDTA at pH 7.4. In one embodiment of the invention, the buffer has a pH that ensures that the pH of the sample is between 6 and 9, e.g. pH between 6 and 8, prior to the heat treatment step, as described in WO 2012/013930, which is incorporated herein in its entirety. . Preferably said pH is 6.5 to 8, 6.8 to 7.8 or 7 to 7.6.

Those skilled in the art are aware of additional suitable buffers described in the art for use in protein extraction.

In a further alternative embodiment, the redox potential of said buffer is maintained below -100 mV, below -200 mV, below -300 mV, or below -400 mV.

The redox potential of the environment is, in particular, important for the dynamics of disulfide bonds between the thiol groups of cysteine residues present in protein molecules. Upon reduction, these disulfide bonds are broken, releasing the corresponding thiol groups (-SH). Antibody molecules have several disulfide bonds that are important for maintaining their structure and thus their biological activity. Some of these disulfide bonds are more accessible to reducing and/or oxidizing compounds present in the environment than others, given their position in the molecule, and thus are more susceptible to rupture.

In relation to the present invention, related impurities are actually fragments of the protein of interest and thus have a smaller size and a simpler structure. Without being limited by theory, it can be said that the cysteine bonds in these related impurities are more accessible and thus more prone to rupture due to the redox potential of the environment.

In a second embodiment of the method of the invention, said heat treatment is carried out in a container and said redox potential is maintained by reducing the amount of oxygen (O ₂ ) present in the gaseous phase in the container during the heat treatment.

In general, the container contains a volume of air (referred to here as the gaseous phase) above the sample, consisting of buffer and host cells. In accordance with the meaning of the invention, reducing the amount of oxygen in the gaseous phase means changing the composition of said gaseous phase in such a way that it contains less oxygen than air. As is known in the art, air typically contains approximately 21% oxygen (by volume).

In order to maintain the redox potential below 0 mV in accordance with the method of the invention, the person skilled in the art is aware of the various means available to prevent oxidation of the extractable sample. This can be done by removing oxygen and other oxidizing gases present in the gaseous phase in the container, usually by replacing them with another inert gas. Said replacement can be carried out by supplying a layer of nitrogen (N2), i. e. maintaining the sample in the container in contact with the gaseous phase containing N2, or by sparging, i.e. bubbling, or passing, of an inert gas through the sample into the container. Other inert gases that can be used to maintain the redox potential in accordance with the method of the invention include argon and helium.

Alternatively, there is a selection of reducing agents available in the art that have varying degrees of activity. Therefore, the person skilled in the art selects the most suitable reducing agent depending on the desired redox potential and the time for which it should be maintained. Known reducing agents in the art include, but are not limited to, glutathione, mercaptoethanol, dithiothreitol, or tris(2-carboxyethyl)phosphine.

Culture of the prokaryotic host cells can take place in any suitable container, such as a shake flask or a fermenter, depending on the scale of production required. Various large fermenters are available with available capacities in excess of 1000 liters and up to approximately 100,000 liters. Preferably, fermenters with a capacity of 1000 to 50000 liters, more preferably 1000 to 25000 liters, 1000 to 20000 liters, 1000 to 15000 liters, 1000 to 10000 liters, 1000 to 5000 liters, with a capacity of 20000, 15000, 12000, 12000 , 5000 or 2000 l. It is also possible to use smaller fermenters having a capacity between 0.5 and 1000 liters.

In a preferred embodiment, said gaseous phase contains less than 20%, 15%, 12%, 10%, 8%, 5%, 2%, or 1% oxygen (by volume) during the heat treatment step.

In a preferred embodiment, said oxygen is actually eliminated from the gaseous phase during the heat treatment step.

In a third embodiment of the method of the invention, nitrogen (N ₂ ) is added to the gaseous phase of the container.

In a fourth embodiment of the method of the invention, said gaseous phase contains at least 80% N2, at least 85%, at least 90% N2, at least 95% N2, or at least 97% or 99% N2. In a further alternative embodiment, said gaseous phase is predominantly N ₂ .

Typically said N2 is added to the gaseous phase of the container as a bed feed and maintained in the headspace of the container containing buffer and host cells by adding 100% N2 gas to the headspace at 0.1-1 rpm (gas volume per minute of liquid, i.e. 1 L/min for 1 L of extraction volume).

In a particular embodiment, the present invention provides a method for producing a protein of interest, wherein said protein is expressed in a host cell, comprising: culturing said host cells under conditions that cause them to express said protein;

collecting host cells from the culture fluid;

adding buffer to host cells; and subjecting the host cells in the buffer to heat treatment in a container, where the gaseous phase of the container is predominantly N2.

Depending on the configuration of the container, said N2 may be added to the gaseous phase of the container, where the container is further sealed, i.e. the inside of the container is isolated from the outside, leaving N2 in the gaseous phase during the heat treatment. Alternatively, a constant flow of N2 through the gaseous phase of the container during the cooking step can be applied, for example for containers that do not allow the possibility of isolating the inside from the outside environment.

In one embodiment, N2 is added or injected into the gaseous phase prior to the heat treatment step to ensure that the desired redox potential is maintained during the heat treatment step.

In a particular embodiment, the method of the invention comprises culturing said host cells under conditions that are conducive to expressing a protein of interest;

collecting host cells from the culture fluid;

adding buffer to the host cells in the container;

adding N2 to the gaseous phase of the container;

sealing said container; and subjecting the host cells in the buffer to heat treatment in the container, where the redox potential of said buffer is maintained below 0 mV during said heat treatment.

Thus, in a further embodiment of the method of the invention, said N ₂ is administered as a layer feed using at least 80%, at least 85%, at least 90%, at least 97%, at least 99 or 100% _N2 .

As an alternative embodiment of the invention, said N2 is introduced by bubbling N2 through the buffer during the heat treatment, preferably by bubbling substantially pure N2.

In a fifth embodiment of the invention, said N2 is added to the container by feeding a bed into the gaseous phase of the container or by bubbling N2 through a buffer.

The method according to the invention includes a heat treatment step which makes it possible to obtain a sample of a soluble, properly folded and assembled protein, such as an antibody fragment, by facilitating the removal of other materials related to the protein.

A protein that is correctly folded and assembled is shown by the presence of a single band corresponding to the expected molecular weight of the assembled protein chain by SDS-PAGE under non-reducing conditions. Another protein related material would be partially degraded fragments of a properly folded and assembled protein.

The heat treatment step in the method of the present invention is the step of maintaining the temperature of the sample at a desired elevated temperature level after that makeable elevated temperature level is reached during the heating step.

The heat treatment step is carried out by subjecting the sample to the desired elevated temperature. Most preferably, the heat treatment step is carried out within the range of 30 to 70°C. The temperature can be chosen as desired and may be dependent on the stability of the purification protein. In another embodiment, the temperature is within the range of 40 to 65°C, or preferably within the range of 40 to 60°C, more preferably within the range of 45 to 60°C, even more preferably within the range of 50 to 60° C and most preferably 55 to 60°C, 58 to 60°C or equal to 59°C. Thus, the minimum temperatures are 30, 35 or 40°C and the maximum temperatures are 60, 65 or 70°C.

In a sixth embodiment of the method of the invention, said heat treatment step is carried out at a temperature of 30 to 70°C.

In a seventh embodiment of the process of the invention, said heat treatment step is carried out at a temperature of 55 to 65°C.

The heat treatment step is preferably carried out over a long period of time. The duration of the heat treatment is preferably in the range of 1 to 24 hours or 1 to 18 hours, more preferably 4 to 18 hours, even more preferably 6 to 16 hours and most preferably 8 to 14 hours or 8 to 12 hours. , for example, within 10 hours. Thus, the minimum time for cooking is 1, 2 or 3 hours and the maximum is 20, 22 or 24 hours.

In the eighth embodiment of the method of the invention, the heat treatment step of the sixth or seventh embodiment is maintained for a period of 1 to 18 hours.

As will be known to those skilled in the art, there is generally an inverse relationship between reaction time and temperature, with the higher the temperature at which the reaction proceeds, the shorter the period of time required for the reaction to proceed.

In a specific embodiment, the heat treatment is carried out at a temperature of 50 to 60°C for a period of time from 10 to 16 hours, and more preferably at a temperature of 59°C for a period of time from 10 to 12 hours. It will be clear to a person skilled in the art that the temperature and time can be chosen as appropriate for the respective sample in relation to the characteristics of the resulting protein of interest, such as an antibody or antibody fragment.

In a further alternative embodiment of the method of the invention, the extraction of said protein is carried out at a temperature of 58 to 60°C for a period of 8 to 12 hours.

The heat treatment step is preferably carried out with sample agitation, such as in a shaker or stirrer, set to a suitable agitation speed, eg 200 revolutions per minute (rpm). Despite this, the speed of agitation will vary depending on the scale of the process.

In a ninth embodiment of the method of the invention, the sample has or undergoes a pH adjustment to 6-9 before the heat treatment step. In a further specific embodiment of the method of the invention, the pH of said sample is between 6.5 and 8, 6.5 and 7.5, or between 6.8 and 7.2 before the heat treatment step.

In a particular embodiment, the pH of the sample is measured or adjusted prior to the cooking step to ensure that the pH of the sample during the cooking step is between 5 and 9, preferably the pH of the sample during the cooking step is between 6 and 7.

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

Any suitable means may be used to correct the pH of the sample. The agent may be a buffer or may be added before and/or after the buffer. Typical pH adjusters include or consist of one or more of the following, NaOH, NH ₄ OH, sulfuric acid, EDTA, Tris buffer.

In a further specific embodiment, the pH of the sample is measured or adjusted prior to the heat treatment step and prior to the addition of an inert gas such as nitrogen to the gaseous phase of the container in order to maintain the redox potential below 0 mV. Alternatively, in another embodiment of the invention, the pH of the sample is measured and/or corrected prior to the heat treatment step and an inert gas such as nitrogen is passed through the sample during the heat treatment step.

The pH measurements referred to in this application are generally given to 20°C.

In the context of the present invention, the phrase before the heat treatment stage means before and includes the point in time at which the sample reaches the desired elevated temperature and the heat treatment (maintaining the elevated temperature) stage begins. In order to achieve

- 6 042536 desired elevated temperature during the stage of heat treatment, the sample is subjected to a heating phase, during which the temperature of the sample is raised to the desired temperature. In one embodiment, the method according to the invention includes subjecting the sample to a heating phase and a heat treatment step. In an embodiment where the method includes subjecting the sample to a heating phase and a cooking step, the sample may have the desired pH prior to the start of the heating phase and/or the desired pH during the heating phase.

In a particular embodiment, N2 is added to the gaseous phase of the container prior to the start of the heating phase. Preferably, the sample has the desired pH prior to said heating phase.

In a tenth embodiment of the method of the invention, the protein of interest is a recombinant protein encoded by an expression vector.

In an eleventh embodiment of the method according to an eighth embodiment of the invention, the protein is a recombinant antibody.

In a twelfth embodiment, said recombinant antibody is a fragment of a recombinant antibody. There are many known recombinant antibody fragments in the art, including Fab, Fab', F(ab') ₂ , Fv, and scFv fragments. Alternatively, recombinant antibodies are selected from diabody, triabody, tetrabody, minibody, domain antibodies, single chain antibodies, bispecific, trispecific, tetraspecific, or multispecific antibodies formed from antibody or antibody fragments, including but not limited to Fab-Fv constructs.

In a thirteenth embodiment of the method of the invention, said recombinant antibody or recombinant antibody fragment specifically binds to TNF-alpha or CD154.

In one embodiment, the present invention provides a method for producing a recombinant antibody, wherein said recombinant antibody is expressed in a host cell, comprising culturing said host cells under conditions allowing them to express said protein, harvesting the host cells from the culture fluid, adding a buffer to the host cells, and subjecting the host cells to a heat treatment to extract a recombinant antibody from said host cells in a container, where the redox potential of said buffer is maintained below 0 mV, below -100 mV, below -200 mV, below -300 mV, or below -400 mV for specified extraction.

In another specific embodiment, the present invention provides a method for producing a recombinant antibody, wherein said recombinant antibody is expressed in a host cell, comprising culturing said host cells under conditions that allow them to express said protein, harvesting host cells from culture fluid, adding buffer to host cells in a container, adding N2 to the gaseous phase of the container, subjecting the host cells to a heat treatment, and reducing said protein.

In another specific embodiment, the present invention provides a method for producing a recombinant antibody, wherein said recombinant antibody is expressed in a host cell, comprising culturing said host cells under conditions that allow them to express said protein, harvesting host cells from culture fluid, adding buffer to host cells in a container, adding N ₂ to the gaseous phase of the container, subjecting the host cells to a heat treatment and reducing said protein, wherein said reduced protein has a lower content of related impurities compared to a protein recovered in a preparation method that includes heat treatment carried out in the presence of N 2 in gaseous phase container.

The method in accordance with the present invention may include one or more additional steps such as purification procedures, such as filtration and/or centrifugation. Fluidized bed chromatography is also included.

Preferred additional purification procedures include ion exchange chromatography, microfiltration, ultrafiltration, diafiltration, fixed bed absorption and expansion bed absorption, as well as a combination of any of these procedures.

The term antibody or antibodies, as used in this application, refers to monoclonal or polyclonal antibodies. The term antibody or antibodies used in this application includes, but is not limited to, recombinant antibodies that are created using recombinant technologies, as is known in the art. The term antibody or antibodies includes antibodies of any species, in particular mammalian species, such as human antibodies of any isotype, including IgA1, IgA2, IgD, IgG1, IgG _2a , IgG _2b , IgG ₃ , IgG ₄ , IgE and IgM, as well as their modified variants, primate antibodies, for example derived from chimpanzees, baboons, rhesus or cynomolgus monkeys;

rodent antibodies, for example, obtained from mice, rats or rabbits;

antibodies of goats or horses; as well as antibodies of the camelid family (for example, derived from camels or llamas, such as Nano-7 042536 bodies™), as well as derivatives of the above substances; or antibodies derived from bird species, such as chicken antibodies, or from fish species, such as shark antibodies.

The term antibody or antibodies also refers to chimeric antibodies in which one portion of at least one antibody heavy and/or light chain sequence is derived from one species and another portion of the antibody heavy and/or light chain sequence is derived from another species. Chimeric antibodies of interest herein include primatized antibodies comprising an antigen-binding region variable region derived from a lower primate (e.g., an Old World monkey such as a baboon, rhesus or cynomolgus monkey) and human constant region sequences. Humanized antibodies are chimeric antibodies that contain a sequence derived from antibodies of animal origin. In many cases, humanized antibodies are human antibodies (recipient antibody) in which residues of the hypervariable region of the recipient are replaced by residues of the hypervariable region [or complementarity determining region, CDR] of a non-human species (donor antibody) such as mouse, rat, rabbit , chicken or lesser primate that have the desired specificity, affinity and activity. In most cases, human (recipient) antibody residues outside the CDR, i.e. in the framework region (FR) are further replaced by corresponding non-human residues. Moreover, humanized antibodies may include residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further improve the performance of the antibody. Humanization reduces the immunogenicity of animal-derived antibodies in humans, thus facilitating the use of antibodies to treat human diseases. Humanized antibodies and several different technologies for making them are well known in the art. The term antibody or antibodies also refers to human antibodies that can be generated as an alternative to humanization. For example, it is possible to create transgenic animals (eg, mice) that are capable, after immunization, of producing the full range of human antibodies in the absence of endogenous murine antibody production. For example, homozygous deletion of the heavy chain of the J segment (JH) gene in chimeric and germline mutated mice has been reported to result in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene template into such a germline mutated mouse will result in the production of human antibodies with specificity for a particular antigen upon immunization of a transgenic animal that carries human germline immunoglobulin genes with said antigen. Technologies for creating such transgenic animals and technologies for extracting and obtaining human antibodies from such transgenic animals are known in the art. Alternatively, in transgenic animals, such as mice, only the immunoglobulin genes encoding the mouse antibody variable regions are replaced with the corresponding human immunoglobulin gene variable sequences. Mouse germline immunoglobulin genes encoding antibody constant regions remain largely unchanged. Thus, the effector functions of the antibody in the immune system of the transgenic mouse, and hence the production of B cells, remain unchanged, which may lead to an improved antibody response to antigen challenge in vivo. Once the genes encoding the particular antibody of interest have been extracted from such transgenic animals, the genes encoding the constant regions can be replaced with human constant region genes to produce a fully humanized antibody. Other methods for producing human antibodies/antibody fragments in vitro are based on display technology, e.g., phage or ribosome display technology, using recombinant DNA libraries that are either generated at least in part artificially or taken from the variable region (V) gene repertoire. donor immunoglobulin. Phage and ribosome display technologies for generating human antibodies are well known in the art. Human antibodies can also be obtained from isolated human B cells that are immunized ex vivo with an antigen of interest and then combined to create hybridomas that can then be tested for the optimal human antibody. The term antibody or antibodies as used herein also refers to an aglycosylated antibody.

In some embodiments, the antibodies are antibodies that are modified by covalent attachment of functional moieties such as water soluble polymers such as polyethylene glycol;

copolymers of polyethylene glycol and polypropylene glycol;

carboxymethylcellulose, dextran, polyvinyl alcohol;

polyvinylpyrrolidone or polyproline, all of which are known to exhibit significantly longer blood half-lives following intravenous administration compared to the corresponding unmodified proteins.

In some embodiments, the antibodies of the present invention are antibodies attached to functional moieties, such as polyethylene glycol (PEG) moieties.

- 8 042536

In one specific embodiment, the antibody is an antibody fragment and the PEG molecules can be attached using any available amino acid side chain or amino acid terminal functional group located in the antibody fragment, for example, using any free amino, imino, thiol, hydroxyl, or carboxyl group. . Such amino acids may occur naturally in the antibody fragment or may be inserted into the fragment using recombinant DNA technology (see, for example, US 5219996, US 5667425, WO 98/25971).

The term antibody or antibodies, as used herein, not only refers to untruncated antibodies of any kind, including those derived from humans (eg, IgG) and other mammalian species, but also refers to an antibody fragment. An antibody fragment includes at least one heavy or one light chain of an immunoglobulin domain, as is known in the art, and binds to one or more antigens. Examples of antibody fragments according to the invention include Fab, Fab', F(ab') ₂ , and Fv, as well as scFv fragments, as well as diabodies, tribodies, tetrabodies, minibodies, domain antibodies (dAbs) such as sdAbs, VHH and V _NAR fragments, single chain antibodies, bispecific, trispecific, tetraspecific or multispecific antibodies formed from fragments of antibodies or antibodies, including but not limited to Fab-Fv or Fab-Fv-Fv constructs. Antibody fragments, as previously defined, are known in the art.

The term redox potential, as used herein, refers to the measurement of the tendency of chemical species to gain electrons and thus to be reduced. Redox potential is measured in volts (V) or millivolts (mV). Each particle has its own internal reduction potential; the more positive the potential, the greater the affinity of the particles for electrons and the tendency to recover.

The term protein extraction or extraction as used herein refers to the removal of a protein expressed in a host cell from the host cell. Extraction may include rupturing the host cell and extracting intracellular fluid from cell debris, or manipulating the host to leak the outer cell membrane of Gram-negative bacteria or other cells having an outer cell membrane to release fluid from the periplasmic space of those cells.

Examples

Ways.

The fermentation process a.

E. coli W3110 transfected with the vector so as to express a Fab' that binds specifically to human TNF-alpha is used as host cells.

A frozen cell bank vial containing these cells is used for inoculation in a shake flask (total volume 700 ml) containing medium with 6x peptone-yeast extract (6xP-Y) and tetracycline. This shake flask is incubated at 30° C. and 230 rpm. At the desired optical density range (OD600=2-3), a shake flask is used to inoculate in an inoculator (total volume 10 L) that contains SM6 medium with a specific chemical composition (Popplewell et al., Expression of Antibody Fragments by Periplasmic Secretion in Escherichia coli , Methods in Molecular Biology, vol.308: Therapeutic Proteins: Methods and Protocols, 2005) and tetracycline with glycerol as a carbon source. The cell culture in the inoculator is maintained at 30°C, the concentration of dissolved oxygen (pO ₂ ) is maintained at 30%, the pH is controlled at 7.0.

At the desired OD range (OD600=30-37), a seed culture is used to inoculate a commercial fermenter (total volume 600 L) containing a chemically defined medium with glycerol as a carbon source. The commercial fermenter is initially maintained at 30° C., 30% pO ₂ , and pH maintained at 7.0, cell growth in batch phase prior to induction. During the batch phase, the temperature is lowered to 25° C. and MgSO ₄ is added to avoid depletion of this metabolite.

With a separate OD range (OD600=43-47), in which glycerol depletion develops, a bolus addition of lactose solution to the culture is performed. Lactose is further utilized by cells as a major carbon source and also acts as an inducer of Fab' expression. The lactose concentration is maintained throughout the induction phase by continuous feeding and the cells are harvested 30 hours after induction.

The fermentation process

A frozen cell bank vial containing E. coli MXE012 Fab' host cells expressing a Fab' that specifically binds to human TNF-alpha is used to inoculate a shake flask (total volume 700 mL) containing medium with 6x peptone yeast extract ( 6xP-Y) and tetracycline. This shake flask is incubated at 30° C. and 230 rpm. With the required range of optical density (OD600=2-3), a shake flask is used to inoculate in an inoculator (total volume 10 L) that contains MD medium with a specific chemical composition (Durany et al., 2004, Studies on the expression of recombinant fuculose- 1-phosphate aldolase in Escherichia coli, Process.

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Biochem., 39:1677-1684) and tetracycline with glycerol as a carbon source. The cell culture in the inoculator is maintained at 30°C, the concentration of dissolved oxygen (pO ₂ ) is maintained at 30%, the pH is controlled at 6.9.

At the desired OD range (OD600=30-37), a seed culture is used to inoculate a commercial fermenter (total volume 600 L) containing a chemically defined medium with glycerol as a carbon source. The production fermenter is initially maintained at 30° C., 30% pO ₂ and the pH is maintained at 6.9 and growth in the batch phase until the glycerol is depleted. MgSO ₄ is added at this time to avoid depletion of this metabolite.

At the end of the batch phase, an exponential glycerol feed is switched on and the culture is fed with a certain amount of glycerol to achieve an OD600 of approximately 70 units. At this point, the glycerol feed is switched from exponential feed to linear feed and Fab' expression is induced by adding IPTG (38.79 g/kg culture). Cells are harvested 40 hours after induction.

The E. coli strain MXE012 is an E. coli metabolic mutant containing the sprH145A mutation in the spr gene as described in WO 2011/086136 (incorporated herein in its entirety).

Recovery and extraction methods.

Cells are harvested by continuous centrifugation using a Westfalia PSC 5 plate centrifuge.

The concentrated cell suspension is resuspended to the initial concentration of harvested cells in 2 liter or 5 liter glass vials by adding water or extraction buffer at pH 7.4 to a final extraction buffer concentration of 10 mM EDTA, 100 mM Tris.

If necessary, the resuspended cells are adjusted to pH 7.2 by adding 2.5 mol Tris base to the extraction buffer prior to protein extraction by the heating step. For heat extraction, cells are heated to 59° C. for 10 h. Temperature and stirring are maintained using Biostat BPlus control units (Sartorius) and Multiple Fermenter Control System (MFCS®) software.

Sample cleaning.

Related Fab' particles are purified from extraction samples to provide an assessment of related impurities. Extraction samples are first purified by centrifugation, collecting the purified supernatant and filtering through a 0.2 µm filter. The purified supernatant is then loaded onto 1 ml Protein L columns (Pierce #89928), washed with low strength phosphate buffer at pH 7.4, and eluted with glycine buffer at pH 2.8. It has been demonstrated that L-protein resin can capture Fab' fragments and that recovery is linear and reproducible over the experimental range.

Sample analysis.

Purified samples are analyzed and counted using cation exchange chromatography. Samples were loaded onto a Dionex Pro Pac SCX-10 analytical column (Thermo Scientific) and eluted with a salt gradient. The eluted proteins are monitored at 280 nm and the chromatographic peaks are counted and interpreted according to the standard procedure.

Example 1

The fermentation is carried out according to process B and the protein is extracted as described above. Redox potential is measured throughout the extraction process in containers using ORP (Oxidation Reduction Potential) probes (Broadley James Corporation, Irvine CA) and monitored using MFCS® software (Sartorius, Bethlehem PA).

To evaluate the effect of maintaining the redox potential of the environment, some extract containers are exposed to nitrogen, others are exposed to air during extraction, and the gas is either passed through the sample into the containers, or its flow is maintained in the space above the product in the containers.

Tab. 1 below shows an overview of container gas-flow conditions. All gas flows are maintained at a flow rate of 1 rpm (gas volume per liquid volume per minute, i.e. 1 L/min for 1 L extraction volume) with a 100% nitrogen or compressed air supply.

Table 1

# extraction Condition 1 Flow layer N ₂ 2 Air layer flow 3 Bubbled N ₂ 4 Bubbled air

Fig. 1 and 2 show the respective redox and temperature extraction profiles (FIG. 1)

- 10 042536 and SDS-PAGE electrophoresis of samples after extraction (Fig. 2).

As can be understood from these figures, maintaining a layer of nitrogen during the cooking step maintains a consistently low redox potential at a minimum value of approximately -435 mV, typically -375 mV while maintaining the temperature at 59°C. A similar low redox potential is observed during nitrogen bubbling. When the buffer containing the host cells is covered with air instead of nitrogen, it can be seen that the redox potential values rise significantly during the heat extraction step and become positive (typically around +45 mV) while holding the temperature at 59°C. A similar high redox potential is observed during air bubbling. Gas sparging is converted to bed feed during extraction due to the observed high levels of foaming.

Fig. 2 shows a significant difference in the concentration of related impurities, as measured by SDS-PAGE electrophoresis. The figure demonstrates that in the presence of nitrogen, the reducing conditions during the heat extraction process are sufficient to provide a significant reduction in the concentration of related impurities compared to extractions carried out in the presence of air.

Example 2

Fermentation according to process A and subsequent extraction are carried out as described previously. The headspace nitrogen flow rate varies from 0.1 rpm to 1 rpm in combination with varying agitation and nominal volumes according to table. 2.

table 2

Extract number Nitrogen layer supply (r/r/min) RPM Nominal volume 1 0, 1 150 1.5 2 1 150 1.5 3 0, 1 450 3 4 1 450 3 5 0.55 300 2.25 6 0.55 300 2.25 7 0.55 300 2.25 8 0 300 2.25

As can be noted in FIG. 3, a clear effect of the nitrogen layer supply on the clearance of related impurities can be observed. In extraction #8, where a nitrogen blanket is not applied, significantly higher concentrations of related impurities are observed. On the other hand, the figure also suggests that the nitrogen layer feed rate may have an effect on the clearance of this contaminant when comparing extractions 1 and 2, and 7 and 8.

Example 3

Fermentation according to Process A and cell harvest were carried out as previously described. Resuspended host cells are adjusted to pH 7.2 by adding 2.5 mol Tris base to the extraction buffer just prior to heat extraction. This heat extraction is carried out either in the presence or absence of a nitrogen blanket. Where applicable, the supply of a layer of nitrogen is maintained by gassing the headspace of the product in the container at 1 rpm using 100% nitrogen.

In FIG. 4, extractions 1-3 are carried out in the presence of a nitrogen blanket, while extractions 4-6 are carried out with air. The results again demonstrate the advantage of the presence of nitrogen, which results in significantly reduced concentrations of related impurities remaining after heat extraction. An additional positive effect is obtained from the pH adjustment carried out before the heat extraction, since the concentrations of related impurities seem to be further reduced compared to the previous examples.

Example 4

The effect of extraction under reducing conditions is also analyzed at smaller volumes, ie. when using shake flasks.

25-100 ml protein-induced cell culture is harvested and resuspended in a 250 ml Erlonmeyer conical flask by adding water and 3x extraction buffer (pH 7.4) to a final 1x concentration of extraction buffer (10 mM EDTA, 100 mM Tris). The flasks have

- 11 042536 sealing plug and made of polycarbonate to minimize the penetration of oxygen into the flask. The nitrogen layer was fed into the gaseous phase of the flasks using a disposable glove bag (Aldrich AtmosBag, cat. #Z530220) with a continuous stream of nitrogen passing through the glove bag. The flasks are sealed prior to removal from the glove bag and heat extraction. Samples are also made on a laboratory bench (with air as a layer in the gaseous phase of the flasks) for comparison. The heat extraction is carried out at 60° C. for 10 hours in a shake flask incubator (New Brunswick Innova42). The redox potential is measured in each flask after extraction using Broadley James ORP samples. The heat treatment step is also carried out on shake flasks of various nominal volumes to evaluate the effect of various surface area to volume ratios on protein extraction. In table. 3 summarizes the experimental conditions under study.

Table 3

# extractions Compound Nominal volume (ml) 1 Nitrogen 25 2 Nitrogen 50 3 Nitrogen 100 4 Air 25 5 Air 50 6 Air 100

Fig. 5 demonstrates that shake flasks that are made in the presence of a nitrogen blanket maintain a more reducing environment in the extract compared to shake flasks that are made in the presence of air. The figure also demonstrates that the larger nominal volume shake flasks are more reducing than the smaller nominal volume flasks, most likely due to the slower rate of extract oxidation due to the lower surface area to volume ratio. All flasks manufactured with air have a positive redox endpoint with no correlation to nominal volume.

Fig. 6 demonstrates that shake flasks, which are made in the presence of nitrogen, have significantly lower concentrations of related impurities after extraction. All flasks made with nitrogen (end point of the ORP range from -10 to -116 mV) show significantly lower concentrations of related impurities compared to flasks made with air (end point of the ORP range from +79 to +97 mV ).

Example 5

Fab' that specifically binds to human TNF-alpha has a higher melting point (Tm) than HCPs (host cell proteins), a property that is exploited during the cooking step. Heating denatures a significant amount of HCP and related impurities, causing them to aggregate and be removed during the extraction steps. Despite this, as described in the previous examples, the removal of these impurities is significantly improved when applying a nitrogen layer supply, taking into account the reduced redox potential. It is believed that the reducing medium facilitates the removal of impurities due to the reduction of intrachain bonds in these compounds during heating, which leads to a decrease in the melting point of impurities.

In order to confirm the above, the melting points of the two main compounds of related impurities are measured under non-reducing and reducing conditions. These reducing conditions are achieved by using TCEP-HCL (tris-(2-carboxyethyl)phosphine hydrochloride) to simulate the environment created by the nitrogen layer flow applied during extraction.

Measurements are made using the Thermofluor assay, which is based on the use of a hydrophobic fluorophore that binds to a protein, provided that all of its hydrophobic regions are accessible. In this particular case, the fluorophore is SYPRO orange (5000x concentration in dimethyl sulfoxide (DMSO, DMSO); Life Technologies S-6650). This allows monitoring of the unfolding process under denaturing conditions (eg, temperature rise) by detecting the fluorescence emitted by the dye when bound to the protein using a real-time PCR system (7900HT fast realtime PCR system from Life Technologies). Samples are incubated at increasing temperature (from 25 to 99° C. with a 30 second hold and an increase of 0.5° C.) and fluorescence emission is recorded. The data is plotted on a chart and the melting point of the protein is determined by finding the inflection point of the curve.

In this case, the two purified related impurities are determined in citrate-phosphate buffer pH 5.0 and 7.0 and sodium phosphate buffer pH 7.4. Apply two different concentrations of TCEP-HCL

- 12 042536 to obtain a molar ratio of protein:reducing agent 2:1 and 10:1 and a sample without

TCEP-HCL is studied in parallel as a control.

Fig. 7 shows thermal stability results for two major related impurity compounds. The correlation between redox potential and thermal stability (melting point) has been demonstrated; the lower the redox potential, the lower the melting point of the protein. A similar effect is described for pH as the pH becomes more neutral. Both indicators provide a greater propensity for related impurities to degrade, thus facilitating their removal.

Conclusions.

Overall, the data demonstrate the importance of maintaining a reducing environment (low redox potential) during the heating step to ensure a significant reduction in the concentration of related impurities during protein extraction. Moreover, the data demonstrate that this reducing environment is provided by applying a nitrogen bed flow and in the absence of a nitrogen bed flow, the redox potential of the environment is significantly less reducing, resulting in higher concentrations of related impurities.

Claims (10)

CLAIM 1. A method for obtaining a protein of interest, where the specified protein is expressed in the periplasm of the host cell, which is E. coli, including a) culturing said host cells under conditions conducive to expressing said protein; b) collecting host cells from the culture fluid; c) adding extraction buffer to host cells; And d) exposing the host cells in buffer to a heat treatment where the redox potential of the buffer is maintained below 0 mV by adding nitrogen to the gaseous phase of the container as a layer stream during said heat treatment step and said heat treatment is carried out at a temperature of 30 to 70°C , wherein the protein of interest is thus extracted from the periplasm. 2. The method of claim 1, wherein the heat treatment is carried out in a container and said redox potential is maintained by reducing the amount of oxygen (O ₂ ) present in the gaseous phase in the container during protein extraction. 3. The method according to claim 1 or 2, wherein nitrogen (N ₂ ) is added to the gaseous phase of the container, said gaseous phase containing at least 50% N ₂ . 4. The method of claim 1, wherein said heating step is carried out at a temperature of 55 to 65°C. 5. The method according to any one of claims 1 to 4, wherein said temperature is maintained for a period of time from 1 to 18 hours. 6. The method according to any one of claims 1 to 5, wherein the pH of the buffer is measured after addition to the host cells and before heat treatment. 7. The method according to claim 6, wherein the pH of the buffer is or adjusted to pH values between 6 and 9. 8. The method according to any one of claims 1 to 7, wherein the protein is a recombinant protein encoded by an expression vector. 9. The method of claim 8, wherein the protein is a recombinant antibody or recombinant antibody fragment. 10. The method of claim 8 or 9, wherein said recombinant antibody or recombinant antibody fragment specifically binds to TNF-alpha or CD154.