FIELD OF THE INVENTION

The present invention relates to methods for inactivating SAR-CoV-2 in samples comprising recombinant protein such as chromatography eluates. In particular, the invention relates to methods comprising a step of treating such samples with a pH of about 3.0 in order to inactivate SARS-CoV-2 in these samples.

BACKGROUND TO THE INVENTION

Biopharmaceutical manufacturing processes involve biological materials such as recombinant DNA/vectors, recombinant host cell lines and cell culture media. The manufacture of such biologically-derived Biopharmaceuticals is a complex process that requires many purification steps to assure safety. Host cells may have chromosomally integrated viral elements encoding endogenous retrovirus-like particles (ERVLPs). Biotherapeutics prepared from patient samples such as whole blood or plasma may be contaminated by blood borne pathogens such as viruses. In addition, adventitious agents might be introduced into manufacturing process intermediates via human contamination. Thus, Biopharmaceutical manufacturing processes may include a low pH treatment step as a means of inactivating enveloped viruses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing the ongoing coronavirus disease 19 (COVID- 19) pandemic presents a challenge to the Biopharmaceutical virus control framework in its potential ability to infect production cells, detectability of virus if present and ability of manufacturing processes to clear the virus. It is unclear as to the effect of low pH treatment on SARS-CoV and SARS-CoV-2 in the context of samples from biopharmaceutical manufacturing processes.

Accordingly, there is a need to provide a method for inactivation of SARS-CoV-2 in samples comprising recombinant protein, such as chromatography eluates.

SUMMARY OF THE INVENTION

The invention provides a method for inactivating SARS-CoV-2 in a sample comprising at least one recombinant protein, wherein the method comprises treating the sample at a pH of between 2.6 and 3.4. In one embodiment, the pH is about 3.0.

The invention further provides a use of a pH of between 2.6 and 3.4 for inactivating SARS-CoV-2 in a sample comprising at least one recombinant protein. The invention also provides a chromatography eluate comprising at least one recombinant protein and inactivated SARS-CoV-2, wherein the eluate has a pH of between 2.6 and 3.4.

The invention further provides a method for purification of a recombinant protein, comprising the steps of: (a) treating a sample comprising at least one recombinant protein and SARS-CoV-2 at a pH of between 2.6 and 3.4 to inactivate SARS-CoV-2; and (b) formulating the recombinant protein for therapeutic use.

DESCRIPTION OF DRAWINGS/FIGURES

FIG. 1 A-E - Susceptibility of SARS-CoV-2 and SARS-CoV to low pH in the presence of protein A eluate. A) SARS-CoV-2 virus samples were spiked into the pH adjusted mAb A (5% v/v), incubated for the indicated time at RT, neutralized, and frozen. Virus titers were determined by TCID50 endpoint titration on Vero E6 cells. Data shown are mean values ± standard deviations (SD) of two independent experiments titrated in duplicate. B) Virus titers of neutral control samples were determined after 120 min. Data shown are technical duplicates of two independent experiments. C) SARS-CoV samples were spiked into the pH adjusted Protein A chromatography eluate (2,5% v/v) and incubated for the indicated time points at RT. Samples were neutralized, frozen and virus titers were determined by TCID50 titration. Data are mean values ± SD of two independent experiments. D) Virus titers of neutral control samples were determined at the indicated time points. Data are technical duplicates of two independent experiments. E) Inactivation of SARS-CoV-2 and SARS-CoV by NaOH. 0.5 M NaOH was spiked with up to 5% v/v of the indicated virus stock and incubated for 60 min at RT. Data are technical duplicates of one (SARS-CoV) or two (SARS-CoV-2) independent experiment and neutral controls.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for inactivating SARS-CoV-2 in a sample comprising at least one recombinant protein. It has surprisingly found that treatment with a pH of about 3.0 effectively inactivates SARS-CoV-2 in samples comprising recombinant protein, whereas it does not have the same effect on SARS-CoV in such samples. Including such a step in biopharmaceutical manufacturing processes provides an advantage in reducing the possibility of SARS-CoV-2 contamination of samples by users of such processes. Accordingly, there is provided a method for inactivating SARS-CoV-2 in a sample comprising at least one recombinant protein, wherein the method comprises treating the sample at a pH of between 2.6 and 3.4.

By "SARS-CoV-2" (severe acute respiratory syndrome coronavirus 2) is meant the virus (a strain of coronavirus) that causes COVID- 19. This definition encompasses all variants of SARS-CoV-2, including, but not limited to, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2) and Omicron (B.1.1529).

By "inactivating" or "inactivation" of SARS-CoV-2 is meant a reduction or elimination of SARS- CoV-2 titer in a sample, or a reduction or elimination in the level of infectious SARS-CoV-2 virus. A suitable reduction includes a reduction in the SARS-CoV-2 viral titre by at least at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% compared to an untreated control (where an untreated control has not been exposed to the pH of 3.0). Preferably, the reduction in SARS-CoV-2 viral titer is at least 50%. Optionally, there may be an elimination of SARS-CoV-2 in the sample, meaning that there is no detectable SARS-CoV-2 viral titer in the sample. Viral titer may be measured in a number of ways known to the skilled person, but in one embodiment, the viral titer is measured by the TCID50 (50% tissue culture infectious dose) of Vero E6 cells as described herein. In one embodiment, the sample comprises SARS-CoV-2 at a titer of between 1.0 x 10 ⁵ and 1.0 x 10 ⁶ TCID50/ml. Preferably, the titer is between 2.5 x 10 ⁵ and 7.5 x 10 ⁵ TCID50/ml, or between 5 x 10 ⁵ and 6 x 10 ⁵ TCID50/ml.

The method described herein uses a pH of 2.6 to 3.4 to inactivate SARS-CoV-2. It has surprisingly been found that this pH inactivates SARS-CoV-2 but does not activate SARS-CoV in the experiments described herein. In one embodiment, the pH is about 3.0. By the term "about" used with reference to pH 3.0 is meant up to +/- 0.1, 0.2, 0.3 or 0.4. As such, the expression "+/- 0.4" includes ranges such as 2.6 to 3.0, 3.0 to 3.4, as well as 2.6 to 3.4. The same principle follows for +/- 0.1, 0.2 or 0.3. For the avoidance of doubt, "about pH 3.0" also includes pH 3.0 per se, to the extent that can be accurately measured through methodology known by the skilled person in the art (e.g. +/- 0.05). Thus, the pH may be 2.7 to 3.3, 2.8 to 3.2, 2.9 to 3.1, or 2.95 to 3.05.

A sample may be any sample that comprises at least one recombinant protein. By the term "recombinant protein" is meant a protein that has been expressed in a host cell from DNA in which the gene sequence that codes for the protein has been recombinantly introduced. Therefore, a recombinant protein may be a protein expressed exogenously by a host cell. Examples of recombinant protein well known to the skilled person include proteins expressed by host cells for biopharmaceutical production, such as monoclonal antibodies and fragments and variants thereof, other proteins for therapeutic application (for example, insulin and analogues thereof, human growth factors, erythropoietin (EPO), granulocyte colony stimulating factor (G- CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), thrombopoietin, cytokines such as interferons, IL-1 and IL-6, monoclonal antibodies, tissue plasminogen activator (TPA), urokinase, serum albumin, blood coagulation factor VIII, leptin, insulin, and stem cell growth factor (SCF), amongst others), immunogenic proteins for vaccines (for example, hepatitis B virus surface antigen). It will be clear to the skilled person that there may be more than one recombinant protein in a sample suitable for use with this method. The sample may further comprise one or more or a combination of Host Cell Protein (HCP), host cell DNA, and process residuals (for example media supplements, flocculants, etc).

The host cell may be any cell suitable for expressing a recombinant protein. Such cells can be eukaryotic or prokaryotic. Such cells may be plant cells, insect cells, bacteria, yeast or mammalian cells. Many examples will be known to the skilled person but examples include CHO cells (Chinese hamster ovary cells), BHK21 cells (baby hamster kidney cells), NSO or Sp2/0 cells (murine myeloma cells), HeLa, HEK293 cells (human embryonic kidney cells), HT-1080 cells (fibrosarcoma cell line), Escherichia coli (E. coli) or Pichia species.

In one embodiment, the sample is a process intermediate. By "process intermediate" is meant a sample produced during steps of the processing and/or purification of a recombinant protein. For example, a process intermediate includes a sample that emerges from any stage of recombinant protein filtration or purification, or a sample from the final stage of processing.

In one embodiment, the process intermediate is a chromatography eluate. The skilled person will understand that a chromatography eluate is a solution that emerges from a chromatography process, and that this eluate will comprise at least one recombinant protein. For example, a chromatography eluate may comprise a recombinant monoclonal antibody. The term "chromatography" will be well known to the skilled person, and will be understood to be a technique for the separation of a mixture by passing it in solution or suspension through a medium in which the components move at different rates. Examples of chromatography include, but are not limited to, affinity chromatography, anion chromatography, cation chromatography, mixed-mode chromatography, hydroxylapatite chromatography, hydrophobic interaction chromatography, reverse-phase HPLC, size exclusion chromatography. Examples of affinity chromatography include, but are not limited to, protein A, protein G or protein L chromatography.

In another aspect of the invention, there is provided a chromatography eluate comprising at least one recombinant protein and inactivated SARS-CoV-2, wherein the eluate has a pH of between 2.6 and 3.4.

By "buffer" is meant a solution which resists changes in pH when acid or alkali is added to it, and is a term that will be known to the skilled person. For the avoidance of doubt, "buffer" and "buffer solution" may be used interchangeably. Examples of suitable buffers that are capable of buffering at pH 3.0 include, but are not limited to, citrate buffer or acetate buffer. Citrate buffer will be known to the skilled person as a buffer solution containing citric acid to achieve a desired pH. Acetate buffer will be known to the skilled person as a buffer solution containing acetic acid to achieve a desired pH.

By "untreated control" is meant a sample that is not exposed to the pH of about 3.0. This may include the sample (e.g. chromatography eluate) before carrying out the method of claim 1. In other words, the method may result in a reduction, of e.g. 50%, of the viral titer present before the sample was exposed to the pH of about 3.0. Viral titer may be measured in a number of ways known to the skilled person, but in one embodiment, the viral titer is measured by the TCID50 of Vero E6 cells as described herein.

In some embodiments, the sample is exposed to the pH of about 3.0 for at least 30 minutes, 60 minutes or 120 minutes. For example, the sample may be exposed for 15 minutes to 240 minutes, 20 minutes to 180 minutes, or 30 minutes to 120 minutes. Alternatively, the sample may be exposed for 30 minutes, 60 minutes or 120 minutes.

In one embodiment, the method is carried out at a temperature of about 18 to about 27°C. This will also be known to the skilled person as "room temperature". By the term "about" used with reference to this temperature, is intended to cover the temperature range to the extent that can be accurately measured through methodology known by the skilled person in the art (e.g. +/- 1°C). Room temperature may also be a narrower range falling in this range, such as about 18 to about 25°C, about 20°C to about 25°C, about 21°C to about 25°C, or, about 18°C to about 21°C. In one embodiment, the temperature is about 18°C to about 21°C. In another aspect of the invention, there is provided a method for purification of a recombinant protein, comprising the steps of: (a) treating a sample comprising at least one recombinant protein and SARS-CoV-2 at a pH of between 2.6 and 3.4 to inactivate SARS-CoV-2; and (b) formulating the recombinant protein for therapeutic use. There is also provided a method for manufacture of a recombinant protein for therapeutic use, comprising the steps of: (a) treating a sample comprising at least one recombinant protein and SARS-CoV-2 at a pH of between 2.6 and 3.4 to inactivate SARS-CoV-2; and (b) formulating the recombinant protein for therapeutic use. There is also provided a method for removal of virus from a sample containing a recombinant protein for therapeutic use comprising treating the sample with a pH of between 2.6 and 3.4 to inactivate SARS-CoV-2; and (b) formulating the recombinant protein for therapeutic use. There is also provided a method for producing a recombinant protein substantially free of SARS-CoV-2, comprising the steps of: (a) treating a sample comprising at least one recombinant protein and SARS-CoV-2 at a pH of between 2.6 and 3.4 to inactivate SARS-CoV-2, and (b) purifying said recombinant protein. Similarly, there is also a method for producing a recombinant protein for therapeutic use which is substantially free of SARS-CoV-2, comprising the steps of: (a) treating a sample comprising at least one recombinant protein and SARS-CoV-2 at a pH of between 2.6 and 3.4 to inactivate SARS-CoV-2, and (b) formulating the recombinant protein for therapeutic use. Formulating the recombinant protein for therapeutic use will comprise steps known to the skilled person working in this field, however may include physical streps such as concentrating, blending, compressing, filtrating, heating, encapsulating, shearing, tableting, granulating, coating and drying. Optionally, such methods may include further upstream or downstream manufacturing steps, including culturing of host cells to express the recombinant protein, preparing the sample for purification of the recombinant protein (e.g. through filtration, lysing, centrifugation and/or clarification), purifying the recombinant protein from the sample, and/or analysis and testing of the resulting sample. It will be clear to the skilled person that the above described embodiments and definitions also apply to these methods.

In another aspect of the invention, there is provided a use of a pH of between 2.6 and 3.4 for inactivating SARS-CoV-2 in a sample comprising at least one recombinant protein.

EXAMPLES

Small scale virus clearance studies are performed with model enveloped viruses to confirm inactivation at the pH range used in the downstream manufacturing process. The studies usually include a retrovirus such as murine leukemia virus (MuLV) to represent the endogenous virus present in rodent cell lines and other relevant model enveloped viruses at later stages of production. Further, as a means of bioburden reduction, chromatography resins may be cleaned and stored using sodium hydroxide. It has been shown that 0.1M and 0.5M NaOH are effective at inactivating enveloped and non-enveloped viruses.

In inactivation studies of viral samples in solution, SARS-CoV-2 does not appear to be susceptible to inactivation at certain pH values , with one study finding that SARS-CoV2 is extremely stable in a wide range of pH values (3-10) at room temperature (Chin et al. 2020. Stability of SARS- CoV-2 in different environmental conditions. Lancet Microbe. l(l):el0), and another study finding that SARS-CoV-2 was still detected after several days of incubation at pH 4 to pH 11 (Chan et al 2020. Factors affecting stability and infectivity of SARS-CoV-2, Journal of Hospital Infection, Volume 106, Issue 2, p226-231) with some sensitivity at pH 2-3 and pH 12-13, but only after several days of incubation (Chin et al 2020, Chan et al 2020). SARS-CoV the causative agent of the outbreak of severe respiratory diseases (SARS) in 2003 has been shown to be effectively inactivated at pH 3.0 at 25°C and 37°C and at pH 12.0 at 4°C, 25°C and 37°C (Darnell et al. 2004. Inactivation of the coronavirus that induces severe acute respiratory syndrome, SARS-CoV. J Virol Methods. 121(1):85-91). At 4°C, however, pH 3 treatment only partially inactivated SARS-CoV.

A study, as outlined in Examples 1-3, was performed to evaluate the effectiveness of the low pH treatment step for the inactivation of SARS-CoV and SARS-CoV-2 and to evaluate the kinetics of inactivation in the presence of a mAb at three pH. In addition, a single sample of SARS-CoV and SARS-CoV-2 was exposed to a 0.5M NaOH for a period of 60 minutes to reflect bioburden reduction and resin column cleaning conditions.

The starting material for the low pH inactivation study was Protein A chromatography eluate in 3.0 mM Sodium Acetate, 27.0 mM Acetic Acid, pH ~ 4.1 - 4.5. Eluate 1 was used for the SARS- CoV-2 low pH studies. SARS-CoV low pH studies were performed with the remaining aliquots of eluate 1 or eluate 2. Starting material samples were provided in frozen aliquots. Aliquots were thawed at room temperature (RT, 18-21°C) on the day of the experiment or at 4°C overnight and were held up to 7 days at 2-8°C after thawing. Low pH titrants were 0.1 M Hydrochloric acid, 0.03 M acetic acid and 0.2 M Citric acid. The neutralization titrant was 1 M Tris.

SARS-CoV-2 (Wetzlar isolate) and SARS-CoV (FFM-1 isolate; GenBank accession number AY310120) were used. SARS-CoV-2 experiments were performed under biosafety level (BSL) 3 conditions whilst SARS-CoV experiments were performed under BSL4 conditions. Virus stocks were propagated on Vero E6 cells in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 3 % fetal calf serum (FCS, Gibco). Cell supernatants were cleared by low- speed centrifugation and stored at -70°C (SARS-CoV-2) or -80°C (SARS-CoV). Virus titers were determined using a 50% tissue culture infectious dose assay (TCID50). The titers of the virus stocks were: SARS-CoV: 1.05 X 10 ⁶ TCID50/ml and SARS-CoV-2: 2.83 X 10 ⁵ TCID50/ml. The virus was spiked into the sample in sufficient amount to allow for demonstration of approximately >4 log reduction in infectivity, but not to exceed 5% volumetric spike. SARS-CoV- 2 was spiked into the sample at 5% v/v and SARS-CoV at 2.5% v/v due to the higher virus titer.

An aliquot of the Protein A chromatography eluate was thawed at RT. 10 mL were removed to a container for pH and temperature adjustment and spiking. The sample was neutralized to pH 6.5-7.5 with IM Tris and the pH and volume of titrant added was recorded. Eluate sample was spiked with up to 5% v/v virus stock (see above). pH was measured and adjusted when needed. A 5 mL sample of the spiked neutral control was removed and stored. The remaining spiked neutral mAb eluate was incubated at RT for 120 minutes. After incubation, the remaining sample (5 ml) was stored as the neutral hold control.

25 mL of eluate was added to a container for pH adjustment and spiking. The sample was equilibrated to RT. The pH of the mAb eluate was adjusted with appropriate titrant to target pH +/- 0.05. The sample was spiked with up to 5% v/v virus stock (SARS-CoV-2 5%; SARS-CoV 2,5%). The pH was checked and readjusted to target pH if necessary. The eluate was incubated at RT for 120 min. No mixing occurred during incubation period. At timepoints 1, 30, 60 and 120 min, a 5 mL sample was removed. The pH was recorded and the sample neutralized immediately with IM Tris. In addition, 5ml of 0.5 M NaOH was spiked with up to 5% v/v virus stock (SARS- CoV-2 5%; SARS-CoV 2.5%) and pH was recorded. After 60 min the sample was neutralized using IM Tris, pH was recorded and samples stored until titration.

Virus titers were determined by 50% tissue culture infectious dose titration (TCID50) on Vero E6 cells. Viral supernatants of SARS-CoV-2 were serial diluted in medium (DMEM without FCS). Each sample was titrated in 4 replicates from 51 to 511. Subsequently, 100 pl of each virus dilution were transferred to Vero E6 cells grown in 96-well plates containing 100 pl medium (DMEM + 3% FCS for SARS-CoV-2) and incubated for 72 h at 37°C. Viral supernatants of SARS-CoV were directly serial titrated (51 to 511) in four replicates on VeroE6 cells containing 160 pl medium (DMEM + 3% FCS). Cells were then incubated for 72 h and viral titers were determined with Spearman and Karber algorithm [6]. EXAMPLE 1 - Low pH inactivation study in SARS-CoV-2

Acetate Buffer Inactivation

SARS-CoV-2 was efficiently inactivated by treatment with the acetic acid titrant pH 3.0 with a reduction of virus titers of 3 log 10 after 30 min treatment and complete inactivation after 60 min treatment (Fig. 1A). In contrast, treatment with the acetic acid titrant pH 3.4 resulted only in a partial inactivation of SARS-CoV-2, causing a ca. 0.5 and 1.5 log 10 reduction in virus titer over 60 min and 120 min, respectively. At pH 3.8, the acetic acid titrant demonstrated no effect on virus inactivation over 120 min. No virus inactivation was observed in the neutral control samples after 120 min incubation at RT (Fig. IB).

Citrate Buffer Inactivation

Treatment of SARS-CoV-2 with the citric acid titrant pH 3.0 caused an efficient inactivation of the virus with a 2 loglO reduction of virus titers over 30 min and a 3.5 loglO reduction over 120 min (Fig. 1A). At pH 3.4, the virus was partially inactivated with a reduction in virus titer of ca. 1.5 loglO, similar to what has been observed for acetic acid titrant pH 3.4 treatment. In contrast, at a pH of 3.8, the citric acid titrant had no effect on SARS-CoV-2 infectivity over 120 min.

Taken together, inactivation occurred more efficiently with acetic acid titrant pH 3.0, demonstrating complete inactivation of SARS-CoV-2 over 60 min, whereas the citric acid titrant pH 3.0 significantly reduced virus infectivity, but was not able to completely inactivate SARS- CoV- 2 after 120 min. In 3 out of 4 experiments, the virus was fully inactivated.

EXAMPLE 2 - Low pH inactivation study in SARS-CoV

SARS-CoV low pH studies were performed with two different eluate preparations, due to limited availability of eluate 1. Acetic acid titrant pH 3.0 and 3.4 treatment was performed with eluate 1, while all other experiments with SARS-CoV were performed with eluate 2. For comparison of both eluates, acetic acid titrant pH 3.4 treatment was performed with both eluates. No significant difference was observed in SARS-CoV inactivation by using eluate 1 compared to eluate 2 (data not shown).

As shown in Fig. 1C, SARS-CoV was less sensitive to inactivation by the different conditions compared to SARS-CoV-2. Acetic acid titrant pH 3.0 caused a 1.5 loglO reduction in virus titer after 1 min treatment but demonstrated no further inactivation over 120 min. Treatment with acetic acid titrant pH 3.4 and 3.8 had no significant effect on SARS-CoV infectivity. A marginal reduction in virus titer was observed over time, however, also the neutral control samples showed a slight reduction in virus titer at timepoint 120 min (Fig. ID). Incubation of SARS-CoV with citric acid titrant at low pH 3.0, 3.4 or 3.8 did not result in virus inactivation.

EXAMPLE 3 - NaOH inactivation of SARS-CoV-2 and SARS-CoV

Treatment of SARS-CoV-2 and SARS-CoV with 0.5 M NaOH (pH ca. 13.4) demonstrated complete inactivation of each virus after 60 min treatment. No virus inactivation was observed in the neutral control samples at 60 min incubation at RT (Fig. IE).

Taking all three examples together, the study demonstrates that SARS-CoV-2 is sensitive to inactivation at pH 3.0 with either acetic acid or citric acid titrants in the presence of protein A eluate. Citric acid titrant was unable to completely inactivate SARS-CoV-2 over 120 min, whereas acetic acid titrant caused complete virus inactivation over 60 min. In addition, SARS-CoV 2 was completely inactivated by 0.5 M NaOH treatment over 60 min.

We have observed that at a slightly higher pH of 3.4 virus inactivation by both titrants was only partial (ca.l log 10 reduction in virus titer over after 120 min in both cases) and no effect on virus infectivity was observed at pH 3.8. In contrast, SARS-CoV infectivity was not significantly affected by the low pH treatments in this study but was completely inactivated by 0.5 M NaOH treatment.

Taken together, the present study suggests that SARS-CoV-2 can be efficiently inactivated by pH 3 treatment conditions. In contrast, low pH inactivation of SARS-CoV was less effective under these conditions.