Shene C, Andrews B A, Asenjo J A
Department of Chemical Engineering, Universidad de La Frontera, Casilla 54-D, Temuco, Chile.
Bioprocess Biosyst Eng. 2003 Jul;25(6):333-40. doi: 10.1007/s00449-002-0313-x. Epub 2003 May 8.
Numerous observations from recombinant systems have shown that properties such as the specific cell growth rate and the plasmid-free cell formation rate are related, not only to the average plasmid content per cell, but also to the plasmid distribution within a population. The plasmid distribution in recombinant cultures can have an effect on the culture productivity that cannot be modelled using average values of the overall culture. The prediction of the behaviour of a plasmid content distribution and its causes and effects can only be studied using segregated models. A segregated model that describes populations of recombinant cells characterized by their plasmid content distribution has been developed. This model includes critical causes of recombinant culture instability such as the plasmid partition mechanism at cell division, plasmid replication kinetics and the effect of the plasmid content on the specific growth rate. The segregated model allows investigation of the effect of each of these causes and that of the plasmid content distribution on the observable behaviour of a recombinant culture. The effect of two partitioning mechanisms (Gaussian distribution and binomial distribution) on culture stability was investigated. The Gaussian distribution is slightly more stable. A small plasmid replication rate constant results in a very unstable culture even after short periods of time. This instability is dramatically improved for a larger value of this constant, hence improving protein synthesis. For a very narrow initial plasmid distribution, a given plasmid replication rate and partitioning mechanism can become broad even after a relatively short period of time. In contrast, a very "broad" initial distribution gave rise to a "Gamma-like" distribution profile. If we compare the results obtained in the simulations of the segregated model with those of the non-segregated one (average model), the latter model predicts much more stable behaviour, thus these average models cannot predict culture instability with the same precision. When compared with the experimental results, the segregated model was able to predict the practical behaviour with accuracy even in a system with a high plasmid content per cell and a high rate of plasmid-free cell formation which could not be achieved with a non-segregated model.
来自重组系统的大量观察表明，诸如比细胞生长速率和无质粒细胞形成速率等特性不仅与每个细胞的平均质粒含量有关，还与群体内的质粒分布有关。重组培养物中的质粒分布会对培养物生产力产生影响，而这种影响无法用整个培养物的平均值来建模。质粒含量分布行为及其因果关系的预测只能使用分离模型来研究。已经开发出一种分离模型，该模型描述了以质粒含量分布为特征的重组细胞群体。该模型包括重组培养物不稳定性的关键原因，如细胞分裂时的质粒分配机制、质粒复制动力学以及质粒含量对比生长速率的影响。分离模型允许研究这些原因中的每一个以及质粒含量分布对重组培养物可观察行为的影响。研究了两种分配机制（高斯分布和二项分布）对培养稳定性的影响。高斯分布略为稳定。即使在短时间后，较小的质粒复制速率常数也会导致培养物非常不稳定。对于该常数的较大值，这种不稳定性会显著改善，从而提高蛋白质合成。对于非常窄的初始质粒分布，即使在相对较短的时间后，给定的质粒复制速率和分配机制也可能会变宽。相反，非常“宽”的初始分布会产生“伽马样”分布曲线。如果我们将分离模型模拟得到的结果与非分离模型（平均模型）的结果进行比较，后者模型预测的行为要稳定得多，因此这些平均模型无法以相同的精度预测培养物的不稳定性。与实验结果相比，即使在每个细胞质粒含量高且无质粒细胞形成速率高的系统中，分离模型也能够准确预测实际行为，而这是非分离模型无法实现的。
