Combined approach of NMR and chemometrics for screening peptones used in the cell culture medium for the production of a recombinant therapeutic protein

The yield of monoclonal antibody (Mab) production processes depends on media formulation, inocula quality, and process conditions. As in industrial processes tight cultivation conditions are used, and inocula quality and viable cell densities are controlled to reasonable levels, media formulation and raw materials lot-to-lot variability in quality will have, in those circumstances, the highest impact on process performance. In the particular Mab process studied, two different raw materials were used: a complex carbon and nitrogen source made of specific peptones and defined chemical media containing multiple components. Using different spectroscopy techniques for each of the raw material types, it was concluded that for the complex peptone-based ingredient, near-infrared (NIR) spectroscopy was more capable of capturing lot-to-lot variability. For the chemically defined media containing fluorophores, two-dimensional (2D)-fluorescence spectroscopy was more capable of capturing lot-to-lot variability. Because in Mab cultivation processes both types of raw materials are used, combining the NIR and 2D-fluorescence spectra for each of the media components enabled predictive models for yield to be developed that out-performed any other model involving either one raw material alone, or only one type of spectroscopic tool for both raw materials. For each particular raw material, the capability of each spectroscopy to detect lot-to-lot differences was demonstrated after spectra preprocessing and specific wavelength regions selection. The work described and the findings reported here open up several possibilities that could be used to feed-forward control the process. These include, for example, enabling specific actions to be taken regarding media formulation with particular lots, and all types of predictive control actions aimed at increasing batch-to-batch yield and product quality consistency at harvest.

Section: Nuclear Magneticmentioning

confidence: 99%

Predicting mab product yields from cultivation media components, using near‐infrared and 2D‐fluorescence spectroscopies

José

Folque

Menezes

et al. 2011

“…Their proposed method demonstrated satisfactory performance with respect to linearity and precision and was able to resolve components with specificity. More recently, Luo and Chen examined use of an approach using a combination of nuclear magnetic resonance (NMR) and chemometrics for screening peptones used in cell culture media (Luo and Chen, 2007). Their approach allowed or rapid screening of peptone lots in order to optimize efficiency and consistency of large-scale protein production.…”

Section: Introductionmentioning

confidence: 99%

Application of near‐infrared (NIR) spectroscopy for screening of raw materials used in the cell culture medium for the production of a recombinant therapeutic protein

Kirdar

Chen

Weidner

et al. 2009

Self Cite

Control of raw materials based on an understanding of their impact on product attributes has been identified as a key aspect of developing a control strategy in the Quality by Design (QbD) paradigm. This article presents a case study involving use of a combined approach of Near-infrared (NIR) spectroscopy and Multivariate Data Analysis (MVDA) for screening of lots of basal medium powders based on their impact on process performance and product attributes. These lots had identical composition as per the supplier and were manufactured at different scales using an identical process. The NIR/MVDA analysis, combined with further investigation at the supplier site, concluded that grouping of medium components during the milling and blending process varied with the scale of production and media type. As a result, uniformity of blending, impurity levels, chemical compatibility, and/or heat sensitivity during the milling process for batches of large-scale media powder were deemed to be the source of variation as detected by NIR spectra. This variability in the raw materials was enough to cause unacceptably large variability in the performance of the cell culture step and impact the attributes of the resulting product. A combined NIR/MVDA approach made it possible to finger print the raw materials and distinguish between good and poor performing media lots.

“…However, there are a few limitations that complicate hydrolysates' utilization. First of all, lot-to-lot variation exists for hydrolysates (Luo and Chen, 2007;Zhang et al, 2003). Prescreening using smallscale performance testing is typically required to eliminate underperforming lots.…”

Section: Introductionmentioning

confidence: 99%

A single nutrient feed supports both chemically defined NS0 and CHO fed‐batch processes: Improved productivity and lactate metabolism

Ellet

Okediadi

et al. 2009

111

A chemically defined nutrient feed (CDF) coupled with basal medium preloading was developed to replace a hydrolysate-containing feed (HCF) for a fed-batch NS0 process. The CDF not only enabled a completely chemically defined process but also increased recombinant monoclonal antibody titer by 115%. Subsequent tests of CDF in a CHO process indicated that it could also replace the hydrolysate-containing nutrient feed in this expression system as well as providing an 80% increase in product titer. In both CDF NS0 and CHO processes, the peak lactate concentrations were lower and, more interestingly, lactate metabolism shifted markedly from net production to net consumption when cells transitioned from exponential to stationary growth phase. Subsequent investigations of the lactate metabolic shift in the CHO CDF process were carried out to identify the cause(s) of the metabolic shift. These investigations revealed several metabolic features of the CHO cell line that we studied. First, glucose consumption and lactate consumption are strictly complementary to each other. The combined cell specific glucose and lactate consumption rate was a constant across exponential and stationary growth phases. Second, Lactate dehydrogenase (LDH) activity fluctuated during the fed-batch process. LDH activity was at the lowest when lactate concentration started to decrease. Third, a steep cross plasma membrane glucose gradient exists. Intracellular glucose concentration was more than two orders of magnitude lower than that in the medium. Fourth, a large quantity of citrate was diverted out of mitochondria to the medium, suggesting a partially truncated tricarboxylic acid (TCA) cycle in CHO cells. Finally, other intermediates in or linked to the glycolytic pathway and the TCA cycle, which include alanine, citrate, isocitrate, and succinate, demonstrated a metabolic shift similar to that of lactate. Interestingly, all these metabolites are either in or linked to the pathway downstream of pyruvate, but upstream of fumarate in glucose metabolism. Although the specific mechanisms for the metabolic shift of lactate and other metabolites remain to be elucidated, the increased understanding of the metabolism of CHO cultures could lead to future improvements in medium and process development.