Viral Safety in Intensified Monoclonal Antibody Bioprocesses

Introduction

Mammalian cells are most frequently used for recombinant monoclonal antibody (mAb) production today. However, there is a certain risk of virus contamination when using mammalian cell culture systems. Furthermore, non-infectious Retrovirus-like particles (RVLP) are detectable in CHO cell culture supernatans by qPCR methods and electron microscopy.1 In order to ensure the viral safety of biopharmaceutical products, the International Conference on Harmonization (ICH) as well as the European Medicines Agency (EMA) expect different strategies to be applied.2

• Virus testing of cell lines and other raw materials
• Virus testing at appropriate steps in the production process
• Demonstrate viral clearance capacity of the production process

To assess the capacity of the downstream process to inactivate or remove viruses, virus spiking studies using downscale models of appropriate process steps have to be performed and validated.

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With regard to the robustness of viral clearance, regulatory agencies expect the understanding of the mechanism by which a process step clears viruses.3 Common model viruses for early-stage phases are the enveloped retrovirus Xenotropic murine leukemia virus (X-MuLV) and the non-enveloped parvovirus minute virus of mice (MVM).4 The adequate overall virus log reduction value (LRV) of the process depends on the RVLP content in the cell culture harvest. A calculation based on the theoretical RVLP content in one dose of the product provides the minimum retrovirus LRV needed. Typically, an additional safety margin of 4-6 logs is applied by the biopharmaceutical industry leading to values of at least 13-14 LRV.3,5 To claim the cumulative viral clearance capacity of the downstream process, only orthogonal operation steps can be included.6 Typical unit operations validated for virus clearance in previous mAb and mAb-related submissions to the FDA include chemical inactivation and virus retentive filtration as well as chromatographic steps like ion exchange (IEX) and Protein A.7

Common Strategies for Virus Clearance in mAb Purification

MAb capture is widely performed by Protein A affinity chromatography. Besides the efficient removal of process-related impurities like host cell proteins and DNA, Protein A can be claimed for virus removal. Different publications have shown that most viruses flow through the column.8 As Protein A is eluted at acidic pH, a chemical inactivation of enveloped viruses by denaturation of the envelope proteins is typically performed.9 Generally this inactivation step is performed at pH 3.0-4.0.3 Slower inactivation kinetics are reported at higher pH and lower temperature.10 Anion exchange chromatography (AEX) provides a high degree of virus removal by electrostatic forces and can be applied as flow-through chromatography as mAbs are typically basic proteins.11 At pH 6-8 viruses are retained by the electropositive ligand whereas most mAbs pass the column. Virus filtration by nanofilters is applied solely as a dedicated virus removal step3. Table 1 shows the virus clearance capacity that we have obtained for different typically claimed unit operations in mAb purification.

Protein A chromatography delivered a mean reduction of 2.6 LRV for the non-enveloped viruses (MVM or PPV) and 3.4 LRV for the enveloped MuLV (Table 1). This trend was also reported by Zhang et al with a 0.7 LRV higher removal of X-MuLV compared to MVM.12 Low pH treatment showed constantly high inactivation of MuLV ranging from 4.5-6.5 LRV. In 47 % of the experiments no residual infectivity was detectable after the incubation period. AEX chromatography showed more variation in the achieved virus reduction with a mean value of 4-5 logs. In contrast, virus filtration delivered at least 2.4 LRV for the smaller non-enveloped viruses and at least 3.7 LRV for MuLV with mean values of nearly 6 LRV. Thus, virus filtration can be considered as a robust virus removal step.

Ensuring Viral Safety in Intensified mAb Purification

Caprylic Acid

Caprylic acid (CA) is described as a standard precipitation method for the isolation of immunoglobulins (IgGs) from plasma.13 More recently, CA-induced impurity precipitation is reported as an alternative method to polishing column chromatography to intensify recombinant mAb purification processes.14-16 Besides product purification, the undissociated and lipophilic form of CA acts as a virus inactivating agent by disrupting the integrity of the lipid coat of enveloped viruses.17

As the routinely used solvent-detergent (S/D) treatment in plasma product manufacturing shows some drawbacks like the removal of the agent and reduction of process yields, CA was evaluated as an alternative virus inactivation method.18 The study revealed that a minimum of 9 mmol L-1 CA (bovine viral diarrhea virus, BVDV) and 12 mmol L-1 (human immunodeficiency virus, HIV-1; pseudorabies virus, PRV) at pH 5.1 (1 h at 25 °C) was sufficient for complete inactivation of the lipid-enveloped viruses in an IgG1 solution. Moreover, the inactivation kinetic of BVDV by 9-19 mmol L-1 CA (pH 5.1) was about 20- to 60-fold faster compared to the S/D set point treatments with 0.3 % TNBP 0.2 % cholate (pH 5.5-5.7).

Complete virus inactivation of four different enveloped viruses was also reported for three IgG preparations in another study.19 The pH value was considered as critical parameter, as the effective non-ionized form of CA (0.005 g kg-1 solution) significantly decrease at pH 8.0 and the inactivation test failed. Furthermore, the study confirmed that non-lipid coated viruses like Reovirus type 3 (Reo3) are not affected by CA treatment. In contrast, another study reported non-enveloped viruses (MVM, ECMV) to be removed moderately by CA precipitation.20

To implement CA-induced virus inactivation into the manufacturing process of albumin, the relationship between pH, temperature and CA:protein ratio was determined.21 It was shown that 16 mmol L-1 CA at pH 4.5 were sufficient for complete BVDV inactivation in a 10 % (w/v) albumin solution after incubation of 1-8 hours at 25-45 °C, but only minimal inactivation was determined at 0 °C. Regarding CA:albumin ratio, a strong dependence of the BVDV inactivation rate was observed. As albumin has at least ten binding sites for CA, it has to be assured that enough free CA is available to act upon the virus membrane.

For the purification process of recombinant mAbs, the operating space for inactivation of the enveloped model virus MuLV was evaluated.14,16 Concentrations of 0.5-1.0 % CA at pH 4.9-5.1 provided X-MuLV clearance of ≥3 LRV already within ten minutes in two purified mAb solutions.14 The other study showed complete inactivation of A-MuLV (≥5.5 LRV) at a concentration of 2.4 mmol L-1 of the protonated CA (total concentration 6.2 mmol L-1). In contrast, experiments at higher pH values with protonated CA concentrations ≤1 mmol L-1 resulted in no significant inactivation (

Overall, the use of CA-induced viral inactivation can provide an additional safety margin when applied as viral clearance step orthogonal to the low pH inactivation. Alternatively, it can replace an S/D treatment in the processing of pH-sensitive antibodies.

Adsorptive Depth Filtration

To remove precipitates and turbidity after neutralization of low pH virus inactivated product intermediates depth filters are commonly used. Moreover, positively charged filters can be applied to improve this process step by additional removal of host cell proteins (HCPs) and DNA.23 Claiming adsorptive depth filters as virus removal step would enable a further intensification of mAb bioprocessing. However, the lack of testing methods to demonstrate post usage filter integrity makes it impossible to claim a size-based sieving of the viruses24 and would also be the same removal mechanism as the robust viral filtration. Thus, virus retention by adsorptive interactions is favored in terms of skipping an AEX chromatography step.

High LRVs of 2.6-5.0 log were reported for the viruses X-MuLV, PRV, MVM and Reo-3 with the Millipore Millistak+® A1HC at 400 L m-2 at pH 5.0 and 4 mS cm-1.25,26 In cell culture media at pH 7.0 and 13 mS cm-1, X-MuLV and PRV removal remained high (3-4 LRV), whereas MVM removal dropped down to 0.8 LRV. Comparable results were reported for the 3M CUNO 90ZA at these conditions.

Another study demonstrated the linkage between PPV clearance and the ionic capacity of the Millistak+® B1HC and X0HC filters with data at different ionic strengths.24 At low conductivity (< 2.5 mS cm-1) PPV removal ranges from 2-4 LRV (pH 5.0 and 8.0), whereas a conductivity of 20 mS cm-1 led to consistently low LRVs of3.26 LRV) leading to the conclusion that mAb products potentially compete with viruses for binding on the filter. Removal of the larger enveloped X-MuLV was >3 LRV for low (

To quantify the contribution of a size based retention of the enveloped model virus X-MuLV, a buffer condition at high ionic strength was included in our study.27 The 3M Zeta PlusTM 90ZA05A and 60ZA05A as well as the synthetic EmphazeTM AEX Hybrid Purifier showed X-MuLV removal of 5-6 LRV at pH 5.5 and 7.0 at low conductivity (4-5 mS cm-1) at 220 L m-2 filter load. MVM removal was 5-7 LRV at pH 5.5 and 2.5-3 LRV at pH 7.0. Additional experiments for X-MuLV at pH 7.0 and 1 M NaCl suppressed virus removal significantly. By subtracting thisresidual LRV at high ionic strength from the LRVs at low conductivity, ZetaPlusTM depth filters delivered 2-3 LRV traced back to adsorption, whereas the synthetic EmphazeTM AEX provided 4-5 LRV. The residual 2.5 LRV of the ZetaPlusTM filters at this high ionic strength were assumed to be attributed to hydrophobic interactions or mechanical retention. In a recent study, a systematic study design including high ionic strength conditions as well as high propylene glycol conditions to suppress hydrophobic interactions was established to get a deeper understanding of the involved virus retention mechanisms.28 Unexpectedly, ionic-based virus retention by Zeta PlusTM filters delivered only 2 LRV (pH 5.5) for MVM and no significant X-MuLV removal in this second study. Thus, leading to the conclusion that the doubled mAb load (competitive binding) in the second study or potential lot-to-lot variability negatively influenced virus retention based on ionic interactions. In contrast, high electrostatically-based virus removal of the EmphazeTM AEX was confirmed by 4-6 LRV at pH 5.5. As propylene glycol did not influence virus clearance significantly, hydrophobic effects were proved to have no impact neither on virus removal by the Zeta PlusTM nor the EmphazeTM AEX Hybrid Purifier. Conditions at 1 M NaCl and 20 % propylene glycol (pH 5.5) were applied to evaluate a size-based virus removal. As expected MVM was not retained by a size exclusion effect, whereas X-MuLV was retained with 2 LRV by the Zeta PlusTM 90ZA05A but not by the Zeta PlusTM 30ZA layer (porosity of 0.8 to 2.0 μm) and the EmphazeTM AEX. Other studies on the viral clearance by the completely synthetic EmphazeTM AEX and a pre-market prototype EmphazeTM ST-AEX were published using bacteriophages.23 The quarternary amine based AEX filter delivered 5.6-6.4 LRV in product-free buffer at low conductivity whereas reduction dropped down with 150 mmol L-1 NaCl suggesting poor salt tolerance. The ST-AEX filter based on a guanidinium ligand showed 5.9-6.5 LRV for low and high conductivity buffer. In mAb containing feedstream the filter delivered >4 LRV at pH of the buffer above the pI of the used bacteriophages.

Summarized, virus removal capability of conventional cellulosebased depth filters was shown by different groups, but much effort is necessary to evaluate the virus retention mechanism. Moreover, it is indicated that the virus removal capacity is subjected to strong dependencies and variations,28 which might be due to the inherit media variations at micron length scale and below.23 Nevertheless, it may provide an additional safety margin in virus clearance capacity of the process. Newly developed synthetic filters seem to be more suitable for a robust virus removal as well as a sole retention mechanism based on ionic interactions.

To meet the demand for an intensified bioprocessing of therapeutic mAbs, each process step has to be optimized regarding the types of contaminants being addressed. One example is the Protein A capture step, which provides a high removal capacity for HCPs, DNA as well as virus removal capability together with a desired concentration of the product. Other steps like virus inactivation by low pH treatment, AEX chromatography and virus filtration are often dedicated for virus clearance only. In order to shorten the purification process, alternative strategies for virus clearance have been reported. By implementing caprylic acid-induced precipitation as an alternative to a polishing chromatography, impurity removal as well as virus inactivation can be achieved in one simple process step. Newly developed synthetic AEX fi lters enable a replacement of conventional AEX column chromatography, concurrently providing the necessary precipitate removal after low pH or caprylic acid treatment. Using these technologies allows the design of intensified mAb processes (Figure 1) competitive regarding safety, robustness and quality.

Author Biography

Anja Trapp is a Scientist in Bioprocessing Technology & Innovation at Rentschler Biopharma SE in Laupheim, Germany (anja.trapp@ rentschler-biopharma.com)

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