Assessing The Impact of HTST Treatment on Cell Culture Media Through Analytical Testing & Characterization

By: Aaron Mack, Daiva Dailide, Chas Hernandez, Erik Klahn, and Bruce Lehr, Sigma-Aldrich Corporation, Cell Sciences and Development, 2909 Laclede Ave., St. Louis, MO, 63103


In pharmaceutical manufacturing, viral contamination can pose a significant risk to patient safety and damage a company’s reputation. Processes that reduce the risk of viral contamination, such as High Temperature Short Time (HTST) treatment, are implemented across the industry to mitigate this risk. This project assessed the amenability of HTST treatment of some of SAFC’s more commonly used proprietary media, concentrates and feeds at the standard treatment temperature and hold time (102°C for 10 seconds) by analytical characterization and cell performance studies. Proprietary customer owned products were also tested at the customer’s request. Cell culture performance and final product quality attributes were not adversely affected by HTST treatment across all products tested, even though analytical testing showed a drop in the concentration of certain components in some products after treatment. Precipitates from certain customer-owned products were analytically tested and identified. Overall, cell tolerance to treatment of media, feeds and concentrates proved each product’s amenability at standard HTST treatment temperatures. Additional studies are ongoing for SAFC’s other catalog media products.


During pharmaceutical production, viral contamination can decimate a company’s proprietary cell lines, cause facility shutdowns, generally result in reduced patient safety, and can even cause insolvency. Processes that reduce the risk of viral contamination are implemented across the industry to mitigate this risk. One such process is High Temperature Short Time (HTST) treatment. In this pasteurization process, the product is quickly heated to a high enough temperature to destroy pathogens and held at that temperature for a set time to ensure treatment efficacy. Once the product is held at temperature for sufficient time, it is quickly cooled to minimize product degradation. HTST has been shown to reduce active pathogens, such as minute mouse virus (MMV), by 4-6 log in cell culture media. The effectiveness and high throughput of the HTST process has led to the implementation of HTST to pretreat cell culture media and feeds; however, media and feeds solutions are not always amenable to the treatment process and can precipitate media components, resulting in heat exchanger fouling and the possibility of reduced media performance.

Materials and Methods

Viral Inactivation Studies

Bench top viral inactivation experiments were performed with a highly viscous 50% w/v glucose product. A solution of glucose was spiked with a known concentration of feline calcivirus stock, then split into 1.2mL aliquots in stainless steel microvials. Two of the vials were thermally processed at the desired inactivation temp and time, while the other two were held at 25°C for the duration of treatment. Upon completion of treatment, all samples were immediately cooled in an ice bath. Two 500μL samples were aliquoted from each vial and stored at -80°C for virus titration studies. The above process was completed for temperatures of 95°C, 102°C and 107°C. Virus titrations were performed with CRFK (ATCC, CCL-94) in 96-well plates. Plates were scored for CPE at days two and three post-infection. TCID50 titer was calculated according to the Reed and Muench method (1938).

HTST Treatment

An Armfield FT74X-90UHT/HTST Processing System (Figure 2) with attached Lauda WK 3200 Chiller were used for all treatments. SAFC’s ImMEDIAte Advantage™ (IA™) lab hydrated all products, and an untreated control was set aside from the batch of media hydrated. HTST treatments were performed at the standard MMV inactivation parameters of 102°C hold temperature with a hold time of 10 seconds. Treated media were immediately sterile filtered, sampled and packaged for use in cell culture studies and for analytical testing. In addition to pH and osmolality measurements, a free amino acid analysis and a water soluble vitamin analysis were run on HPLC. Inductively coupled plasma mass spectroscopy (ICP-MS) was used to assess trace and macro elements in each sample, including any observed precipitates. FTIR was also used to characterize observed precipitates.

Cell Culture Studies

HTST processed samples and control samples were used as processed, as in the case of 1X media, or added at an appropriate concentration as a feed or supplement. Duplicate TPP (30 mL working volume) were inoculated with CHOZN®GS-/- ZFN-modified CHO cells at 3.0 x 105 cells/mL. Cultures were counted daily using a Beckman Coulter Vi-CELL™ cell counter to determine growth and viability (trypan blue exclusion method). Cultures were maintained in a Multitron incubator at 37 °C, 5% CO2 until viabilities dropped below 70%. Spent media samples were collected at days 7, 10, 12 and 14 for IgG productivity and were analyzed with a ForteBio Octet Interferometer system using Protein A biosensors.


Figure 1. HTST Process Flow Diagram


Figure 2. Armfield HTST Processing System

Viral Inactivation

The thermal heating profile for the bench top HTST unit (Figure 3) was similar to theoretical heating ramp up times calculated for the pilot scale HTST. Results from the viral inactivation study on 50% w/v glucose show that treatment at 102°C or greater for 10 seconds provides sufficient log reduction of feline calcivirus. (Table 1).

Condition Ave. Titer SD % CV Virus Log Reduction
97˚C Pre 7.10 0.20 3.0 N/A
97˚C Post 3.23 0.12 4.0 3.87
102˚C Pre 7.17 0.23 3.0 N/A
102˚C Post 2.50 0.00 0.0 ≥4.67
107˚C Pre 7.17 0.23 3.0 N/A
107˚C Post 2.50 0.00 0.0 ≥4.67

Table 1. Viral Load Reduction Results for 50% w/v Glucose



Figure 3. Thermal Profile for Bench Top HTST Runs

Precipitate Identification

As an initial assessment of a precipitation event, the shell side hot water temperature is monitored during each run. An increase in the shell side hot water temperature, during the run indicates less efficient heat transfer, resulting from precipitate formation on the heat exchanger surfaces. After the run, a calculation of log mean temperature difference (LMTD, Figure 4) for the hot heat exchanger with precipitating product has a positive slope over time.

When a precipitation event occurs, samples of precipitate are collected and characterized with ICP-MS elemental analysis and FTIR. The ICP-MS results show that many of the observed precipitates are mainly composed of calcium, phosphate and iron salts, and these results correlate with FTIR spectra. At this point, only media from customer projects have precipitated (Figure 5, Table 2).

Element Precipitate
HTST Treated [g/L]
Ca 4.05 0.043 0.040
Fe 0.57 0.0027 0.0025
P 5.95 0.034 0.030

Table 2. ICP-MS Results from Customer Owned Media


Figure 4. LMTD Across 'Heating' Heat Exchanger During Precipitation Event


Figure 5. Observed precipitate in A) HTST Tubing Pre- 10% CIP-100 B) Post-10% CIP-100 and C) in 10% CIP-100

Analytical Characterization

Analysis of water soluble vitamin results show that HTST treatment at higher temperatures can cause a drop in vitamin concentration (Figure 7, DME/F12). Amino acid results from the initial testing with multiple media, feeds and concentrates showed that HTST treatment at 102°C for 10 seconds has little effect on amino acid concentration (Figures 8&9). HTST treatment did not impact the pH and osmolality of any of the materials tested (data not shown).


Figure 6. Water Soluble Vitamin Analysis (Feeds and Concentrates)


Figure 7. Water Soluble Vitamin Analysis (Media)


Figure 8. Amino Acid Analysis (Feeds & Concentrates)


Figure 9. Amino Acid Analysis (Media)

Cell Performance Testing

Overall, HTST treatment at all temperatures tested did not detrimentally impact viable cell density, percent viability or cell doubling time in tested products. Treated and untreated conditions reached similar viable cell densities with the viabilities of ≥90%. Examples of cell performance data from tests performed on ExCell 302 are shown in the Figure 10 below.


Figure 10. Compilation of Cell Performance Testing Results for ExCell 3020


  • HTST treatment of all SAFC media, feeds and concentrates tested thus far showed no signs of detrimental impact to cell culture performance
  • Observed precipitate mainly consisted of calcium, phosphate, iron and other metal salts
  • Thiamine and other vitamin concentration in some treated media decreased as a result of treatment, as in figure 7 HTST conditions of 102°C for a hold time of 10 seconds were well tolerated by products tested.
  • Tables 3 & 4 summarize the results of HTST amenability testing

Table 3. Summary of HTST Impact Assessment Testing for Catalogue Media

Media ExCell 302 Media (Normal 1X Media) ExCell CD CHO Fusion (Chemically Defined Media) CHOZN Media (for use with ZFN) DMEM/F12 (Normal 1X Media) CHO 4X4 Kit Customer’s Proprietary Media Formulation 1 IA ExCell Pro II (Stem Cell Media)
Final Product Spec Pass Pass Pass Pass Pass Pass Fail (Precip.)
Analytical Testing Pass Pass Pass Pass TBD Pass TBD
Biological Testing Pass Pass Pass Pass TBD Pass TBD


Table 4. Summary of HTST Impact Assessment Testing for Feeds and Concentrates

Fail (Precip.) 50% w/v Glucose (High Viscosity, High Glucose) 20X ExCell CD Hydrolysate Fusion, (High Hydrolysate Feed) 50X Soy Hydrolysate UF, (High Hydrolysate, High Viscosity) 50X MEM Amino Acids, (High Amino Acids) Fatty Acid Supplement, (High Fatty Acids) 100X MEM Vitamins, (High Water Soluble Vitamins) ExCell CHOZN Platform Feed NF4 DPM (4X Concentrate)
Final Product Spec Pass Pass Pass Pass Pass Pass Pass
Analytical Testing Pass Pass Pass Pass TBD Pass Pass
Biological Testing Pass Pass Pass Pass Pass Pass Pass

Criteria for pass/fail is defined by final product specification (pH/OSMO), precipitation events, and cell performance testing comparability across treated and untreated samples


All media, feeds and concentrates tested were considered amenable to the HTST process. The HTST process did not impact the pH and osmolality of the materials tested. Analytical results showed that there was a decrease thiamine concentration in the DME/F12 formulation, but that did not negatively affect cell performance in the cell line tested. Cell performance data was within SAFC’s historical experience with respect to productivity at small scales. Process performance and quality indicate that HTST treatment conditions were tolerated. Future testing will include additional SAFC catalog media and customer owned media upon request.


Special thanks to Joe Schuler, Sheila Stock, and Andy Feldman in the ImMEDIAte Advantage™ lab at SAFC, Guy Matthews, formerly of SAFC, and Philip Montgomery.


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