Patent Application
20230391823
The current invention is in the field of protein purification. Especially, the current invention relates to methods for purifying or producing proteins wherein the same depth filter is used multiple times. In particular, the methods of the invention involve contacting the depth filter with a regeneration solution prior to the re-use of the depth filter for removing impurities.
Commercial production processes for the manufacture of therapeutic biopharmaceuticals, such as monoclonal antibodies (mAbs), often utilize various clarification technologies for the removal of whole cells, cellular debris, large particulates, and colloidal matter. These clarification technologies can include centrifugation, depth filtration, chemical flocculation, and tangential or normal flow filtration methods and may be located at multiple steps within the downstream purification process. Often, the primary role of the clarification step is for the harvest of the mAb product from the cell culture broth and this may be accomplished by a combination of centrifugation followed by depth filtration. The harvest clarification step removes insoluble matter and protects the subsequent sterile filter and chromatography columns from plugging. Further downstream, depth filtration steps are also employed for secondary clarification and haze removal applications. Depth filter have also been used for achieving some level of impurity clearance, especially for the removal of process-related impurities such as host cell proteins (HCPs) and DNA, as well as product-related impurities such as aggregated mAb species (Nguyen et al, Biotechnol. J. 2019, 14, 1700771, Yigzaw et al, Biotechnol. Prog. 2006, 22, 288-296).
Generally, depth filters utilizes its depth, or thickness, for carrying out the filtration and are typically manufactured employing a material structured with a gradient density, generally having larger pores near the top and smaller pores at the bottom (when view in the direction of flow). Depth filters retain particles throughout the porous media, allowing for retention of particles both larger and smaller than the pore size. Particle retention is thought to involve both size exclusion and adsorption through hydrophobic, ionic and other interactions. Depth filters come in several different forms. A common design consists of a layer of cellulose, a porous filter aid such as diatomaceous earth (DE), and a charged polymeric resin that binds the two together. Based on those major components, depth filters remove impurities and particulate material, which is essential protection for membrane filters downstream. Several depth filtration systems are commercially available. All process-scale models e.g., Millistak+ Pod from Millipore Sigma, Stax from Pall Corporation, 3M Zeta Plus from Cuno Inc., and Sartoclear P from Sartorius Stedim Biotech can separate cells and prepare culture fluid for downstream chromatographies (Schmidt et. al, Bioprocess International, 2017).
WO 2015/031899 discloses a synthetic depth filtration media comprised of polyacrylic fibers, a precipitated silica filter aid, and a charged polymeric binder resin. This synthetic depth filter is known as the Millistak HC Pro XOSP depth filter and is commercially available as single use filter from Millipore Sigma (Bedford, MA). The XOSP depth filter has a nominal pore size rating of 0.1 microns and is intended for secondary clarification applications. Millistak+® HC Pro is a high capacity synthetic medium.
Generally, depth filters are sold for single-use. Thereby allegedly certain advantages are offered, such as, e.g., no shut down of the system is required for CIP (O'Brian et al. Bioprocess International 10, 50-67), a prerequisite in a GMP environment.
However, the filtration costs increase with single use systems compared to multi-use systems. Moreover, there is a strong need besides overall cost-reduction for ecological saving of resources.
However, the fouling mechanisms of a depth filter including pore blockage, cake formation and/or pore constriction, require an efficient regeneration protocol to get rid of the filtered impurities and to re-generate an at least equally effective depth filter.
Sodium hydroxide has become the standard for cleaning and sanitizing chromatography columns. However, some chromatography media are not compatible with sodium hydroxide. Examples of chromatography media sensitive to sodium hydroxide are: 1) chromatography media employing a protein ligand, and 2) chromatography media based on silica or glass (Application note 28-9845-64 AA GE Life Sciences). That NaOH might not be suitable for silica matrices, had also been implicated by Claesson et al., Chromatogr. A; 728 (1996) 259-270, discussing that in the case of silica based materials, NaOH treatments that render the pH value to be above pH 10 concomittantly have an intrinsic risk of hydrolyzing siloxane bonds in the silica matrix, which are the backbone of the porous structure.
In breweries, diatomaceous earth depth filter material accumulates in considerable amounts during the filtration of the wort and the stored beer. EP 0 253 233 describes a time consuming tedious regeneration protocol for the used kieselguhr employing a multistep-procedure with high temperatures and high concentrations of NaOH. Further, NaOH solutions were used in order to sterilize/sanitize Millipore depth filter prior to use US 2016/0114272. Normally, the single-use filters sold are not sanitized and need pre-flush sanitization.
Another method commonly used for the purification of depth filter is backflushing with e.g. buffer or water (e.g. in swimming pools).
The underlying invention provides methods allowing the multiple use or re-use of a depth filter, especially silica-comprising depth filters. This is achieved by regenerating the filter material with, e.g., an acidic or alkaline solution or by pre-treating, i.e. conditioning, the depth filter prior to its first use, as well as combinations thereof. In more detail, the current invention also provides methods for increasing the efficacy of a depth filter by flushing or pre-treating it with an alkaline solution prior to its first use. By using the method according to the current invention, the efficacy of a depth filter can be increased as well as at the same time regenerated. This offers considerable economic and ecologic advantages over the single-use of depth filters.
Herein is reported a method for the purification or production of a therapeutic polypeptide using the same depth filter multiple times, i.e. wherein the same depth filter has been used at least twice and has been regenerated in between the uses. The present invention is based, at least in part, on the unexpected finding that a depth filter (such as those that contain silica as filter aid and which are intended to be a single-use, disposable depth filter) can be used multiple times, i.e. it can be regenerated, by contacting/washing/regenerating said depth filter for example with an acidic or alkaline solution according to the method of the invention. It has been found that the regenerated depth filter surprisingly maintains its ability to remove process-related impurities, such as, e.g., amongst others host cell proteins (HCPs), which are hydrolytically active, while keeping the ability to recover the main product (in comparable purity and yield as in the first use of the depth filter).
Thus, one aspect of the invention is a method for purifying a therapeutic polypeptide, characterized in that the method comprises the following steps:
Another aspect according to the invention is a method for producing a therapeutic polypeptide, characterized in that the method comprises the following steps:
In certain embodiments of the above aspects and the other embodiments, the acidic (regeneration) solution of step b) is a solution comprising phosphoric acid.
In certain embodiments of the above aspects and the other embodiments, the acidic (regeneration) solution of step b) is a solution comprising phosphoric acid and acetic acid.
In certain embodiments of the above aspects and the other embodiments, the acidic (regeneration) solution of step b) comprises phosphoric acid in a concentration of about 0.1 M to about 0.8 M, or about 0.2 M to about 0.7 M, or about 0.4 M to about 0.6 M.
In certain embodiments of the above aspects and the other embodiments, the acidic (regeneration) solution of step b) comprises phosphoric acid in a concentration of about 0.3 M, or about 0.4 M or about 0.5 M or about 0.6 M.
In certain embodiments of the above aspects and the other embodiments, the acidic (regeneration) solution of step b) comprises acetic acid in a concentration of about 10 mM to about 2 M, or about 20 mM to about 1.5 M, or about 50 mM to about 1 M, or about 80 mM to about 800 mM.
In certain embodiments of the above aspects and the other embodiments, the acidic (regeneration) solution of step b) comprises acetic acid in a concentration of about 10 mM, or about 20 mM, or about 50 mM, or about 100 mM, or about 120 mM, or about 140 mM, or about 160 mM, or about 180 mM, or about 200 mM, or about 500 mM, or about 1 M, or about 2 M.
In certain embodiments of the above aspects and the other embodiments, the acidic (regeneration) solution of step b) comprises phosphoric acid in a concentration of about 300 mM and acetic acid in a concentration of about 167 mM.
In certain embodiments of the above aspects and the other embodiments, the acidic (regeneration) solution of step b) has a pH value of about 1 to about 5.5, or about 1 to about 5, or about 1 to about 4.5, or about 1 to about 4, or about 1 to about 3.5, or about 1 to about 3.
In certain embodiments of the above aspects and the other embodiments, the acidic (regeneration) solution of step b) has a pH value of about 1, or about 1.3, or about 1.5, or about 1.7, or about 1.9.
In certain embodiments of the above aspects and other embodiments, the acidic (regeneration) solution of step b) is an acidic aqueous buffered solution.
It has further been found that also alkaline regeneration solutions can be used in method according to the current invention.
Thus, one aspect of the invention is a method for purifying a therapeutic polypeptide, characterized in that the method comprises the following steps:
One further aspect according to the invention is a method for producing a therapeutic polypeptide, characterized in that the method comprises the following steps:
It has further been found that alkaline solutions besides being used as regeneration solutions can also be used in a pre-treatment of the depth filter (prior to its first use) resulting in a beneficial effect.
Thus, one aspect according to the invention is a method for purifying a therapeutic polypeptide, characterized in that the method comprises the following steps:
One further aspect according to the invention is a method for producing a therapeutic polypeptide, characterized in that the method comprises the following steps:
It has further been found that aqueous buffered solutions can be used as regeneration solutions in the method according to the invention in combination with a pre-treatment of the depth filter with an alkaline solution, which results in a beneficial effect.
Thus, one aspect according to the invention is a method for purifying a therapeutic polypeptide, characterized in that the method comprises the following steps:
One further aspect according to the invention is a method for producing a therapeutic polypeptide, characterized in that the method comprises the following steps:
It has further been found that water in combination with/followed by aqueous buffered solutions can be used as regeneration solutions in the method according to the invention in combination with a pre-treatment of the depth filter with an alkaline solution, which results in a beneficial effect.
Thus, one aspect according to the current invention is a method for purifying a therapeutic polypeptide, characterized in that the method comprises the following steps:
One further aspect according to the current invention is a method for producing a therapeutic polypeptide, characterized in that the method comprises the following steps:
It has further been found that water can be used as regeneration solution in combination with a pre-treatment of the depth filter with an alkaline solution, which results in a beneficial effect.
Thus, one aspect according to the current invention is a method for purifying a therapeutic polypeptide, characterized in that the method comprises the following steps:
One further aspect according to the invention is a method for producing a therapeutic polypeptide, characterized in that the method comprises the following steps:
One further aspect according to the invention is the use of an acidic solution for regeneration of a depth filter (comprising silica) that is being used at least two times in the purification of a therapeutic polypeptide.
One further aspect according to the invention is the use of an alkaline solution for regeneration of a depth filter (comprising silica) that is being used at least two times in the purification of a therapeutic polypeptide.
In certain embodiments of the above aspects and the other embodiments, the depth filter comprises silica (as filter aid).
In certain embodiments of the above aspects and the other embodiments, the method reduces the (HCP-based/originating) enzymatic hydrolytic activity/hydrolysis activity rate.
In certain embodiments of the above aspects and the other embodiments, the method reduces/removes HCPs (host cell proteins) with enzymatic hydrolytic activity. In certain embodiments of the above aspects and the other embodiments, the method reduces the enzymatic hydrolysis activity rate in the purified therapeutic polypeptide compared to the aqueous composition (of step a) prior to the application to the filter).
In certain embodiments of the above aspects and the other embodiments, the method reduces the enzymatic hydrolysis activity rate in the purified therapeutic polypeptide by at least 25%, or by at least 30%, or by at least 35%, or by at least 40% compared to the aqueous composition (of step a) prior to the application of the filter).
In certain embodiments of the above aspects and the other embodiments, the enzymatic hydrolysis activity rate is determined by a lipase activity assay (as described herein in Example 21).
In certain embodiments of the above aspects and the other embodiments, the enzymatic hydrolysis activity rate is determined by the enzymatic hydrolysis of a substrate. In certain embodiments of the above aspects and the other embodiments, enzymatic hydrolysis activity rate is determined by monitoring the conversion of a substrate. In certain embodiments of the above aspects and the other embodiments, enzymatic hydrolysis activity rate is determined by monitoring the conversion of a fluorogenic substrate 4-Methylumbelliferyl Caprylate (4-MU-C8) by cleavage of the ester bond by hydrolases present in the sample/the purified therapeutic polypeptide into a fluorescent moiety, i.e. 4-Methylumbelliferyl (4-MU).
In certain embodiments of the above aspects and the other embodiments, the enzymatic hydrolysis activity rate is determined by the enzymatic hydrolysis of a substrate. In one embodiment, the hydrolysis rate is the hydrolysis rate of 4-Methylumbelliferyl Caprylate or a nonionic surfactant, e.g. a polysorbate, (in one embodiment polysorbate 20 or polysorbate 80).
In certain embodiments of the above aspects and the other embodiments, a pharmaceutical formulation comprising the purified/produced therapeutic polypeptide and a nonionic surfactant, e.g. a polysorbate, shows reduced hydrolysis of the nonionic surfactant, e.g. the polysorbate, compared to an identical pharmaceutical formulation comprising the aqueous composition instead of the purified/produced therapeutic polypeptide.
In certain embodiments of the above aspects and the other embodiments, the yield of the (monomeric) therapeutic polypeptide obtained in step a) using the depth filter treated according to step b) (i.e. after regeneration) is at least 80%, or at least 85%, or at least 90%, or at least 95% of the yield obtained in step a) using the depth filter for the first time, i.e. in the first filtration step.
In certain embodiments of the above aspects and the other embodiments, the yield of the (monomeric) therapeutic polypeptide obtained in step a) after performing step b) (i.e. regeneration) is at least 90% of the yield obtained in step a) using the depth filter for the first time, i.e. in the first filtration step.
In certain embodiments of the above aspects and the other embodiments, the depth filter comprises a material that is selected from the group consisting of
In certain embodiments of the above aspects and the other embodiments, the depth filter is selected from the group consisting of an X0SP depth filter, or a PDD1 depth filter, or aVR02 depth filter.
In certain embodiments of the above aspects and the other embodiments, the depth filter is an X0SP depth filter, or a PDD1 depth filter.
In certain embodiments of the above aspects and the other embodiments, the depth filter is contacted with the (regeneration) solution of step b) for about 20 min., for 20 min. or more, for 30 min. or more, for 40 min. or more, for 50 min. or more, or for 60 min. or more.
In certain embodiments of the above aspects and the other embodiments, the depth filter is used before a first chromatography step/prior to applying the aqueous composition to a chromatography material.
In certain embodiments of the above aspects and the other embodiments, the filter is used after the first chromatography step/after applying the aqueous composition to a chromatography material, i.e. the aqueous solution is a chromatographically purified aqueous solution.
In certain embodiments of the above aspects and the other embodiments, the therapeutic polypeptide is a recombinantly produced protein. In certain embodiments of the above aspects and the other embodiments, the therapeutic polypeptide is a recombinantly produced protein that is being formulated with a nonionic surfactant e.g. a polysorbate. In certain embodiments of the above aspects and the other embodiments, the therapeutic polypeptide is selected from the group of therapeutic polypeptides consisting of antibodies, antibody fragments, antibody fusion polypeptides, Fc-region fusion polypeptides, interferons, blood factors, cytokines, and enzymes.
In addition to the various aspects and embodiments depicted and claimed, the invention is also encompassing other embodiments having other combinations of the aspects and embodiments disclosed and claimed herein. As such, the particular features presented herein, especially presented as aspects or embodiments, can be combined with each other in other manners within the scope of the disclosed subject matter such that the disclosed subject matter includes any suitable combination of the features disclosed herein. The description of specific embodiments of the disclosed subject matter has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosed subject matter to those embodiments disclosed.
Herein is reported a method for the purification or production of a therapeutic polypeptide using the same depth filter multiple times, i.e. using a depth filter, which has been used before and has been regenerated.
The present invention is based, at least in part, on the unexpected finding that the functionality of a depth filter can be maintained after reaching its capacity limit when an acidic or alkaline solution in a method according to the invention is applied to the depth filter after its use, i.e. when the depth filter is regenerated thereby.
The regeneration of depth filters for re-use according to the current invention follows a general principle. The regeneration can be performed with regeneration solutions of different nature. In different aspects of the invention the regeneration is effected by applying
The acidic regeneration solution or the alkaline regeneration solution can be acidic aqueous buffered solutions or alkaline aqueous buffered solutions.
The invention is further based, at least in part, on the finding that the treatment with one of the above solutions can be effected or improved, if it is combined with an alkaline pre-treatment of the depth filter.
In the alkaline pre-treatment, the depth filter is incubated with an alkaline solution for a defined period of time prior to its first use and the regeneration is effected by one of the above regeneration solutions and in addition an aqueous buffered regeneration solution, or water, or water in combination with an aqueous buffered regeneration solution can be used.
The invention is further based, at least in part, on the finding that after alkaline pre-treatment of the depth filter regeneration of the depth filter can also be achieved by applying
The method according to the invention can be used to purify as well as to produce a therapeutic polypeptide, such as an antibody.
In more detail, it has been found that the use of regeneration solutions enable the re-use of depth filters for multiple times (cycles) for the purification of compositions comprising therapeutic polypeptides. The current inventors have shown the general applicability of the method according to the current invention with several different depth filters, different regeneration solutions as well as different antibodies/therapeutic polypeptides (in different formats) as exemplary implementation and embodiments of the invention.
In a first setup (see Example 11) the solution was an acidic solution (phosphoric acid; grey bars). It was surprisingly found that the depth filter can be re-used multiple times without significantly impairing its function when the depth filter was contacted with an acidic solution in between the cycles. The hydrolytic activity remains at very low levels throughout all cycles compared to the load (before depth filtration). Thus, with an acidic solution an efficient regeneration of the depth filter could be achieved.
In a second setup (see Example 10) an alkaline solution (sodium hydroxide; black dotted bars) was applied to the depth filter in between the cycles. Additionally the depth filter was per-treated before its first use by incubation with sodium hydroxide for approximately 30 minutes. Again, it was surprisingly found that the depth filter can be re-used multiple times without significantly impairing its function. The hydrolytic activity is very low compared to the load (before depth filtration) and it remains on low levels over multiple filtration cycles, i.e. the depth filter could be regenerated multiple times for re-use.
In a third setup the depth filter was treated with water and a buffered solution between the filtration cycles. No pre-treatment with an alkaline solution was done (see Example 9; grey dotted bars). After reaching the binding capacity limit a substantial increase of the hydrolytic activity in the filtrate can be observed (cf. #2 and #3 in
In
This unexpected effect of the method according to the current invention as shown in
For instance in example 1 a bispecific antibody is used and the depth filter is regenerated with an alkaline solution without pre-treatment. As can be seen in the Tables of Example 1, the hydrolytic activity is significantly reduced and remains on a low level. Interestingly, at first the yield decreases and it increases after four filtration cycles again, reaching higher levels than after the first filtration cycle. This drop in mainpeak yield can be circumvented by the pre-treatment of the filter with an alkaline solution (see e.g. Example 7 and Example 10 (=setup 2 in
A skilled person will acknowledge that the depth filter has to be equilibrated (with equilibration buffer) before the aqueous composition is applied, i.e. before it can be used. Without explicitly mentioning this, step a) includes the sub-step of contacting the depth filter with an equilibration buffer.
Regeneration with an Acidic Solution
It has been found that an acidic regeneration solutions can be used in method according to the invention.
One aspect according to the invention is a method for purifying a therapeutic polypeptide, characterized in that the method comprises the following steps:
One aspect according to the invention is a method for purifying a therapeutic polypeptide, characterized in that the method comprises the following steps:
A further aspect according to the invention is a method for producing a therapeutic polypeptide, characterized in that the method comprises the following steps:
In certain embodiments of the above aspects and other embodiments, the acidic regeneration solution of step b) is a solution comprising phosphoric acid.
In certain embodiments of the above aspects and other embodiments, the acidic regeneration solution of step b) is a solution comprising acetic acid.
In certain embodiments of the above aspects and other embodiments, the acidic regeneration solution of step b) is a solution comprising citric acid.
In certain embodiments of the above aspects and other embodiments, the acidic regeneration solution of step b) is a solution comprising phosphoric acid and acetic acid.
In certain embodiments of the above aspects and other embodiments, the acidic regeneration solution of step b) is a solution comprising phosphoric acid, acetic acid and benzyl alcohol.
In certain embodiments of the above aspects and other embodiments, the acidic regeneration solution of step b) comprises phosphoric acid in a concentration of about 0.1 M to about 0.8 M, or about 0.2 M to about 0.7 M, or in one preferred embodiment about 0.4 M to about 0.6 M.
In certain embodiments of the above aspects and other embodiments, the acidic regeneration solution of step b) comprises phosphoric acid in a concentration of about 0.3 M, or about 0.4 M, or about 0.5 M, or about 0.6 M.
In certain embodiments of the above aspects and other embodiments, the acidic regeneration solution comprises acetic acid in a concentration of about 10 mM to about 2 M, or about 20 mM to about 1.5 M, or about 50 mM to about 1 M, or in one preferred embodiment about 80 mM to about 800 mM.
In certain embodiments of the above aspects and other embodiments, the acidic regeneration solution of step b) comprises acetic acid in a concentration of about 10 mM, or about 20 mM, or about 50 mM, or about 100 mM, or about 120 mM, or about 140 mM, or about 160 mM, or about 180 mM, or about 200 mM, or about 500 mM, or about 1 M, or about 2 M.
In one preferred embodiment of the above aspects and other embodiments, the acidic regeneration solution of step b) comprises phosphoric acid in a concentration of about 300 mM and acetic acid in a concentration of about 167 mM.
In certain embodiments of the above aspects and other embodiments, the acidic regeneration solution of step b) is a solution comprising acetic acid in a concentration of about 50 mM to about 200 mM.
In certain embodiments of the above aspects and other embodiments, the acidic regeneration solution of step b) is a solution comprising citric acid in a concentration of about 10 mM to about 100 mM.
In certain embodiments of the above aspects and other embodiments, the acidic regeneration solution has a pH value of about 1 to about 5.5, or about 1 to about 5, or about 1 to about 4.5, or about 1 to about 4, or about 1 to about 3.5, or in one preferred embodiment about 1 to about 3.
In certain embodiments of the above aspects and other embodiments, the acidic regeneration solution of step b) has a pH value of about 1, or about 1.3, or about 1.5.
In certain embodiments of the above aspects and other embodiments, the acidic regeneration solution of step b) is an acidic aqueous buffered solution.
Regeneration Using an Alkaline Solution
It has been found that an alkaline regeneration solutions can be used in method according to the invention.
One aspect according to the invention is a method for purifying a therapeutic polypeptide, characterized in that the method comprises the following steps:
One further aspect according to the invention is a method for producing a therapeutic polypeptide, characterized in that the method comprises the following steps:
In certain embodiments of the above aspects and other embodiments, the alkaline regeneration solution of step b) is a solution comprising sodium hydroxide (NaOH) or potassium hydroxide (KOH).
In certain embodiments of the above aspects and other embodiments, the alkaline solution of step b) comprises NaOH in a concentration of about 0.1 M to about 1.5 M, about 0.2 M to about 1.4 M, about 0.3 M to about 1.2 M, about 0.4 M to about 1.1 M, or about 0.5 M to about 1 M.
In certain embodiments of the above aspects and other embodiments, the alkaline solution of step b) comprises NaOH in a concentration of at least about 0.01 M, at least about 0.05 M, at least about 0.1 M, at least about 0.2 M, at least about 0.3 M, at least about 0.4 M, at least about 0.5 M, at least about 0.6 M, at least about 0.7 M, at least about 0.8 M, at least about 0.9 M, at least about 1 M, at least about 1.1 M, at least about 1.2 M, at least about 1.3 M, at least about 1.4 M or at least about 1.5 M. In one preferred embodiment, the alkaline solution of step b) comprises at least about 1 M NaOH.
In certain embodiments of the above aspects and other embodiments, the alkaline regeneration solution of step b) is an alkaline aqueous buffered solution.
In certain embodiments of the above aspects and other embodiments, the alkaline solution of step b) additionally comprises sodium chloride (NaCl).
In certain embodiments of the above aspects and other embodiments, the alkaline solution of step b) comprises NaCl in a concentration of about 0.5 M to about 2.5 M, about 0.6 M to about 2.3 M, about 0.7 M to 2 M, about 0.8 M to about 1.8 M, or in one preferred embodiment about 0.9 M to about 1.5 M.
In certain embodiments of the above aspects and other embodiments, the alkaline solution of step b) comprises NaCl in a concentration of about 0.5 M, about 0.7 M, about 0.8 M, or in one preferred embodiment about 1 M.
In certain embodiments of the above aspects and other embodiments, the alkaline (regeneration) solution of step b) has a pH value of about 9 to 14, about 9.5 to 14, or in one preferred embodiment about 10 to 14.
In certain embodiments of the above aspects and other embodiments, the alkaline regeneration solution of step b) has a pH value of about 9 or more, about 9.5 or more, in one preferred embodiment about 10 or more, about 10.5 or more, or about 11 or more.
In certain embodiments of the above aspects and other embodiments, the method further includes prior to step a) the step a0) of incubating a depth filter comprising silica (as filter aid) with an alkaline solution.
The skilled person understands that the pre-incubation/pre-treatment time (contact time) with the alkaline solution can vary depending on the concentration and/or the pH value and/or the flow of the alkaline solution.
In certain embodiments of the above aspects and other embodiments, the pre-incubation is with an NaOH solution of 100 mM to 1.2 M. In certain embodiments of the above aspects and other embodiments, the pre-incubation is with an NaOH solution for 30 minutes to 4.5 hours. In one embodiment of the above aspects and other embodiments, the pre-incubation is with an at least 100 mM NaOH alkaline solution for at least about 30 minutes. In one preferred embodiment of the above aspects and other embodiments, the pre-incubation is with a 1 M NaOH alkaline solution for about 4 hours.
In certain embodiments of the above aspects and other embodiments, the pre-incubation is with at least 50 L, or 50 L to 200 L, or 100 L to 150 L of the alkaline solution per m2 of depth filter area. In one preferred embodiment of the above aspects and other embodiments, the pre-incubation is with 100 L of the alkaline solution per m2 of depth filter area (100 L/m2).
Regeneration with an Alkaline Solution and Alkaline Pre-Treatment
It has been found that alkaline solutions can be used as regeneration solutions and that a pre-treatment of the depth filter with an alkaline solution has a beneficial effect.
One aspect according to the invention is a method for purifying a therapeutic polypeptide, characterized in that the method comprises the following steps:
One aspect according to the invention is a method for purifying a therapeutic polypeptide, characterized in that the method comprises the following steps:
One further aspect according to the invention is a method for producing a therapeutic polypeptide, characterized in that the method comprises the following steps:
In certain embodiments of the above aspects and other embodiments, the alkaline solution of step a0) is a solution comprising NaOH or KOH.
In certain embodiments of the above aspects and other embodiments, the alkaline regeneration solution of step b) is a solution comprising NaOH or KOH.
In certain embodiments of the above aspects and other embodiments, the alkaline solution of step a0) or step b) comprises NaOH in a concentration of about 0.1 M to about 1.5 M, about 0.2 M to about 1.4 M, about 0.3 M to about 1.2 M, about 0.4 M to about 1.1 M, about 0.5 M to about 1 M. In one preferred embodiment, the alkaline solution of step a0) and b) comprises NaOH in a concentration of about 0.9 M to about 1.1 M.
In certain embodiments of the above aspects and other embodiments, the alkaline solution of step a0) or step b) comprises NaOH in a concentration of at least about 0.05 M, at least about 0.1 M, at least about 0.2 M, at least about 0.3 M, at least about 0.4 M, at least about 0.5 M, at least about 0.6 M, at least about 0.7 M, at least about 0.8 M, in one preferred embodiment at least about 0.9 M, at least about 1 M, at least about 1.1 M, at least about 1.2 M, at least about 1.3 M, at least about 1.4 M, or at least about 1.5 M.
In certain embodiments of the above aspects and other embodiments, the pre-incubation is with an NaOH solution of 100 mM to 1.2 M. In certain embodiments of the above aspects and other embodiments, the pre-incubation is with an NaOH solution for 30 minutes to 4.5 hours. In one embodiment of the above aspects and other embodiments, the pre-incubation is with an at least 100 mM NaOH alkaline solution for at least about 30 minutes. In one preferred embodiment of the above aspects and other embodiments, the pre-incubation is with a 1 M NaOH alkaline solution for about 4 hours.
In certain embodiments of the above aspects and other embodiments, the pre-incubation is with at least 50 L, or 50 L to 200 L, or 100 L to 150 L of the alkaline solution per m2 of depth filter area. In one preferred embodiment of the above aspects and other embodiments, the pre-incubation is with 100 L of the alkaline solution per m2 of depth filter area (100 L/m2).
In certain embodiments of the above aspects and other embodiments, the alkaline regeneration solution of step b) is an alkaline aqueous buffered solution.
In certain embodiments of the above aspects and other embodiments, the alkaline solution of step a0) and/or step b) additionally comprises NaCl.
In certain embodiments of the above aspects and other embodiments, the alkaline solution of step a0) or step b) comprises NaCl in a concentration of about 0.5 M to about 2.5 M, about 0.6 M to about 2.3 M, about 0.7 M to about 2 M, about 0.8 M to about 1.8 M, or in one preferred embodiment about 0.9 M to about 1.5 M.
In certain embodiments of the above aspects and other embodiments, the alkaline solution of step a0) or step b) comprises NaCl in a concentration of about 0.5 M, about 0.7 M, about 0.8 M, or in one preferred embodiment about 1 M.
Regeneration with an Aqueous Buffered Solution and Alkaline Pre-Treatment
It has been found that aqueous buffered solutions can be used as regeneration solutions in the method according to the invention and that a pre-treatment of the depth filter with an alkaline solution has a beneficial effect.
One aspect according to the invention is a method for purifying a therapeutic polypeptide, characterized in that the method comprises the following steps:
One further aspect as reported herein is a method for producing a therapeutic polypeptide, characterized in that the method comprises the following steps:
In certain embodiments of the above aspects and other embodiments, the aqueous buffered regeneration solution of step b) is the equilibration buffer/the buffer used for equilibrating the depth filter.
In certain embodiments of the above aspects and other embodiments, the equilibration buffer has a pH value of about 3.5 to about 8, or about 3.5 to about 6, or in a preferred embodiment about 4 to about 5.5. In one preferred embodiment, the equilibration buffer has a pH value of pH 4+/−0.2. In one preferred embodiment, the equilibration buffer has a pH value of pH 5.5+/−0.2.
In certain embodiments of the above aspects and other embodiments, the equilibration buffer comprises 150 mM acetic acid/tris (hydroxymethyl) amino methane.
In certain embodiments of the above aspects and other embodiments, the alkaline solution of step a0) is a solution comprising NaOH or KOH.
In certain embodiments of the above aspects and other embodiments, the alkaline solution of step a0) comprises NaOH in a concentration of about 0.1 M to about 1.5 M, about 0.2 M to about 1.4 M, about 0.3 M to about 1.2 M, about 0.4 M to about 1.1 M, about 0.5 M to about 1 M. In one preferred embodiment, the alkaline solution of step a0) comprises NaOH in a concentration of about 0.9 M to about 1.1 M.
In certain embodiments of the above aspects and other embodiments, the alkaline solution of step a0) comprises NaOH in a concentration of at least about 0.05 M, at least about 0.1 M, at least about 0.2 M, at least about 0.3 M, at least about 0.4 M, at least about 0.5 M, at least about 0.6 M, at least about 0.7 M, at least about 0.8 M, in one preferred embodiment at least about 0.9 M, at least about 1 M, at least about 1.1 M, at least about 1.2 M, at least about 1.3 M, at least about 1.4 M, or at least about 1.5 M.
In certain embodiments of the above aspects and other embodiments, the pre-incubation is with an NaOH solution of 100 mM to 1.2 M. In certain embodiments of the above aspects and other embodiments, the pre-incubation is with an NaOH solution for 30 minutes to 4.5 hours. In one embodiment of the above aspects and other embodiments, the pre-incubation is with an at least 100 mM NaOH alkaline solution for at least about 30 minutes. In one preferred embodiment of the above aspects and other embodiments, the pre-incubation is with a 1 M NaOH alkaline solution for about 4 hours.
In certain embodiments of the above aspects and other embodiments, the pre-incubation is with at least 50 L, or 50 L to 200 L, or 100 L to 150 L of the alkaline solution per m2 of depth filter area. In one preferred embodiment of the above aspects and other embodiments, the pre-incubation is with 100 L of the alkaline solution per m2 of depth filter area (100 L/m2).
In certain embodiments of the above aspects and other embodiments, the alkaline regeneration solution of step b) is an alkaline aqueous buffered solution.
In certain embodiments of the above aspects and other embodiments, the alkaline solution of step a0) additionally comprises NaCl.
In certain embodiments of the above aspects and other embodiments, the alkaline solution of step a0) comprises NaCl in a concentration of about 0.5 M to about 2.5 M, about 0.6 M to about 2.3 M, about 0.7 M to about 2 M, about 0.8 M to about 1.8 M, or about 0.9 M to about 1.5 M. In one preferred embodiment, the alkaline solution of step a0) comprises NaOH in a concentration of about 0.9 M to about 1.1 M.
In certain embodiments of the above aspects and other embodiments, the alkaline solution of step a0) comprises NaCl in a concentration of at least about 0.5 M, at least about 0.7 M, at least about 0.8 M, at least about 0.9 M, or in one preferred embodiment at least about 1 M.
In certain embodiments of the above aspects and other embodiments, the alkaline regeneration solution of step a0) has a pH value of about 9 to 14, about 9.5 to 14, or about 10 to 14.
In certain embodiments of the above aspects and other embodiments, the alkaline regeneration solution of step a0) has a pH value of about 9 or more, about 9.5 or more, in one preferred embodiment about 10 or more, about 10.5 or more, or about 11 or more.
Regeneration with Water Followed by an Aqueous Buffered Solution and Alkaline Pre-Treatment
It has been found that water in combination with/followed by aqueous buffered solutions can be used as regeneration solutions in the method according to the invention and that a pretreatment of the depth filter with an alkaline solution has a beneficial effect.
One aspect as reported herein is a method for purifying a therapeutic polypeptide, characterized in that the method comprises the following steps:
One further aspect as reported herein is a method for producing a therapeutic polypeptide, characterized in that the method comprises the following steps:
In certain embodiments of the above aspects and other embodiments, the alkaline solution of step a0) is a solution comprising NaOH or KOH.
In certain embodiments of the above aspects and other embodiments, the alkaline solution of step a0) comprises NaOH in a concentration of about 0.1 M to about 1.5 M, about 0.2 M to about 1.4 M, about 0.3 M to about 1.2 M, about 0.4 M to about 1.1 M, about 0.5 M to about 1 M. In one preferred embodiment, the alkaline solution of step a0) comprises NaOH in a concentration of about 0.9 M to about 1.1 M.
In certain embodiments of the above aspects and other embodiments, the alkaline solution of step a0) comprises NaOH in a concentration of at least about 0.05 M, at least about 0.1 M, at least about 0.2 M, at least about 0.3 M, at least about 0.4 M, at least about 0.5 M, at least about 0.6 M, at least about 0.7 M, at least about 0.8 M, in one preferred embodiment at least about 0.9 M, at least about 1 M, at least about 1.1 M, at least about 1.2 M, at least about 1.3 M, at least about 1.4 M, or at least about 1.5 M.
In certain embodiments of the above aspects and other embodiments, the pre-incubation is with an NaOH solution of 100 mM to 1.2 M. In certain embodiments of the above aspects and other embodiments, the pre-incubation is with an NaOH solution for 30 minutes to 4.5 hours. In one embodiment of the above aspects and other embodiments, the pre-incubation is with an at least 100 mM NaOH alkaline solution for at least about 30 minutes. In one preferred embodiment of the above aspects and other embodiments, the pre-incubation is with a 1 M NaOH alkaline solution for about 4 hours.
In certain embodiments of the above aspects and other embodiments, the pre-incubation is with at least 50 L, or 50 L to 200 L, or 100 L to 150 L of the alkaline solution per m2 of depth filter area. In one preferred embodiment of the above aspects and other embodiments, the pre-incubation is with 100 L of the alkaline solution per m2 of depth filter area (100 L/m2).
In certain embodiments of the above aspects and other embodiments, the alkaline regeneration solution of step b) is an alkaline aqueous buffered solution.
In certain embodiments of the above aspects and other embodiments, the alkaline solution of step a0) additionally comprises NaCl.
In certain embodiments of the above aspects and other embodiments, the alkaline solution of step a0) comprises NaCl in a concentration of about 0.5 M to about 2.5 M, about 0.6 M to about 2.3 M, about 0.7 M to about 2 M, about 0.8 M to about 1.8 M, or about 0.9 M to about 1.5 M. In one preferred embodiment, the alkaline solution of step a0) comprises NaOH in a concentration of about 0.9 M to about 1.1 M.
In certain embodiments of the above aspects and other embodiments, the alkaline solution of step a0) comprises NaCl in a concentration of at least about 0.5 M, at least about 0.7 M, at least about 0.8 M, at least about 0.9 M, or in one preferred embodiment at least about 1 M.
In certain embodiments of the above aspects and other embodiments, the alkaline regeneration solution of step a0) has a pH value of about 9 to 14, about 9.5 to 14, or about 10 to 14.
In certain embodiments of the above aspects and other embodiments, the alkaline regeneration solution of step a0) has a pH value of about 9 or more, about 9.5 or more, in one preferred embodiment about 10 or more, about 10.5 or more, or about 11 or more.
In certain embodiments of the above aspects and other embodiments, the aqueous buffered regeneration solution of step b) is the equilibration buffer/the buffer used for equilibrating the depth filter.
In certain embodiments of the above aspects and other embodiments, the equilibration buffer has a pH value of about 3.5 to about 8, or about 3.5 to about 6, or in a preferred embodiment about 4 to about 5.5. In one preferred embodiment, the equilibration buffer has a pH value of pH 4+/−0.2. In one preferred embodiment, the equilibration buffer has a pH value of pH 5.5+/−0.2.
In certain embodiments of the above aspects and other embodiments, the equilibration buffer comprises 150 mM acetic acid/tris (hydroxymethyl) amino methane.
Regeneration with Water and Alkaline Pre-Treatment
It has been found that water can be used a regeneration solution and that a pre-treatment of the depth filter with an alkaline solution has a beneficial effect.
One aspect according to the invention is a method for purifying a therapeutic polypeptide, characterized in that the method comprises the following steps:
One further aspect according to the invention is a method for producing a therapeutic polypeptide, characterized in that the method comprises the following steps:
In certain embodiments of the above aspects and other embodiments, the alkaline solution of step a0) is a solution comprising NaOH or KOH.
In certain embodiments of the above aspects and other embodiments, the alkaline solution of step a0) comprises NaOH in a concentration of about 0.1 M to about 1.5 M, about 0.2 M to about 1.4 M, about 0.3 M to about 1.2 M, about 0.4 M to about 1.1 M, about 0.5 M to about 1 M. In one preferred embodiment, the alkaline solution of step a0) comprises NaOH in a concentration of about 0.9 M to about 1.1 M.
In certain embodiments of the above aspects and other embodiments, the alkaline solution of step a0) comprises NaOH in a concentration of at least about 0.05 M, at least about 0.1 M, at least about 0.2 M, at least about 0.3 M, at least about 0.4 M, at least about 0.5 M, at least about 0.6 M, at least about 0.7 M, at least about 0.8 M, in one preferred embodiment at least about 0.9 M, at least about 1 M, at least about 1.1 M, at least about 1.2 M, at least about 1.3 M, at least about 1.4 M, or at least about 1.5 M.
In certain embodiments of the above aspects and other embodiments, the pre-incubation is with an NaOH solution of 100 mM to 1.2 M. In certain embodiments of the above aspects and other embodiments, the pre-incubation is with an NaOH solution for 30 minutes to 4.5 hours. In one embodiment of the above aspects and other embodiments, the pre-incubation is with an at least 100 mM NaOH alkaline solution for at least about 30 minutes. In one preferred embodiment of the above aspects and other embodiments, the pre-incubation is with a 1 M NaOH alkaline solution for about 4 hours.
In certain embodiments of the above aspects and other embodiments, the pre-incubation is with at least 50 L, or 50 L to 200 L, or 100 L to 150 L of the alkaline solution per m2 of depth filter area. In one preferred embodiment of the above aspects and other embodiments, the pre-incubation is with 100 L of the alkaline solution per m2 of depth filter area (100 L/m2).
In certain embodiments of the above aspects and other embodiments, the alkaline regeneration solution of step b) is an alkaline aqueous buffered solution.
In certain embodiments of the above aspects and other embodiments, the alkaline solution of step a0) additionally comprises NaCl.
In certain embodiments of the above aspects and other embodiments, the alkaline solution of step a0) comprises NaCl in a concentration of about 0.5 M to about 2.5 M, about 0.6 M to about 2.3 M, about 0.7 M to about 2 M, about 0.8 M to about 1.8 M, or about 0.9 M to about 1.5 M. In one preferred embodiment, the alkaline solution of step a0) comprises NaOH in a concentration of about 0.9 M to about 1.1 M.
In certain embodiments of the above aspects and other embodiments, the alkaline solution of step a0) comprises NaCl in a concentration of at least about 0.5 M, at least about 0.7 M, at least about 0.8 M, at least about 0.9 M, or in one preferred embodiment at least about 1 M.
In certain embodiments of the above aspects and other embodiments, the alkaline regeneration solution of step a0) has a pH value of about 9 to 14, about 9.5 to 14, or about 10 to 14.
In certain embodiments of the above aspects and other embodiments, the alkaline regeneration solution of step a0) has a pH value of about 9 or more, about 9.5 or more, in one preferred embodiment about 10 or more, about 10.5 or more, or about 11 or more.
One further aspect according to the invention is the use of an acidic solution for regeneration of a depth filter (comprising silica) that is being used at least two times in the purification of a therapeutic polypeptide.
One further aspect according to the invention is the use of an alkaline solution for regeneration of a depth filter (comprising silica) that is being used at least two times in the purification of a therapeutic polypeptide.
Effects and Uses of the Methods According to the Invention
In certain embodiments of all above aspects and embodiments, the method reduces the (HCP-based/originating) enzymatic hydrolytic activity/hydrolysis activity rate.
In certain embodiments of all above aspects and embodiments, the method reduces/removes HCPs (host cell proteins) with enzymatic hydrolytic activity.
In certain embodiments of all above aspects and embodiments of the invention, the method reduces the enzymatic hydrolysis activity rate in the produced/purified therapeutic polypeptide compared to the aqueous composition (of step a) prior to the application to the filter).
In certain embodiments of all above aspects and embodiments of the invention, the method reduces the enzymatic hydrolysis activity rate in the produced/purified therapeutic polypeptide compared to the aqueous composition (of step a) prior to the application to the filter).
In certain embodiments of all above aspects and embodiments of the invention, the method reduces the enzymatic hydrolysis activity rate in the produced/purified therapeutic polypeptide by at least 25%, or by at least 30%, or by at least 35%, or by at least 40% compared to the aqueous composition (of step a) prior to the application to the filter).
In certain embodiments of the above aspects and the other embodiments, the enzymatic hydrolysis activity rate is determined by a lipase activity assay (as described herein).
In certain embodiments of the above aspects and the other embodiments, enzymatic hydrolysis activity rate is determined by the enzymatic hydrolysis of a substrate. In certain embodiments of the above aspects and the other embodiments, enzymatic hydrolysis activity rate is determined by monitoring the conversion of a substrate. In certain embodiments of the above aspects and the other embodiments, enzymatic hydrolysis activity rate is determined by monitoring the conversion of a fluorogenic substrate ‘4-Methylumbelliferyl Caprylate’ (4-MU-C8) by cleavage of the ester bond by hydrolases present in the sample/the purified therapeutic polypeptide into a fluorescent moiety, i.e. 4-Methylumbelliferyl (4-MU).
In certain embodiments of all above aspects and embodiments, the enzymatic hydrolysis activity rate is determined by the enzymatic hydrolysis of a substrate. In one embodiment, the hydrolysis rate is the hydrolysis rate of 4-Methylumbelliferyl Caprylate or polysorbate (in one embodiment polysorbate 20).
In certain embodiments of all above aspects and embodiments, a pharmaceutical formulation comprising the purified/produced therapeutic polypeptide and a polysorbate shows reduced hydrolysis of the polysorbate compared to an identical pharmaceutical formulation comprising the aqueous composition instead of the purified/produced therapeutic polypeptide. In certain embodiments of all above aspects and embodiments of the invention, the yield of the (monomeric) therapeutic polypeptide obtained when using a depth filter after regeneration is at least 80%, or at least 85%, or at least 90%, or at least 95% of the yield obtained when using the depth filter for the first time, i.e. without regeneration/after the first filtration with the depth filter.
In certain embodiments of all above aspects and embodiments of the invention, the yield of the (monomeric) therapeutic polypeptide obtained using a depth filter after regeneration is at least 90% of the yield obtained when using the depth filter for the first time, i.e. without regeneration/after the first filtration with the depth filter.
In certain embodiments of all above aspects and embodiments of the invention, the depth filter comprises a substrate comprising one or more of a diatomaceous earth composition, a silica composition, a cellulose fiber, a polymeric fiber, a cohesive resin, or/and an ash composition.
In certain embodiments of all above aspects and embodiments of the invention, the depth filter comprises (a substrate comprising) one or more selected from a diatomaceous earth material (composition), a silica material (composition), a cellulose fiber, and a polymeric fiber.
In certain embodiments of all above aspects and embodiments of the invention, at least a portion of the substrate of the depth filter comprises a surface modification.
In certain embodiments of all above aspects and embodiments of the invention, at least a portion of the substrate of the depth filter comprises one or more surface modification(s) selected from a quaternary amine surface modification, charged surface group modifications (a cationic surface modifications or an anionic surface modifications). In one preferred embodiment, the surface modification is a cationic surface modification.
In certain embodiments of all above aspects and embodiments of the invention, the depth filter comprises a material that is selected from the group of
In certain embodiments of all above aspects and embodiments of the invention, the depth filter is selected from the group consisting of an X0SP depth filter (Millistak+® HC Pro X0SP), or a PDD1 depth filter (SUPRAcap™-50 (SC050PDD1)), or aVR02 depth filter (Zeta Plus™ Biocap VR02).
In certain embodiments of all above aspects and embodiments of the invention, the depth filter is an X0SP depth filter (Millistak+® HC Pro X0SP), or a PDD1 depth filter (SUPRAcap™-50 (SC050PDD1)).
In certain embodiments of all above aspects and embodiments of the invention, the depth filter is contacted with the regeneration solution for about 20 min. or more, about 30 min. or more, about 40 min. or more, about 50 min. or more, or about 60 min. or more.
It is understood that the depth filter can be used before or after a first chromatography step. It can also be used before or after a second, third, fourth or any further chromatography step. In one preferred embodiment of all aspects and other embodiments according to the invention, the depth filter is before or after a first chromatography step performed with the aqueous composition. One especially preferred use is before the first chromatography step (i.e. after the harvesting of the cells from cell cultivation).
In certain embodiments of all above aspects and embodiments of the invention, the method according to the invention comprises as first step or as last step a chromatography step. In one preferred embodiment of all above aspects and embodiments of the invention, the method comprises as first step a chromatography step and the aqueous composition of step a) is the eluate (fraction) of the chromatography step comprising the therapeutic polypeptide.
In one preferred embodiment of all above aspects and embodiments of the invention, the method comprises as last step a chromatography step and the produced/purified therapeutic polypeptide obtained in step a) is used in the chromatography step/applied to the chromatography material.
In certain embodiments of the above aspects and the other embodiments, the therapeutic polypeptide is a recombinantly produced protein. In certain embodiments of the above aspects and the other embodiments, the therapeutic polypeptide is a recombinantly produced protein that is being formulated with a nonionic surfactant like e.g. a polysorbate. In certain embodiments of the above aspects and the other embodiments, the therapeutic polypeptide is selected from the group of therapeutic polypeptides consisting of antibodies, antibody fragments, antibody fusion polypeptides, Fc-region fusion polypeptides, interferons, blood factors, cytokines, proteins for vaccination, and enzymes.
For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing quantities of ingredients, percentages or proportions of materials, reaction conditions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about” whether or not explicitly indicated. The term “about” denotes a range of +/−20% of the thereafter following numerical value. In one embodiment, the term about denotes a range of +/−10% of the thereafter following numerical value. In one embodiment the term about denotes a range of +/−5% of the thereafter following numerical value.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Moreover, all ranges disclosed herein are to be understood to encompass all sub ranges subsumed therein. For example, a range of “1 to 10” includes any and all sub ranges between (and including) the minimum value of 1 and the maximum value of 10, that is, any and all sub ranges having a minimum value of equal to or greater than 1 and a maximum value of equal to or less than 10, e.g., 5.5 to 10.
Useful methods and techniques for carrying out the current invention are described in e.g. Ausubel, F. M. (ed.), Current Protocols in Molecular Biology, Volumes I to III (1997); Glover, N. D., and Hames, B. D., ed., DNA Cloning: A Practical Approach, Volumes I and II (1985), Oxford University Press; Freshney, R. I. (ed.), Animal Cell Culture—a practical approach, IRL Press Limited (1986); Watson, J. D., et al., Recombinant DNA, Second Edition, CHSL Press (1992); Winnacker, E. L., From Genes to Clones; N. Y., VCH Publishers (1987); Celis, J., ed., Cell Biology, Second Edition, Academic Press (1998); Freshney, R. I., Culture of Animal Cells: A Manual of Basic Technique, second edition, Alan R. Liss, Inc., N.Y. (1987).
The use of recombinant DNA technology enables the generation of derivatives of a nucleic acid. Such derivatives can, for example, be modified in individual or several nucleotide positions by substitution, alteration, exchange, deletion or insertion. The modification or derivatization can, for example, be carried out by means of site directed mutagenesis. Such modifications can easily be carried out by a person skilled in the art (see e.g. Sambrook, J., et al., Molecular Cloning: A laboratory manual (1999) Cold Spring Harbor Laboratory Press, New York, USA; Hames, B. D., and Higgins, S. G., Nucleic acid hybridization—a practical approach (1985) IRL Press, Oxford, England).
It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth. As well, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein. It is also to be noted that the terms “comprising”, “including”, and “having” can be used interchangeably.
The term “aqueous solution” or “aqueous composition”, as used herein, relates to any liquid preparation wherein the concentration of water (H2O) in the solvent is at least 50% (w/v), in an embodiment at least 75% (w/v), in a further embodiment at least 90% (w/v), in a further embodiment at least 95% (w/v), in a further embodiment at least 98% (w/v), in a further embodiment at least 99% (w/v), in a further embodiment is at least 99.5% (w/v). Thus, the term aqueous solution encompasses liquid preparations comprising up to 50% (w/v) D20 or PEG (poly ethylene glycol).
Further, as used herein, the term “solution” indicates that at least a fraction of the compounds (solutes) in the solution is dissolved in the solvent. Methods for preparing solutions are known in the art. Thus, the term aqueous solution, in an embodiment, relates to a liquid preparation comprising a therapeutic polypeptide, which is, at least partially, dissolved in a solvent comprising, in an embodiment consisting of, a buffered solution.
The term “comprising” also encompasses the term “consisting of”.
Antibody Production and Purification
Depth filters can be used at various stages of monoclonal antibody (mAb) production/purification processes. Such processes can comprise one or more of the following steps in the following or a different order:
Depth filtration can be used, for example, prior to or after viral inactivation, ion exchange chromatography, viral filtration, or/and ultrafiltration. Depth filtration can be used to reduce (process-related) impurities. Depth filtration can be used to reduce (process-related) impurities that are hydrolytically active or possess hydrolytic activity. In other words, depth filtration can reduce the enzymatic hydrolysis activity rate. This effect/rate can be determined/measured by methods known to the skilled person, some of which are described herein (e.g. Lipase activity assay (LEAP) in Example 21). In some embodiments, depth filtration is used to reduce the hydrolytic activity of an aqueous composition. In some embodiments, depth filtration is used to reduce host cell DNA in an aqueous solution.
Depth filtration can also be used in further downstream stages of the purification process for secondary clarification and haze removal, and for further removal of process-related impurities as disclosed herein.
As used herein the term “depth filter” denotes a filter that achieves filtration, i.e. separation of material, within the depth of the filter material. In some embodiments, the depth filter comprises a porous filtration medium capable of retaining portions of a sample, such as, e.g., cell components and debris, wherein filtration occurs, e.g., within the depth of the filter material. A common class of such filters is those that comprise a (random) matrix of bonded fibers (or otherwise fixed), to form a complex, tortuous maze of flow channels. Particle separation in these filters generally results from entrapment by or adsorption to the filter material. Frequently used depth filter media for bioprocessing of cell culture broths and other feedstocks consists of cellulose fibers (as matrix) and a filter aid such as diatomaceous earth (DE). Another depth filter used in the context of the invention is a depth filter comprising silica and polyacrylic fiber. In some embodiments, the depth filter is a synthetic filter. In some embodiments, the depth filter comprises a silica filter aid, and/or polyacrylic fiber. In some embodiments, the depth filter comprises a silica filter aid, and/or polyacrylic fiber, and/or non-woven material. In some embodiments, the depth filter comprises silica and polyacrylic fiber as non-woven material. Depth filter media, unlike absolute filters, retain particles and other impurities throughout the porous media allowing, e.g., for retention of particles both larger and smaller than the pore size. Particle and impurity retention is thought to involve size exclusion and adsorption through hydrophobic, ionic and other interactions. Depth filters are advantageous because they remove contaminants/impurities. The depth filter may be a multi-layer depth filter comprising multiple levels of depth filter media, which are layered in series. Employing multiple depth filters ensures that more of the filtrate stream efficiently contacts the depth filter media, enabling a better adsorption profile for the impurities.
In some embodiments, the depth filter comprises synthetic material, non-synthetic material, or a combination thereof. In some embodiments, the depth filter comprises a substrate comprising one or more of a diatomaceous earth composition, a silica composition, a cellulose fiber, a polymeric fiber, a cohesive resin, and an ash composition. In some embodiments, the depth filter is selected from the group consisting of an X0SP depth filter (Millistak+® HC Pro X0SP), a PDD1 depth filter (Pall/3M PDD1 SUPRAcap™-50 (SC050PDD1)), or a VR02 depth filter (Zeta Plus™ Biocap VR02).
In some embodiments, the depth filter comprises cellulose fibers, diatomaceous earth, and perlite. In some embodiments, the depth filter comprises two layers, wherein each layer comprises a cellulose filter matrix, and wherein the cellulose filter matrix is impregnated with a filter aid comprising one or more of diatomaceous earth or perlite. In some embodiments, the depth filter comprises two layers, wherein each layer comprises a cellulose filter matrix, wherein the cellulose filter matrix is impregnated with a filter aid comprising one or more of diatomaceous earth or perlite, and wherein each layer further comprises a resin binder. In some embodiments, the depth filter is a PDD1 depth filter.
In some embodiments, the depth filter comprises a silica, such as a silica filter aid, and a polyacrylic fiber. In some embodiments, the depth filter comprises two layers of filter media, wherein a first layer comprises a silica, such as a silica filter aid, and a second layer comprises a polyacrylic fiber, such as a polyacrylic fiber pulp. In some embodiments, the depth filter is a depth filter comprising synthetic material and does not comprise diatomaceous earth and/or perlite. In some embodiments, the depth filter is a X0SP depth filter.
In some embodiments, the depth filter comprises cellulose fibers (as matrix) and charged surface groups (ionic charge modifications). In some embodiments, the depth filter comprises cellulose fibers (as matrix) and a cationic charge modifier that is chemically bound to the matrix components. In some embodiments, the depth filter is a VR02 depth filter.
In some embodiments, the silica filter aid is a precipitated silica filter aid. In some embodiments, the filter aid is an aspect of the filter, such as a layer, that aids with performing the filter function. In some embodiments, the silica filter aid is a silica gel filter aid. In some embodiments, the depth filter has a pore size of about 0.05 μm to about 0.2 μm, such as about 0.1 μm. In some embodiments, the depth filter has a surface area in the range of about 0.1 m2 to about 1.5 m2, such as at least about 22 cm2, at least about 23 cm2, or at least about 25 cm2, at least about 0.11 m2, at least about 0.55 m2, or of at least about 1.1 m2 or greater.
It is understood that depth filters have a certain capacity (binding capacity). This binding capacity defines the upper limit of the amount of therapeutic polypeptide molecules per surface that can be applied to the filter without impairing the filter properties (separation efficiency and yield). The skilled person knows how to determine said binding capacity limit for each depth filter and for each molecule. This can be done using standard methods.
When exceeding the capacity limit of a given depth filter, the ability of the filter to effectively remove impurities from the load and, thus, for example, the obtainable yield and/or the purity of the molecule of interest will decrease.
The skilled person understands that the depth filter functions well until reaching its binding capacity limit and there will be no need for regenerating of the depth filter beforehand.
The term “incubating” in connection with the pre-treatment of a depth filter prior to its first use includes different types of contact of the solution used for pre-treatment with the depth filter. This can for example be in the form of contacting the depth filter with a solution by flowing the solution through the depth filter for a period of time, i.e. washing the depth filter with the solution with a certain flow rate (e.g. 7 or 10 mL/min). The depth filter can also be contacted by placing it into a (pre-treatment) solution and storing it in the solution for a certain time. It is also possible that the flow-through of the solution/washing is performed first, followed by a pausing of the flow and therefore resting the depth filter in the solution; or vice-versa (i.e. storing before and/or after washing).
The invention encompasses the purification and production of therapeutic polypeptides. The therapeutic polypeptide can be of different nature. The therapeutic polypeptide is designed and suitable for therapeutic as well as for diagnostic purposes. In certain embodiments of the above aspects and the other embodiments, the therapeutic polypeptide is a recombinantly produced protein. In certain embodiments of the above aspects and the other embodiments, the therapeutic polypeptide is a recombinantly produced protein that is being formulated with a nonionic surfactant like e.g. a polysorbate. In certain embodiments of the above aspects and the other embodiments, the therapeutic polypeptide is selected from the group of therapeutic polypeptides consisting of antibodies, antibody fragments, antibody fusion polypeptides, Fc-region fusion polypeptides, interferons, blood factors, cytokines, proteins for vaccination, and enzymes. In a preferred embodiment of the above aspects and the other embodiments, the therapeutic polypeptide is an antibody.
The term “antibody” includes full-length antibodies and antigen-binding fragments thereof. In some embodiments, a full-length antibody comprises two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light chain (LC) CDRs including LC-CDR1, LC-CDR2, and LC-CDR3, heavy chain (HC) CDRs including HC-CDR1, HC-CDR2, and HC-CDR3). CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDRs of the heavy or light chains are interposed between flanking stretches known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several of the major antibody classes are divided into subclasses such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain). In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a human, humanized, or chimeric antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a semi-synthetic antibody. In some embodiments, the antibody is a diabody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a multispecific antibody, such as a bispecific antibody. In some embodiments, the antibody is a bispecific antibody. In some embodiments, the antibody is linked to a fusion protein. In some embodiments the antibody is linked to an immunostimulating protein, such as an interleukin. In some embodiments the antibody is linked to a protein which facilitates the entry across the blood brain barrier.
The term “multispecific antibodies” as used herein refer to monoclonal antibodies that have binding specificities for at least two different sites, i.e., different epitopes on different antigens or different epitopes on the same antigen. In certain aspects, the multispecific antibody has two binding specificities (bispecific antibody). In certain aspects, the multispecific antibody has three or more binding specificities. Multispecific antibodies may be prepared as full length antibodies or antibody fragments.
The term “semi-synthetic” in reference to an antibody or antibody moiety means that the antibody or antibody moiety has one or more naturally occurring sequences and one or more non-naturally occurring (i.e., synthetic) sequences.
The following Examples and Figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.
Materials and Methods
Antibodies:
General information regarding the nucleotide sequences of human immunoglobulins light and heavy chains is given in: Kabat, E. A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). Amino acids of antibody chains are numbered and referred to according to numbering according to Kabat (Kabat, E. A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)).
The current invention is exemplified using a number of exemplary antibodies, including: an anti-CD20/TfR bispecific antibody as reported in WO 2017/055542 and therein in SEQ ID NO: 01 to 03 and 10; an anti-C5 antibody (Crovalimab) as reported in WO2017/104779 and therein in SEQ ID NO: 106 to 111; a fusion protein comprising an inert antibody conjugated at the C-terminus of the heavy chain to a human IL-2 (interleukin 2) as reported in WO2015/118016 and therein in SEQ ID NO: 19 and 50; an anti-GPRC5D/anti-CD3 bispecific antibody in 2+1 format as reported in WO2021/018859A2.
Synthetic Depth Filter Media:
Herein the synthetic depth filter Millistak+® HC Pro X0SP is used. It is commercially available from MilliporeSigma (Bedford, MA). The X0SP depth filter has a nominal pore size rating of 0.1 microns and is intended for secondary clarification applications.
A cellulose/diatomaceous earth-comprising (also containing silica) PDD1 depth filter (Pall PDD1 SUPRAcap™-50 (SC050PDD1)), was also used.
A cellulose-comprising (also containing silica) VR02 depth filter (Zeta Plus™ Biocap VR02) was also used.
Depth Filtration Device:
All testing was performed using 22 cm2, 23 cm2 or 25 cm2 μPod1 scale devices.
The depth filter devices were fabricated using two layers of the depth filtration media encapsulated in single-use, over-molded device housing.
Recombinant DNA Techniques:
Standard methods were used to manipulate DNA as described in Sambrook, J. et al., Molecular Cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. The molecular biological reagents were used according to the manufacturer's instructions.
Gene Synthesis:
Desired gene segments were prepared from oligonucleotides made by chemical synthesis. The long gene segments, which were flanked by singular restriction endonuclease cleavage sites, were assembled by annealing and ligating oligonucleotides including PCR amplification and subsequently cloned via the indicated restriction sites. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing. Gene synthesis fragments were ordered according to given specifications at Geneart (Regensburg, Germany).
DNA Sequence Determination:
DNA sequences were determined by double strand sequencing performed at MediGenomix GmbH (Martinsried, Germany) or SequiServe GmbH (Vaterstetten, Germany).
DNA and protein sequence analysis and sequence data management: The GCG's (Genetics Computer Group, Madison, Wisconsin) software package version 10.2 and Infomax's Vector NT1 Advance suite version 8.0 was used for sequence creation, mapping, analysis, annotation and illustration.
Expression Vectors:
For the expression of the described bispecific antibodies, expression plasmids for transient expression (e.g. in HEK293 cells) based either on a cDNA organization with or without a CMV-intron A promoter or on a genomic organization with a CMV promoter can be applied.
Beside the antibody expression cassette the vectors contain:
The transcription unit of the antibody gene is composed of the following elements:
The fusion genes encoding the antibody chains are generated by PCR and/or gene synthesis and assembled by known recombinant methods and techniques by connection of the according nucleic acid segments e.g. using unique restriction sites in the respective vectors. The subcloned nucleic acid sequences are verified by DNA sequencing. For transient transfections larger quantities of the plasmids are prepared by plasmid preparation from transformed E. coli cultures (Nucleobond AX, Macherey-Nagel).
For all constructs knob-into-hole heterodimerization technology was used with a typical knob (T366W) substitution in the first CH3 domain and the corresponding hole substitutions (T366S, L368A and Y407V) in the second CH3 domain (as well as two additional introduced cysteine residues S354C/Y349′C) (contained in the respective corresponding heavy chain (HC) sequences depicted above).
Cell Culture Techniques:
Standard cell culture techniques as described in Current Protocols in Cell Biology (2000), Bonifacino, J. S., Dasso, M., Harford, J. B., Lippincott-Schwartz, J. and Yamada, K. M. (eds.), John Wiley & Sons, Inc., are used.
Transient Transfections in HEK293 System:
The bispecific antibodies are produced by transient expression. Therefore a transfection with the respective plasmids using the HEK293 system (Invitrogen) according to the manufacturer's instruction is done. Briefly, HEK293 cells (Invitrogen) growing in suspension either in a shake flask or in a stirred fermenter in serum-free FreeStyle™ 293 expression medium (Invitrogen) are transfected with a mix of the respective expression plasmids and 293Fectin™ or fectin (Invitrogen). For 2 L shake flask (Corning) HEK293 cells are seeded at a density of 1.0*106 cells/mL in 600 mL and incubated at 120 rpm, 8% CO2. On the next day the cells are transfected at a cell density of approx. 1.5*106 cells/mL with approx. 42 mL of a mixture of A) 20 mL Opti-MEM medium (Invitrogen) comprising 600 μg total plasmid DNA (1 μg/mL) and B) 20 ml Opti-MEM medium supplemented with 1.2 mL 293 fectin or fectin (2 μl/mL). According to the glucose consumption glucose solution is added during the course of the fermentation. The supernatant containing the secreted antibody is harvested after 5-10 days and antibodies are either directly purified from the supernatant or the supernatant is frozen and stored.
Optical Density Determination:
The protein concentration of purified antibodies and derivatives was determined by determining the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence according to Pace, et al., Protein Science 4 (1995) 2411-1423.
Protein Concentration Determination (yield):
Photometric Determination:
Protein concentrations were determined by UV spectroscopy using a Cary® 50 UV-Vis Spectrophotometer (Varian). Protein samples were diluted in their respective buffers and measured as duplicates. Concentrations were determined according to the following equation deriving from Lambert-Beer law: c=(A280 nm-A320 nm)/ε·d. F with c protein concentration [mg/ml], A absorbance, ε extinction coefficient [ml/(mg·cm)], d cell length [cm] and F dilution factor. The anti-C5 antibody-specific extinction coefficient is 1.44 ml/(mg·cm), the bispecific anti-GPRC5D antibody specific extinction coefficient is 1.43 ml/(mg·cm), the bispecific anti-CD20/TfR specific extinction coefficient is 1.57 ml/(mg·cm) and the antibody-IL2 fusion polypeptide specific extinction coefficient is 1.25 ml/(mg·cm).
Chromatographic Determination:
The concentration of the antibodies was quantitatively measured by affinity HPLC chromatography. Briefly, solution containing antibodies that bind to protein A are applied, e.g., to an Applied Biosystems Poros A/20 column in 200 mM KH2PO4, 100 mM sodium citrate, pH 7.4 and eluted with 200 mM NaCl, 100 mM citric acid, pH 2.5 on an Agilent HPLC 1100 system. The eluted antibody is quantified by UV absorbance and integration of peak areas. A purified standard IgG1 antibody served as a standard.
ELISA Determination:
Alternatively, the concentration of antibodies and derivatives in solutions is measured by Sandwich-IgG-ELISA. Briefly, StreptaWell High Bind Streptavidin A-96 well microtiter plates (Roche Diagnostics GmbH, Mannheim, Germany) are coated with 100 μL/well biotinylated anti-human IgG capture molecule F(ab′)2<h-FcT>BI (Dianova) at 0.1 μg/mL for 1 hour at room temperature or alternatively overnight at 4° C. and subsequently washed three times with 200 μL/well PBS, 0.05% Tween (PBST, Sigma). Thereafter, 100 L/well of a dilution series in PBS (Sigma) of the respective antibody containing solution is added to the wells and incubated for 1-2 hour on a shaker at room temperature. The wells are washed three times with 200 μL/well PBST and bound antibody is detected with 100 μl F(ab′)2<hFcγ>POD (Dianova) at 0.1 μg/mL as the detection antibody by incubation for 1-2 hours on a shaker at room temperature. Unbound detection antibody is removed by washing three times with 200 μL/well PBST. The bound detection antibody is detected by addition of 100 μL ABTS/well followed by incubation. Determination of absorbance is performed on a Tecan Fluor Spectrometer at a measurement wavelength of 405 nm (reference wavelength 492 nm).
Preparative Antibody Purification:
Antibodies were purified from filtered cell culture supernatants referring to standard protocols. In brief, antibodies were applied to a protein A Mab Select SuRe column (GE healthcare) and washed with buffer. Elution of antibodies was achieved at low pH followed by immediate neutralization. Antibody fractions were pooled, frozen and stored at −20° C., −40° C. or −80° C.
Hydrolytic Activity Determination—Lipase Activity Assay (LEAP):
The lipase activity was determined by monitoring the conversion of a substrate, such as a nonfluorescent substrate, to a detectable product of the hydrolytic enzyme, such as a fluorescent product. An exemplary method is described, e.g., in WO 2018/035025, which is hereby incorporated by reference in its entirety.
In more detail, with the LEAP assay hydrolase activity in samples was determined.
This was done by monitoring the conversion of a fluorogenic substrate ‘4-Methylumbelliferyl Caprylate’ (4-MU-C8, available from Chem Impex Int'l Inc Art. Nr. 01552) by cleavage of the ester bond by hydrolases present in the sample into a fluorescent moiety, i.e. 4-Methylumbelliferyl (4-MU). Cleaved 4-MU-C8, i.e. 4-MU, was excited with a light of wavelength 355 nm. The emitted radiation at a different wavelength of 460 nm was recorded on Tecan Infinite® 200 PRO device as readout. The determination was performed at 37° C. for 2 hours with recording every 10 mins to calculate the rate of substrate hydrolysis.
The sample to be analyzed was at first buffer exchanged to 150 mM Tris-Cl, pH 8.0, by using Amicon Ultra-0.5 ml centrifugal filter units (10,000 Da cut-off, Merck Millipore, Art. Nr. UFC501096). The assay reaction mixture constituted of 80 μL reaction buffer (150 mM Tris-Cl, pH 8.0, 0.25% (w/v) Triton X-100, 0.125% (w/v) Gum Arabic), 10 μL 4-MU-C8 substrate solution (1 mM in DMSO), and 10 μL protein containing sample. The protein samples' concentration were adjusted to be in the range between 1-30 g/L and 2-3 dilution series were performed for each determination. Each reaction was set up at least in duplicates in 96-well half-area polystyrene plates (black with lid and clear flat bottom, Corning Incorporated Art. Nr. 3882).
Host Cell Protein (CHOP) Determination:
The residual CHO HCP content in process samples is determined by an electrochemiluminescence immunoassay (ECLIA) on cobas e 411 immunoassay analyzer (Roche Diagnostics).
The assay is based on a sandwich principle using polyclonal anti-CHO HCP antibody from sheep.
First incubation: Chinese hamster ovary host cell protein (CHO HCP) from 15 μL sample (neat and/or diluted) and a biotin conjugated polyclonal CHO HCP specific antibody form a sandwich complex, which becomes bound to streptavidin-coated microparticles via interaction of biotin with streptavidin.
Second incubation: After addition of polyclonal CHO HCP-specific antibody labeled with ruthenium complex (Tris(2,2′-bipyridyl)ruthenium(II)-complex) a ternary sandwich complex is formed on the microparticles.
The reaction mixture is aspirated into the measuring cell where the microparticles are magnetically captured onto the surface of the electrode. Unbound substances are then removed in a washing step. Application of a voltage to the electrode then induces chemiluminescent emission which is measured by a photomultiplier.
The concentration of CHO HCP in the test sample is finally calculated from a CHO HCP standard curve of known concentration.
Host Cell DNA Determination:
The residual Chinese Hamster Ovary (CHO) deoxyribonucleic acid (DNA) in process samples is determined on FLOW FLEX System (Roche Diagnostics GmbH, Mannheim, Germany).
The FLOW FLEX System consists of three modules: FLOW PCR SETUP Instrument, MagNA Pure 96 Instrument and LightCycler®480.
The FLOW PCR SETUP Instrument module is used as PSH (FLOW Primary Sample Handling) for sample transfer from primary tubes into a 96 well processing plate, and as PSU (FLOW PCR SETUP Instrument) for transfer of extracted DNA from the 96 well output plate into the PCR plate.
The MagNA Pure 96 Instrument module is used for automated isolation of nucleic acids. To release the DNA, the sample material is incubated under denaturing conditions. The released DNA is separated from the other buffer and sample components by binding to magnetic glass particles via a magnet, and the bound DNA is then eluted with buffer. Up to 96 samples can be processed simultaneously.
The LightCycler®480 module (microplate LightCycler®) is used for quantification of DNA or RNA based on PCR technology. The Residual DNA CHO Kit uses specific PCR of highly conserved regions within the DNA of CHO. The highly specific forward primers and reverse primers bind specifically to the ends of the target sequence of single-stranded DNA. The CHO DNA probe, labeled with a fluorescent reporter dye (FAM) at the 5′ end and a quencher dye at the 3′ end, hybridizes between the primers and the target sequence of single-stranded DNA. As long as the probe is intact, the proximity of the Quencher dye suppresses the fluorescence of the reporter dye. Upon amplification, the Taq polymerase, due to its 5′→3′exonuclease activity, disrupts the probe attached to the target sequence. This releases the reporter dye and the fluorescence increases. The increase in fluorescence is directly proportional to the amount of PCR product. The amount of CHO DNA in the samples is quantified with a standard curve.
Size Exclusion High Performance Liquid Chromatography (SE-HPLC):
Size exclusion chromatography (SEC) for the determination of the aggregation and oligomeric state of antibodies was performed by HPLC chromatography. Briefly, protein A purified antibodies were applied to a Tosoh TSK-Gel G3000SWXL (7.8×300 mm; 5 μm (TOSOH Bioscience Nr. 08541)) in 250 mM KCl, 200 mM KH2PO4/K2HPO4 buffer (pH 7.0) on an Dionex Ultimate® system (Thermo Fischer Scientific). The eluted antibody was quantified by UV absorbance and integration of peak areas. BioRad Gel Filtration Standard 151-1901 served as a standard.
MCE (Caliper):
Purity and antibody integrity were analyzed by CE-SDS using microfluidic Labchip technology (PerkinElmer, USA). Therefore, 5 μl of antibody solution was prepared for CE-SDS analysis using the HT Protein Express Reagent Kit according manufacturer's instructions and analyzed on LabChip GXII system using a HT Protein Express Chip. Data were analyzed using LabChip GX Software.
Filtration of a T-Cell Bispecific Anti-GPRC5D Antibody Solution with a Silica-Containing Millipore Millistak+® HC Pro X0SP Filter, Regenerated with Alkaline Treatment
Materials:
Filter Conditioning:
Following steps were performed sequentially:
Experimental Setup:
Following steps were performed sequentially:
Analyses and Results:
The following analyses were performed with the respective final filtration pools:
The results are shown in the following Table X-1:
indicates data missing or illegible when filed
Filtration of a Bispecific Anti-CD20/TfR Antibody Solution with a Silica-Containing Millipore Millistak+® HC Pro X0SP Filter, Regenerated with Alkaline Treatment
Materials:
Filter Conditioning:
Following steps were performed sequentially:
Experimental Setup:
Following steps were performed sequentially:
Analyses and Results:
The following analyses were done:
The results are shown in the following Table X-2:
Filtration of a Bispecific Anti-CD20/TfR Antibody Solution with a Silica-Containing Millipore Millistak+® HC Pro X0SP Filter, Regenerated with an Acidic Treatment
Materials:
Filter Conditioning:
Following steps were performed sequentially:
Experimental Setup:
Following steps were performed sequentially:
Analyses and Results:
The following analyses were performed with the respective final filtration pools:
The results are shown in the following Table X-3:
Filtration of an IgG Antibody IL-2 Fusion Protein Solution with a Silica-Containing Millipore Millistak+® HC Pro X0SP Filter, Regenerated with an Acidic Treatment
Materials:
Filter Conditioning:
Following steps were performed sequentially:
Following steps were performed sequentially:
Analyses and Results:
The following analyses were performed with the respective final filtration pools:
The results are shown in the following Table X-4:
Filtration of an IgG-IL2 Fusion Polypeptide Solution with a Silica-Containing Pall PDD1 SUPRAcap 50 Filter, Regenerated with an Acidic Treatment
Materials:
Filter Conditioning:
Following steps were performed sequentially:
Following steps were performed sequentially:
Analyses and Results:
The following analyses were performed with the respective final filtration pools:
The results are shown in the following Table X-5:
Filtration of Crovalimab (Anti-C5 Antibody) Solution with a Silica-Containing Millipore Millistak+® HC Pro X0SP Filter Unit, with Water and Buffer Application (without Alkaline or Acidic Filter Regeneration)—Comparative Example
Materials:
Filter Conditioning:
Following steps were performed sequentially:
Following steps were performed sequentially:
Analyses and Results:
The following analyses were performed with the respective final filtration pools:
The results are shown in the following Table X-6:
Filtration of Crovalimab (Anti-C5 Antibody) Solution with a Silica-Containing Millipore Millistak+® HC Pro X0SP Filter, Derivatized with an Alkaline Pre-Treatment, Regenerated with an Alkaline Treatment
Materials:
Filter Derivatization:
Following steps were performed sequentially:
Following steps were performed sequentially:
Analyses and Results:
The following analyses were performed with the respective final filtration pools:
The results are shown in the following Table X-7:
Filtration of Crovalimab (Anti-C5 Antibody) Solution with a Silica-Containing Millipore Millistak+® HC Pro X0SP Filter, Regenerated with an Acidic Treatment
Materials:
Filter Conditioning:
Following steps were performed sequentially:
Experimental Setup:
Following steps were performed sequentially:
Analyses and Results:
The following analyses were performed with the respective final filtration pools:
The results are shown in the following Table X-8:
Filtration of a Crovalimab (Anti-C5 Antibody) Solution with a Silica-Containing Pall PDD1 SUPRAcap 50 Filter, with Water and Buffer Application (without Alkaline or Acidic Filter Regeneration)—Comparative Example
Materials:
Filter Conditioning:
Following steps were performed sequentially:
Following steps were performed sequentially:
The feed flow was adjusted to 191 LMH (liter per square meter per hour; L*m−2*h−1). This resulted in a calculated feed flow of 7.67 mL/min. The maximum feed pressure was 5.0 bar.
Analyses and Results:
The following analyses were performed with the respective final filtration pools:
The results are shown in the following Table X-9:
Filtration of a Crovalimab (Anti-C5 Antibody) Solution with a Silica-Containing Pall PDD1 SUPRAcap 50 Filter, Derivatized with an Alkaline Pre-Treatment, Regenerated with an Alkaline Treatment
Materials:
Filter Derivatization:
Following steps were performed sequentially:
Following steps were performed sequentially:
Analyses and Results:
The following analyses were performed with the respective final filtration pools:
The results are shown in the following Table X-10:
Filtration of a Crovalimab (Anti-C5 Antibody) Solution with a Silica-Containing Pall PDD1 SUPRAcap 50 Filter, Regenerated with an Acidic Treatment
Materials:
Filter Conditioning:
Following steps were performed sequentially:
Experimental Setup:
Following steps were performed sequentially:
Analyses and Results:
The following analyses were performed with the respective final filtration pools:
The results are shown in the following Table X-11:
Filtration of a Crovalimab (Anti-C5 Antibody) Solution with a Silica-Containing Millistak+® HC Pro Synthetic Depth Filter X0SP (Reference Example)
Materials:
Filter Conditioning:
Following steps were performed before the first sample application:
Experimental Setup:
Following steps were performed sequentially:
Analyses and Results:
The following analyses were performed with the respective final filtration pools:
The results are shown in the following Table X-12:
Filtration of a Crovalimab (Anti-C5 Antibody) Solution with a Silica-Containing Millistak+® HC Pro Synthetic Depth Filter X0SP, Regenerated with an Acidic Treatment.
Materials:
Filter Conditioning:
Following steps were performed sequentially:
Following steps were performed sequentially:
Analyses and Results:
The following analyses were performed with the respective final filtration pools:
The results are shown in the following Table X-13:
Filtration of a Crovalimab (Anti-C5 Antibody) Solution with a Silica-Containing Millistak+® HC Pro Synthetic Depth Filter X0SP, with Water and Buffer Regeneration (without Alkaline or Acidic Filter Regeneration), with a Pre-Incubation with 1 M NaOH for Four Hours Prior to First Use
Materials:
Filter Derivatization:
Following steps were performed sequentially:
Following steps were performed sequentially:
Analyses and Results:
The following analyses were performed with the respective final filtration pools:
The results are shown in the following Table X-14:
Filtration of a Crovalimab (Anti-C5 Antibody) Solution with a Silica-Containing Pall PDD1 SUPRAcap 50 Filter (Reference Example)
Materials:
Filter Conditioning:
Following steps were performed before the first sample application:
Following steps were performed sequentially:
Analyses and Results:
The following analyses were performed with the respective final filtration pools:
The results are shown in the following Table X-15:
Filtration of a Crovalimab (Anti-C5 Antibody) Solution with a Silica-Containing Pall PDD1 SUPRAcap 50 Filter, Regenerated with an Acidic Treatment
Materials:
Filter Conditioning:
Following steps were performed sequentially:
Following steps were performed sequentially:
Analyses and Results:
The following analyses were performed with the respective final filtration pools:
The results are shown in the following Table X-16:
Filtration of a Crovalimab (Anti-C5 Antibody) Solution with a Silica-Containing Pall PDD1 SUPRAcap 50 Filter, Regenerated with an Acidic Treatment, without Intermediate Water Flush for Reducing Process Time
Materials:
Filter Conditioning:
Following step was performed:
Experimental Setup:
Following steps were performed sequentially:
Analyses and Results:
The following analyses were performed with the respective final filtration pools:
The results are shown in the following Table X-17:
Filtration of a Crovalimab (Anti-C5 Antibody) Solution with a Zeta Plus™ Biocap VR02 without Intermediate Flush (Reference Example)
Materials:
Filter Conditioning:
Following steps were performed before the first sample application:
Following steps were performed sequentially:
Analyses and Results:
The following analyses were performed with the respective final filtration pools:
The results are shown in the following Table X-18:
Filtration of a Crovalimab (Anti-C5 Antibody) Solution with a Zeta Plus™ Biocap VR02 Filter, with Water and Buffer Application (without Alkaline or Acidic Filter Regeneration)—Comparative Example
Materials:
Filter Conditioning:
Following steps were performed sequentially:
Following steps were performed sequentially:
Analyses and Results:
The following analyses were performed with the respective final filtration pools:
The results are shown in the following Table X-19:
Filtration of a Crovalimab (Anti-C5 Antibody) Solution with a Zeta Plus™ Biocap VR02 Filter, Regenerated with an Acidic Treatment
Materials:
Filter Conditioning:
Following steps were performed sequentially:
Experimental Setup:
Following steps were performed sequentially:
Analyses and Results:
The following analyses were performed with the respective final filtration pools:
The results are shown in the following Table X-20:
Hydrolytic Activity Determination—Lipase Activity Assay (LEAP):
The lipase activity was determined by monitoring the conversion of a substrate, such as a nonfluorescent substrate, to a detectable product of the hydrolytic enzyme, such as a fluorescent product.
In more detail, with the LEAP assay hydrolase activity in samples was determined. This was done by monitoring the conversion of a fluorogenic substrate ‘4-Methylumbelliferyl Caprylate’ (4-MU-C8, available from Chem Impex Int'l Inc Art. Nr. 01552) by cleavage of the ester bond by hydrolases present in the sample into a fluorescent moiety, i.e. 4-Methylumbelliferyl (4-MU). Cleaved 4-MU-C8, i.e. 4-MU, was excited with a light of wavelength 355 nm. The emitted radiation at a different wavelength of 460 nm was recorded on Tecan Infinite® 200 PRO device as readout. The determination was performed at 37° C. for 2 hours with recording every 10 mins to calculate the rate of substrate hydrolysis.
The sample to be analyzed was at first buffer exchanged to 150 mM Tris-Cl, pH 8.0, by using Amicon Ultra-0.5 ml centrifugal filter units (10,000 Da cut-off, Merck Millipore, Art. Nr. UFC501096). The assay reaction mixture constituted of 80 μL reaction buffer (150 mM Tris-Cl, pH 8.0, 0.25% (w/v) Triton X-100, 0.125% (w/v) Gum Arabic), 10 μL 4-MU-C8 substrate solution (1 mM in DMSO), and 10 μL protein containing sample. The protein samples' concentration were adjusted to be in the range between 1-30 g/L and 2-3 dilution series were performed for each determination. Each reaction was set up at least in duplicates in 96-well half-area polystyrene plates (black with lid and clear flat bottom, Corning Incorporated Art. Nr. 3882).
| Number | Date | Country | Kind |
|---|---|---|---|
| 21159023.7 | Feb 2021 | EP | regional |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/EP2022/054210 | Feb 2022 | US |
| Child | 18454131 | US |
