Human Cytomegalovirus (HCMV), is a herpesvirus with worldwide prevalence that naturally infects more than 50% of the adult population by the age of 40 and 33% of children by age 5 in the United States ($author1$ et al., 1. </id><collab>CDC</collab><maintitle>Cytomegalovirus (CMV) and Congenital CMV Infection. 18Aug2020 25Jan2022]</maintitle><comment>Available from</comment><e-link>https://www.cdc.gov/cmv/index.html</e-link>). Primary HCMV infection is usually asymptomatic in healthy adults, but infections can pose significant health challenges in immunocompromised individuals and in utero ($author1$ et al., 1. </id><collab>CDC</collab><maintitle>Cytomegalovirus (CMV) and Congenital CMV Infection. 18Aug2020 25Jan2022]</maintitle><comment>Available from</comment><e-link>https://www.cdc.gov/cmv/index.html</e-link>). In immunocompromised individuals, such as organ transplant recipients, neonates, or people living with human immunodeficiency virus (HIV), HCMV is a well-known cause of morbidity and mortality (Emery, 2001, Khare and Sharland, 2001, Gerna and Lilleri, 2019, Lilleri and Gerna, 2017). Congenital HCMV, via infection during pregnancy, can lead to a variety of developmental disabilities in newborns, such as intellectual disability, hearing loss, and vision impairment (Rawlinson et al., 2017, Ross and Boppana, 2005). The United States Food and Drug Administration (FDA) acknowledges HCMV as an unmet medical need and has published guidance for the purpose of aiding in the development of drugs and vaccines to treat and/or prevent HCMV infection (FDA, Hodowanec et al., 2020).

As of September 2018, multiple candidate vaccines were in development for the prevention of HCMV infection and disease (Plotkin et al., 2020). One such candidate, known as V160, is in development at the research laboratories of Merck & Co., Inc., Kenilworth, NJ, USA and utilizes an anchorage dependent cell line known as ARPE-19 (Adult Retinal Pigment Epithelial cells) for the propagation of the HCMV vaccine strain (Adler et al., 2019, Wang et al., 2016, Li et al., 2021a). Epithelial cells play a key role in natural infection as the point for entry and transmission of HCMV in the body, and the pentameric glycoprotein complex, gH/gL/pUL128/pUL130/pUL131, is an important neutralizing epitope associated with receptor-mediated entry into epithelial cells (Wang and Shenk, 2005, Rustandi et al., 2021). Extensive propagation of laboratory HCMV strains in fibroblasts has been shown to result in the loss of the UL131-128 gene locus, since it is not required for viral entry in fibroblasts. Therefore, strains propagated on fibroblasts often no longer have the capacity to infect epithelial cells effectively and efficiently (Wang and Shenk, 2005, Hahn et al., 2004, Prichard et al., 2001), potentially making them less optimal choices as vaccine candidates. The V160 vaccine strain has been genetically repaired for preservation of UL131-128 and is propagated on a cell substrate selective for epithelial-tropic virus replication, preserving epitopes that may be important for effective neutralizing responses against epithelial cell entry (Adler et al., 2019, Wang et al., 2016, Li et al., 2021b).

Bioprocess development for live virus vaccines typically involves the use of anchorage dependent cell substrates as host cells, resulting in the widespread adoption of microcarriers for the production of live virus vaccine candidates at large scales (hundreds to thousands of liters). Scale-up of adherent cell culture processes is primarily limited by surface area availability, and the common use of tissue culture plasticware vessels quickly highlights this limitation when there is a need for substantially increasing virus production output. Microcarriers are small biocompatible beads that can be suspended via agitation in a stirred tank bioreactor format to provide large available surface area for cells to adhere and grow without the need to scale out to large numbers of individual static tissue culture vessels. Scaling up and intensifying a microcarrier bioreactor process can follow similar schemes as a standard suspension cell process by increasing culture volume and/or microcarrier (i.e., cell) concentration. One such microcarrier, Cytodex-1 (Cytiva), has often prevailed as the growth support of choice for vaccine manufacturing in stirred tank bioreactors (Kiesslich and Kamen, 2020, Mattos et al., 2015, Rourou et al., 2007, Arifin et al., 2010).

Until recently, Cytodex-1 microcarriers were not available in a pre-sterilized “ready to use” format. Instead, the dry powder beads required hydration, washing, and heat sterilization. These steps added complications to large scale adherent cell culture processes, especially with the bioprocess field widely moving toward full integration of single use processing solutions (Tapia et al., 2014, Lesch et al., 2021, Yang et al., 2019). Heat sterilization validation packages can also be costly and time consuming to implement, while a gamma irradiated option offers logistical benefits for larger scale manufacturing operations. Here we have evaluated a strategy to allow the use of gamma irradiated Cytodex-1 microcarriers as a direct replacement for the traditional heat-sterilized Cytodex-1 microcarrier within the context of the V160 HCMV vaccine candidate production process.