Viral contamination is an inherent risk during the manufacture of biopharmaceutical products. Whether introduced from raw materials or during manufacturing operations, unmitigated viral contamination has led to serious health implications and plant shutdowns. Moreover, Type C Retrovirus Like Particles (RVLP) are endogenously produced during CHO cell expression1. As such, regulatory agencies require proof that downstream process steps can effectively remove or inactive retrovirus.Historically, this has been demonstrated through the use of a model mammalian virus, Xenotropic Murine Leukemia Virus (XMuLV). This is accomplished by conducting live viral clearance “spiking” studies which require specialized contract research organizations (CRO’s) and trained personnel resulting in high costs and complex logistics. These hurdles deter companies from analyzing viral clearance during the years of small-scale process development. Instead, companies spend considerable resources optimizing manufacturing processes before gaining any knowledge of their viral clearance efficacy. Unfortunately, this increases the risk of validation failure, forcing companies to invest additional time and money redeveloping process steps, which in turn, could postpone regulatory approvals and delay patients’ timely access to therapies.
In September of 2022, the FDA (as part of the ICH) approved a draft revision to Q5A which provides guidance to the biotechnology industry on viral safety and evaluation2. This document stated that “For CHO cell-derived products, CHO-derived endogenous virus particles [RVLP] can also be used for viral clearance experiments. ”In response, Cygnus Technologies has developed a novel MockV® RLVP approach to enable the utility of RVLP for downstream spiking applications. Packaged as a complete, ready-to-use solution, these CHO-derived, non-infectious RVLP provide a BSL-1 compatible spiking agent for viral clearance testing. The MockV RVLP approach overcomes the barriers imposed by live XMuLV studies as RVLP can be safely handled on the benchtop to predict the outcomes of CRO-led spiking studies (Table 1). This technology allows the incorporation of viral clearance into QbD, DOE and HTS approaches.
Why delay until late phase clinical manufacturing before testing if your downstream purification process steps provide sufficient viral clearance? Instead, you can now gain actionable insights early in process development. Instead of relying solely on a CRO-provided model retrovirus (XMuLV), process development groups can now independently spike and assess the removal of the original retrovirus particle of regulatory concern1.
Table 1: RVLP vs. XMuLV Comparison Summary1
Non-infectious RVLP were produced during CHO cell cultivation and purified via multiple modes of chromatography before being concentrated to a final stock solution of 1 x 1010 particles/mL (Figure 1).
Figure 1: TEM Image of RVLP Particles
RVLP and XMuLV Characteristic Comparison
RVLP diameter and net-surface charge were assessed alongside XMuLV (produced by Texcell, N.A.) via Dynamic Light Scattering (DLS) and Zeta Potential. The DLS results demonstrated that RVLP, are monodisperse and exhibit an average diameter of 193 nm while XMuLV are also monodisperse but exhibit a slightly larger diameter.
Figure 2: DLS Size Comparison
Zeta Potential results indicated that the surface charge of each particle were slightly acidic.
Table 1: Net Surface Charge
To analyze the concentration of noninfectious RVLP in samples, an RT-qPCR method modified from De Wit, 20003 was employed. In short, samples were added to a 96 deep-well plate and were treated with Endonuclease to degrade CHO-endogenous RVLP DNA sequences. RNA was then extracted from RVLP and precipitated with a set of proprietary buffers (Cygnus Technologies, LLC).The plate was then stored at -20 °C for 30 minutes and RNA was pelleted via centrifugation at 3,000 x g for 20 minutes at 4 °C. After washing and final pelleting, the RNA was resuspended in a proprietary buffer. 10 μL of sample was transferred from each well of the 96 deep-well plate to a qPCR plate containing TaqMan primers/probe directed against the pol region of the CHO-RVLP genome. To determine the quantity of particles in unknown samples, threshold cycle (Ct) values were interpolated into a standard curve generated by including a dilution series of a known sRNA standard. From those concentration values, RVLP LRVs for each experiment were calculated.
Figure 3: Example sRNA standard curve
Cygnus introduced the MockV® RLVP Kit (Cygnus Technologies Cat # M230) to realize the advantages of an RVLP approach and to enable biopharmaceutical manufacturers the ability to gain viral clearance insights early in process development. The MockV® RLVP Kit includes a vial of RVLP stock solution, a 96-well plate for sample analysis, RNA extraction and qPCR reagents, and a well-controlled RNA standard for accurate and reliable RVLP quantification. By following the kit’s easy-to-use protocol, scientists can detect as little as 1 x 103 RVLP/mL, enabling LRV of ~ 5.0 to be determined.Each kit contains 2.0 mL of RVLP Stock Solution at a concentration of 1 x 1010 RVLP/mL sufficient for spiking up to 200 mL of load material to 1% (v/v) and conducting analysis on 23 samples in triplicate in less than one day. A real-time qPCR instrument is required along with standard laboratory equipment. Minimal experience with RNA extraction or qPCR protocols is required. Compatible purification steps include Protein A chromatography, virus filtration, anion and cation exchange chromatography, mixed-mode, hydrophic-interaction and size- exclusion chromatography. MockV® RLVP Kit achieves LRV accuracy to within ± 0.5 for these modes of separation as compared to traditional viral clearance studies with XMuLV model virus.
Now, using Cygnus’s BSL-1 compatible viral clearance kits, you can easily and economically quantify viral clearance for downstream process steps right in your own lab and on your timeline.
To learn more about Cygnus MockV RVLP and MockV MVM Kits, visit www.cygnustechnologies.com/MockV
QbD = Quality by Design
DOE = Design of Experiment
HTS = High-throughput Screening
David Cetlinis Senior Director, MockV Products, at Cygnus Technologies. He can be contacted at firstname.lastname@example.org
Alla Zilberman is Vice President, Technical Marketing at Cygnus Technologies. She can be contacted at email@example.com