Thursday, January 9, 2014

Modeling of Inactivation vs. Temperature for Interpreting HTST results

by Dr. Ray Nims

Inactivation vs. temperature modeling data have been used to help interpret the results of recent studies1,3 of the inactivation of the parvovirus murine minute virus through high-temperature short-time (HTST) treatment. The rationale for pursing the approach for modeling heat inactivation susceptibility of viruses, and the approach itself, have been described in detail previously. In brief, this approach consists of assigning a power function line fit directly to a plot of D (the time required to inactivate one log10 of virus) vs. temperature. As with the more traditionally used z value approach, the power function approach requires that inactivation must have been assessed and D values obtained at three or more different temperatures. The power function line fit may be obtained using Excel as:
 where a and b are constants assigned during the line fit process.

Once these power function parameters have been obtained, the equation may be solved for D at a given temperature, as in equation (3). For modeling the inactivation of viruses under conditions approximating high-temperature short-time treatment (i.e., 102 °C for 10 seconds or 115 °C for 30 seconds), the following equations were used:
where D102 °C and  D115 °C are the times (in minutes) required to inactivate 1 log10 of virus at 102 °C  or 115 °C, respectively, determined from the available literature D results using equation (3).

These estimates are subject to the following assumptions: 1) first-order kinetics apply to inactivation over multiple log10 reductions in titer; and 2) the power function equations are valid for predicting inactivation at 102-115 °C even in cases where empirical results were obtained at lower temperatures. The data underlying these assumptions for the available inactivation references for parvoviruses are shown in Table 1. They were most nearly satisfied in the analyses performed for the parvoviruses murine minute virus and canine parvovirus, for which inactivation temperatures as high as 100 °C were evaluated.

Table 1. Satisfaction of assumptions related to modeling of HTST data

Virus studied
Temperatures evaluated (°C)
First-order kinetics through multiple log10 inactivation
45, 60, 100
70, 80, 90
canine parvovirus
56, 80, 100
bovine parvovirus
75, 80, 85, 90

Results of modeling of viral inactivation under HTST conditions
Recently, the potential of high-temperature short-time treatment for mitigating the risk of introducing viral contaminants into biologicals manufacturing processes via cell culture reagents has been explored.1-3  These have assessed short-time (10 s) treatment at 102 °C and/or 115 °C or 30 s at 115 °C.  Murphy, et al. reported3 that treatment at 102 °C for 10 s was able to inactivate completely a suspension of murine minute virus at low titer (10 TCID50/ml) but not a higher titer suspension (100 TCID50/ml). The predictive modeling (Table 2) based on the mean D102 °C  value obtained from two heat inactivation studies performed for MMV and one each for bovine parvovirus and canine parvovirus  indicates that ~ 0.3 log10 of inactivation may be expected for parvoviruses under these conditions. If this prediction is reflective of the actual conditions used for the empirical HTST experiments,3 then perhaps it is not surprising that  100 TCID50/ml of murine minute virus was not completely inactivated. Schleh, et al. reported1 that heating at 115 °C for 30 seconds inactivated 4.9 log10 of murine minute virus in their HTST experiment. Modeling of these conditions (Table 2) suggests that at least1.9 log10 inactivation of parvoviruses might be expected. If the result of Harris, et al. is removed from the calculation (their data indicated an atypically high heat resistance for murine minute virus compared to other experimental results reported for this and other parvoviruses), this modeled inactivation value increases to 6.2 log10

  These modeling data suggest that a temperature of 102 °C for 10 s is probably insufficient for assuring substantial inactivation of a parvovirus (Table 2). On the other hand, exposure to 115 °C for 30 s (Table 2) should provide greater, albeit not total, assurance of inactivation of this virus family. It is important to keep in mind that inactivation efficacy is reported in terms of log10 inactivation, implying that 100% (i.e., complete) inactivation may not be attainable. As with other inactivation modalities, mitigation of the risk of introducing viral contaminants through heat inactivation is maximized by attaining the highest possible log10 inactivation result compatible with maintaining the required performance characteristics of the process material (inactivation matrix) being treated.

1.      Schleh M, Romanowski P, Bhebe P, Zhang L, Chinniah S, Lawrence B, Bashiri H, Gudah A, Rajurs V, Rasmussen B, Chuck A, Dehghani H. Susceptibility of mouse minute virus to inactivation by heat in two cell culture media types. Biotechnol Prog. 2009;25:854-860.
2.      Weaver B, Rosenthal S. Viral risk mitigation for mammalian cell culture media. PDA J Pharm Sci Technol.2010;64:436-439.
3.      Murphy M, Quesada GM, Chen D. Effectiveness of mouse minute virus inactivation by high temperature short time treatment technology: A statistical assessment. Biologicals. 2011;39:438-443.