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Events of viral contaminations occurring during the production of biopharmaceuticals have been publicly reported by the biopharmaceutical industry. Upstream raw materials were often identified as the potential source of contamination. Viral contamination risk can be mitigated by inactivating or eliminating potential viruses of cell culture media and feed solutions. Different methods can be used alone or in combination on raw materials, cell culture media, or feed solutions such as viral inactivation technologies consisting mainly of high temperature short time, ultraviolet irradiation, and gamma radiation technologies or such as viral removal technology for instance nanofiltration. The aim of this review is to present the principle, the advantages, and the challenges of high temperature short time (HTST) technology. Here, we reviewed effectiveness of HTST treatment and its impact on media (filterability of media, degradation of components), on process performance (cell growth, cell metabolism, productivity), and product quality based on knowledge shared in the literature. K E Y W O R D S contamination, flash pasteurization, high temperature short time, precipitation, virus 1 | INTRODUCTION Viral contaminations are a severe issue in the frame of the production of biopharmaceuticals by mammalian cells. The production of therapeutic proteins from living organisms, such as mammalian cells, requires a set of components essential for cell survival and cell growth. Those components are provided by cell culture media. Formerly, bovine serum was widely used in cell culture media as a source of amino acids, proteins, vitamins, carbohydrates, lipids, hormones, growth factors, inorganic salts, trace elements, and other compounds. The use of serum or in general of any animal or animal derived raw materials is associated with a higher risk of viral contaminations. For safety and economic reasons, serum-free media, sometimes supplemented with protein hydrolysates, have been developed to replace the use of serum. 1 However, animal component free raw materials also carry a viral contamination risk if they were in contact with animal or animal-derived material during cultivation, manufacturing process, storage, or shipment. This risk of viral contamination by cell culture media raw materials was pointed out early in 1977 by Nuttall and al. 2 They reported the high risk of occurrence of bovine viral diarrhea virus (BVDV) contamination in untreated fetal bovine serum. Some examples of viral contamination in the pharmaceutical industry were publicly reported and are presented in Table 1. Those virus contamination events become rare as only 26 virus contaminations were reported over the past 36 years. 3 Raw materials were the likely source of contamination in most cases. In 11 of the 12 reported

A challenging aspect with the use of protein hydrolysates in commercial manufacturing processes of recombinant therapeutic proteins is their impacts on the protein production due to a lack of understanding of batch-to-batch variability. Soy hydrolysates variability and its impact on fed-batch production of a recombinant monoclonal antibody (mAb) expressed in Sp2/0 cells were studied using 37 batches from the same vendor. The batch-to-batch variability of soy hydrolysates impacted cell growth, titer and product quality. Physicochemical characterization of batches confirmed that soy hydrolysates are mainly a source of amino acids and peptides containing lower amounts of other components such as carbohydrates and chemical elements in cell culture media. Soy hydrolysates composition of different batches was consistent except for trace elements. Statistical analyses identified iron as a potential marker of a poor process performance. To verify this correlation, two forms of iron, ferric ammonium citrate and ferrous sulfate, were added to a batch of soy hydrolysates associated to a low level of iron during cell culture. Both forms of iron reduced significantly cell growth, mAb titer and increased level of the acidic charge variants of the mAb. Consequently, trace element composition of soy hydrolysates or of all incoming raw materials might lead to significant impacts on process performance and product quality and therefore need to be tightly controlled.

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