Guan Y, Evans P M, Kemp R B
Institute of Biological Sciences, The University of Wales, Aberystwyth, Ceredigion SY23 3DA, United Kingdom.
Biotechnol Bioeng. 1998 Jun 5;58(5):464-77. doi: 10.1002/(sici)1097-0290(19980605)58:5<464::aid-bit2>3.0.co;2-b.
One of the requirements for enhanced productivity by the animal culture systems used in biotechnology is the direct assessment of the metabolic rate by on-line biosensors. Based on the fact that cell growth is associated with an enthalpy change, it is shown that the specific heat flow rate is stoichiometrically related to the net specific rates of substrates, products, and indeed to specific growth rate, and therefore a direct reflection of metabolic rate. Heat flow rate measured by conduction calorimetry has a technical advantage over estimates for many material flows which require assays at a minimum of two discrete times to give the rate. In order to make heat flow rate specific to the amount of the living cellular system, it would be advantageous to divide it by viable biomass. This requirement has been fulfilled by combining a continuous flow microcalorimeter ex situ with a dielectric spectroscope in situ, the latter measuring the viable cell mass volume fraction. The quality of the resulting biosensor for specific heat flow rate was illustrated using batch cultures of Chinese hamster ovary cells (CHO 320) producing recombinant human interferon-gamma (IFN-gamma) during growth in a stirred tank bioreactor under fully aerobic conditions. The measuring scatter of the probe was decreased significantly by applying the moving average technique to the two participant signals. It was demonstrated that the total metabolic rate of the cells, as indicated by the specific heat flow rate sensor, decreased with increasing time in batch culture, coincident with the decline in the two major substrates, glucose and glutamine, and the accumulation of the by-products, ammonia and lactate. Furthermore, the specific heat flow rate was an earlier indicator of substrate depletion than the flow rate alone. The calorimetric-respirometric ratio showed the intensive participation of anaerobic processes during growth and the related IFN-gamma production. Specific heat flow rate was monotonically related to specific cell growth rate and associated with specific IFN-gamma production. Specific heat flow rate is potentially a valid control variable for the growth of genetically engineered cell lines producing target proteins.
生物技术中使用的动物培养系统提高生产力的要求之一是通过在线生物传感器直接评估代谢率。基于细胞生长与焓变相关这一事实，研究表明比热流率与底物、产物的净比速率以及比生长速率在化学计量上相关，因此是代谢率的直接反映。通过传导量热法测量的热流率相对于许多物质流的估算具有技术优势，后者需要至少在两个离散时间进行测定才能得出速率。为了使热流率与活细胞系统的量相关，将其除以活生物质是有利的。通过将异位连续流动微量热计与原位介电光谱仪相结合满足了这一要求，后者测量活细胞质量体积分数。在完全有氧条件下，在搅拌罐生物反应器中生长期间，使用产生重组人干扰素 -γ（IFN -γ）的中国仓鼠卵巢细胞（CHO 320）的分批培养来说明所得比热流率生物传感器的质量。通过对两个参与信号应用移动平均技术，显著降低了探头的测量散射。结果表明，比热流率传感器所示的细胞总代谢率在分批培养中随时间增加而降低，这与两种主要底物葡萄糖和谷氨酰胺的减少以及副产物氨和乳酸的积累相一致。此外，比热流率比单独的流速更早地指示底物耗尽。量热 - 呼吸比表明生长和相关的IFN -γ产生过程中厌氧过程的强烈参与。比热流率与比细胞生长速率单调相关，并与特定的IFN -γ产生相关。比热流率可能是生产目标蛋白的基因工程细胞系生长的有效控制变量。
