Elsevier

Biotechnology Advances

Volume 27, Issue 2, March–April 2009, Pages 177-184
Biotechnology Advances

Research review paper
The application of SELDI-TOF mass spectrometry to mammalian cell culture

https://doi.org/10.1016/j.biotechadv.2008.10.007Get rights and content

Abstract

Surface Enhanced Laser Desorption/Ionisation Time-of-Fight Mass Spectrometry (SELDI-TOF MS) is a technique by which protein profiles can be rapidly produced from a wide variety of biological samples. By employing chromatographic surfaces combined with the specificity and reproducibility of mass spectrometry it has allowed for profiles from complex biological samples to be analysed. Profiling and biomarker identification have been employed widely throughout the biological sciences. To date, however, the benefits of SELDI-TOF MS have not been realised in the area of mammalian cell culture. The advantages in identifying markers for cell stresses, apoptosis and other culture parameters mean that these tools could help greatly to enhance monitoring and control of bioreaction process and improve the production of therapeutics. Better characterisation of culture systems through proteome analysis will allow for improved productivity and better yields.

Introduction

Mammalian cell-based bioprocesses have been used in the production of viral vaccines, and diagnostic and therapeutic proteins, for nearly half a century and mammalian cells are now the workhorse of the industry (Griffin et al., 2007). With a constantly growing demand for biopharmaceutical therapeutics, the role of modern -omics technologies in upstream development is becoming ever more necessary (Lawrence, 2005).Advancements in both genomic and proteomic technologies such as the recent increase in availability of mass spectrometry instrumentation, allow us to examine biological systems with greater depth than ever before. The application of technologies such as these to the production of therapeutics from mammalian cell systems will allow us to work toward meeting modern medical requirements. One of the major areas of biopharmaceutical output is monoclonal antibodies. The production of monoclonal antibodies (MAbs) is vitally important to modern medicine and as such has become the second largest class of biopharmaceutical products in development (Walsh, 2003). The majority of MAbs are generated in Chinese hamster ovary (CHO) and murine myeloma (NS0) cell systems. These production systems can correctly fold and post-translationally modify the products, thus the functionality of these proteins in the body is not compromised.

The need for antibodies with unique specificity in large quantities has advanced the development of new technologies for the production of diagnostic and therapeutic proteins. The high affinity and specificity of antibodies and related recombinant molecules have been widely exploited with the emergence of therapeutic antibodies being particularly relevant to the medical industry. Hundreds of monoclonal antibodies are in clinical trials and 18 monoclonal antibody based therapeutics have reached the market, although only three of them are murine/mouse antibodies and eight anti-cancer MAbs have received FDA approval (van Dijk and van de Winkel, 2001, Brekke and Sandlie, 2003, Hale, 2006, Zafir-Lavie et al., 2007). The majority of therapeutic MAbs are engineered “human-like” antibodies (chimeric or CDR-grafted), which represented the first attempt to reduce immunogenicity (Reichert, 2001). An ever-increasing demand for increased productivity of MAbs is representative of the biopharmaceutical industry, in general. Using recent advances in technology will allow us to help meet the shortfall.

To enhance production, cultures have to be run more efficiently and this can be achieved through nutrient optimisation, feeding, cell engineering or by manipulation of the cellular environment. The effect of the bioreactor environment on cell growth and productivity has been well studied. Alterations to growth conditions offer an economical and reliable way to ensure optimal productivity. Reduced cultivation temperature and increased osmolarity have been shown to have a significant positive effect on productivity, with up to three-fold increases in productivity obtainable (Trummer et al., 2006, Swiderek and Al-Rubeai, 2007, Yoon et al., 2005, Miller et al., 2000). Other conditions such as dissolved oxygen, pH and media components have also been studied and shown to have significant effects on gene regulation, physiology, viability and productivity (Zanghi et al., 1999, Trummer et al., 2006, Swiderek et al., 2008, Ishaque and Al-Rubeai, 2002, Simpson et al., 1998). Deviations from optimum culture conditions can result in a dramatic loss in viability, growth and productivity. The ability to predict when cultures will deviate from the optimal conditions or to rapidly identify a biomarker of stress that would allow for process prediction and subsequently an increased productivity as preventative measures could be taken to avoid a loss of yield. Premature cell death during production is a known response to the stressful environment that prevails during extended bioreactor cultures.

It has been shown that the primary means of cell death in the bioreactor is apoptosis (Cotter and al-Rubeai, 1995, Singh et al., 1994). However, apoptosis can be delayed or prevented by manipulating the environment or by genetic modification of cells to overexpress an anti-apoptotic protein such as Bcl-2 and Bcl-xL (Simpson et al., 1998, Tey et al., 2000, Figueroa et al., 2003). In these studies the ability of the anti-apoptotic protein to protect cells from a range of insults was demonstrated. While altering the agitation speed pH and oxygen levels can reduce apoptosis and increase cell number, the most significant factor in the cellular environment is the growth media. Supplementation with amino acids, survival/growth factors, peptones and hydrolysates can be a highly effective technique in prolonging cell viability in a culture.

While some significant advances have already been made using the strategy of genetic manipulation, our understanding of the intracellular processes pertaining to stress sensing in a bioreactor environment still needs to be expanded. While much is known about individual stresses such as hypoxia, nutrient limitation, shear stress, metabolite accumulation and pH changes, the interaction between these factors and their early signalling pathways is poorly understood (Halliwell, 2003, Lara et al., 2006, Lao and Toth, 1997).

The identification of markers that could predict the onset of apoptosis in a bioreactor would allow for more effective monitoring of the culture process. Monitoring of cell cultures is generally achieved through routine analysis of parameters such as cell number, viability, glucose and lactate. While this type of analysis yields vital information about the culture, its predictive value is extremely limited. Protein markers secreted from mammalian cells under various stress conditions would be ideal candidates for monitoring. It is very likely that the secreted proteome of cell lines under different stress conditions would be unique and that markers for these conditions would be readily available in the supernatant media. Up- or down-regulated proteins in cells or culture may then indicate the response of cells to depletion of a certain nutrient or a change in dissolved oxygen, for example. Recent advancements in biomarker technology, generally used for diagnostic purposes and in techniques for rapid proteome analysis, make it an excellent option for the identification and monitoring of such biomarkers and offer great prospects for mammalian cell culture as outlined here.

Section snippets

Surface Enhanced Laser Desorption/Ionisation Time-of-Flight Mass Spectrometry (SELDI-TOF MS)

SELDI-TOF MS is a protein analysis technique, which was introduced by Hutchens and Yip (1993) and is a modification of the standard matrix assisted laser desorption–ionisation time-of-flight MALDI technology. The ProteinChip Biology System (PBS) uses SELDI-TOF MS to retain proteins on a solid-phase chromatographic surface that are subsequently ionized and detected by TOF MS (Ciphergen Biosystem Inc., Freemont, CA, USA). The versatility of the machine allows the analysis of biochemical reactions

Sample preparation

The standard procedure for initial biomarker discovery via SELDI-TOF analysis is termed Expression Difference Mapping (EDM). The basic procedure for EDM applications with the PBS is quite simple and outlined here. One of the most powerful aspects of the PBS is that virtually any type of protein-containing solutions can be directly applied to the spots of ProteinChip Arrays for subsequent analysis. These spots present either chromatographic surfaces with certain physicochemical characteristics

SELDI and cell culture

While many exogenous nutrient supplements, physical parameters, chemical agents and genes have been examined for their ability to improve culture longevity and productivity, few have been examined in detail through multiple studies. This clearly highlights the need to identify possible gene/protein candidates to target for improving culture processes. Methods that have proven effective in this regard have been discussed already in the Introduction. In recent years the availability of new

Conclusion

While some concerns have been raised over the reproducibility of SELDI-TOF data, there is no doubt as to the usefulness of the technique in the identification of proteins from biological fluids. The ability to rapidly screen and to examine complex biological samples means this method can easily be incorporated into cell culture process and would certainly help in the identification of potential protein markers, from both supernatant media or lysed cells, to be exploited for process monitoring

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