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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References and Notes

Fetal bovine serum (FBS) is the most widely used growth supplement for cell cultures, primarily because of its high levels of growth stimulatory factors and low levels of growth inhibitory factors. Maintaining successful and consistent cell fermentations can be difficult, as FBS is a complex natural product and may vary from lot to lot even from a single manufacturer. The quality and concentration of both bulk and specific proteins can affect cell growth. Quality control tools for FBS are relatively primitive and expensive given the complexity of the sample and the large amounts of FBS used. We undertook this study to examine whether proteomics could be used as a tool to analyze the variability of different fermentation processes. We hypothesized that inconsistent cell growth in fermentations could be due to the quality of FBS and that different lots of FBS had varying concentrations of proteins such as growth stimulatory factors, growth inhibitory factors, and/or other proteins that may correlate with cellular growth rate. To investigate whether this was the case, we grew three batches of adult retinal pigment epithelial cells (ARPE-19) using three different lots of fetal bovine serum (FBS-Ia, FBS-Ib, and FBS-II). We found that the growth rate of the culture was significantly and consistently higher in the FBS-II lot. To determine why the other lots promoted different growth properties, we used proteomic techniques to analyze the protein composition of the three lots. We then performed a time course study to monitor specific changes in individual proteins in the fermentation medium. The amount of several extracellular matrix and structural proteins, which are indicators of cell growth, increased over time. Alternatively, components supplied by the FBS addition, such as nutritional-related and cell-spreading-related proteins, decreased over time.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References and Notes

For high yield cell culture, such as used when producing a protein pharmaceutical, one needs a medium that supplies the necessary nutrients and growth factors. The chemical composition of the medium is very complex but essentially consists of four groups of components:  inorganic salts, carbohydrates, amino acids, and various supplements (i.e., vitamins, fatty acids, lipids, and growth factors).

For most cell lines, the primary media supplement is fetal bovine serum (FBS). It provides several important biological molecules such as albumin, antichymotrpsin, apolipoproteins, biotin, and growth supporting factors, which are required for optimal growth of cells (4). Serum changes the physiochemical properties of the cell culture media, including viscosity, osmolality, buffering capacity, and diffusion rates. It helps to protect the cells from mechanical damage, which may occur in stirred cultures or while using a cell scraper. A further advantage of serum is that it can be used in a wide variety of cultures despite the varying growth requirements of different cells. Many factors, such as the quality, type, and concentration of the components in the different FBS lots, can affect the cellular growth rate in a fermentation process (5). Therefore, it is plausible to screen batches of serum for the factors necessary for consistent cell cultures. However, current approaches for screening raw materials (such as testing osmolality, presence of endotoxins, and growth promoters) are potentially imprecise and/or expensive. With the recent growth in international manufacture of biopharmaceuticals there is a need for more powerful screening technologies. We propose to use information generated from the sequencing of the human genome and subsequent proteomic studies.

In this study, we evaluated the growth rates of adult retinal pigment epithelial cells (ARPE-19) using three different lots of FBS. Then we used proteomic techniques in an attempt to discover whether the different FBS serum lots varied in important proteins, such as growth stimulatory factors, growth inhibitory factors, or other proteins that may correlate with cell culture growth rates. We then did a follow-up study to monitor the change in amount of certain proteins in the cell culture medium over time.

Proteomics is the global analysis of protein expression patterns. In the past, proteomic analysis was accomplished by two-dimensional gel electrophoresis coupled with mass spectrometry. In our study, we chose to use the newer approach, reversed phase liquid chromatography coupled with ion-trap tandem mass spectrometry analysis (LC-MS/MS) of the corresponding tryptic digest, with the goal of identifying a number of significant proteins with reasonable throughput. In addition, quantitation of selected proteins in complex samples can be achieved via the measurement of peptide ions (6, 7). Regardless of the proteomics approach, abundant proteins present in a sample limit the dynamic range of the measurement. We added an additional step to deplete the most abundant proteins using a molecular weight cutoff centrifugal filter, which then allowed us to analyze the low molecular weight fraction in greater depth.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References and Notes

Materials. Three different pools of fetal bovine serums (FBS-Ia, FBS-Ib, and FBS-II) were obtained from two different suppliers:  I and II. FBS-Ia and FBS-Ib were from supplier I; FBS-II was from supplier II. Microcon centrifugal filter devices were obtained from Millipore (Billerica, MA). Sequencing grade-modified trypsin was purchased from Promega (Madison, WI). Acetonitrile, dl-dithiothreitol (DTT), iodoacetamide (IAA), and ammonium bicarbonate were obtained from Sigma (St. Louis, MO). Formic acid (FA) was obtained from ICN Biomedicals (Aurora, OH). HPLC grade acetonitrile was purchased from Fisher (Fair Lawn, NJ). LCMS grade water was purchased from Mallinckrodt Baker (Philipsburg, NJ). Dulbecco's modified Eagle's medium/Ham's nutrient mixture (DMEM/F12) was from Invitrogen (Carlsbad, CA). Adult retinal pigment epithelial cells (ARPE-19) cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). The cells were maintained in DMEM/F12 supplemented with fetal bovine serum at a final concentration of 10%. Three aliquots of cell culture media supernatant were collected over several days and in 3 time-points as T0 (control), T5 (5 days), and T8 (8 days) for proteomic analysis.

Trypsin Digestion of Unfractionated FBS Fraction. The procedure for enzymatic digest of the fetal bovine serum (FBS) was modified from a method found in the literature (8). One hundred micrograms of FBS was diluted into 100 μL of 100 mM ammonium bicarbonate buffer, pH = 8.20. FBS was denatured by adding 400 μL of 6 M GuCl in 100 mM ammonium bicarbonate buffer, pH = 8.20. Then FBS was reduced by adding dl-dithiothreitol (DTT) with a final concentration of 5 mM and heated at 75 °C for 1 h. After cooling to room temperature, the FBS proteins were alkylated by adding iodoacetamide (IAA) with a final concentration of 20 mM and then incubating at room temperature for 1 h in dark. Then the diluted FBS solution was transferred to a Microcon centrifugal filter device (MWCO 10 KDa) to remove GuCl and excess DTT and IAA. The buffer exchange step was repeated six times. The volume of the retentate was maintained at 200 μL. Finally, trypsin was added to the reduced and alkylated FBS with a protein-to-trypsin ratio of 100:1, followed by incubation overnight at room temperature. In order to ensure maximum proteins digestion, a second aliquot of trypsin was added to the serum solution with the same protein-to-trypsin ratio, with additional incubation at room temperature for 8 h. About 100 μg of T0, T5, and T8 was prepared by the same procedure of unfractionated FBS fraction digestion.

Trypsin Digestion of Enriched Low Molecular Weight FBS ProteinsFraction. One hundred micoliters of FBS-Ia, or FBS-Ib, or FBS-II sample was denatured with 6 M GuCl in 100 mM ammonium bicarbonate buffer, pH = 8.20, then reduced with dl-dithiothreitol (DTT), and alkylated with iodoacetamide (IAA) as above. The FBS solution was transferred to a Microcon centrifugal filter device (MWCO 30 KDa) to collect LMW (<30 KDa) proteins. Approximately 400 μL out of 500 μL of alkylated FBS solution was collected. Then GuCl, DTT, and IAA in the filtrate were removed by ultrafiltration with a Microcon centrifugal filter device (MWCO 3 KDa). The desalting step was repeated six times. The volume of concentrate was maintained at 200 μL. The protein concentration of desalted LMW proteins was measured by bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL). Then trypsin was added to the desalted LMW FBS with a protein-to-trypsin ratio of 100:1 twice as for trypsin digestion of unfractionated serum.

LC-MS/MS Analysis. NanoLC-MS/MS analysis was performed on an Ettan MDLC system (GE Healthcare, Piscataway, NJ) and LTQ mass spectrometer (ThermoFinnigan, San Jose, CA). The capillary column (150 mm × 0.075 mm) used for all LC-MS/MS analyses was from New Objective (Woburn, MA) and was slurry-packed in house with 5 μm, 200 Å pore size magic C18 stationery phase (Michrom Bioresources, Auburn, CA). The LC mobile phase A was 0.1% formic acid in water. The mobile phase B for LC was 0.1% formic acid in acetonitrile. Approximately 2 μg of each sample was loaded onto a Peptide Captrap column (Michrom Bioresources, Auburn, CA) at a flow rate of 10,000 nL/min in 2 min. Sample desalting was achieved by washing the Peptide Captrap column at the same flow rate (10,000 nL/min) with 98% A for 5 min. Then the sample was eluted out of the Peptide Captrap column and directed to the capillary reverse phase column, where the sample was separated with a gradient. The gradient was programmed with a linear increase from 2% B to 40% B in 70 min and from 40% B to 90% B in 5 min. The gradient was maintained at 90% B for 15 min. The spray voltage was set at 1.8 kV, and the normalized collision energy was set at 35% for MS/MS. The temperature of the ion transfer tube was 185 °C. Data-dependent ion selection was performed by using the most abundant 8 ions from a full MS scan for MS/MS analysis. A precursor ion was excluded from further LTQ MS/MS analysis for 3 min if it was analyzed twice in 0.5 min. The exclusion list size was 200.

Data Processing and Analysis. Protein/peptide identifications were obtained through a database search against bovine proteomic database using the SEQUEST algorithm (Version C1) incorporated in Bioworks software (Version 3.1, ThermoFinnigan, San Jose, CA). The database used was extracted from nr database on 4/25/2005 (ftp://ftp.ncbi.nih.gov/blast.db/FASTA/). Creation of the subset database was achieved by extracting proteins that contain the words bovine and/or bos taurus in their description line in the nr database. Raw data from the digest of media supernatant was also searched against a human proteomic database to monitor the appearance of host cell proteins. The final subset database contained 3583 proteins. Among them, 30 proteins were growth factors or growth factor binding proteins. Search parameters used were as follows:  trypsin, static modification of 57.0215 on cysteine residues, peptide mass tolerance was 1.5, fragment ion mass tolerance was 0.0, and the number of results scored was 250. The search was limited to tryptic peptides. For a protein to be considered a potentially positive identification, its unified score was at least 4000 (9).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References and Notes

In this study, we have chosen adult cultured retinal pigment epithelial cells (ARPE) as a model system. Fetal bovine serum (FBS) was used as a supplement to the growth media used in the cell culture, and three lots from different manufacturers were examined in terms of their ability to promote effective growth. The growth properties of the FBS lots were examined in replicate fermentation studies. Figure 1 shows a comparison of two FBS lots used in the culture of ARPE cells (FBS-Ia and FBS-II). It can be seen that in this comparison FBS-II gave significantly increased growth rates after a 1 day period. For comparative purposes, another lot of FBS (FBS-Ib) was also observed to give low growth rates similar to those of FBS-Ia. The protein concentration was also measured and found to be similar for all lots (see Table 1).

Table Table 1. Protein Concentrationa of Each Lot of FBS and Yield of LMW Proteins Enriched by Ultrafiltration
 lot of FBS
 FBS- IaFBS- IbFBS-II
  1. a Concentration was determined by bicichoninic acid (BCA) protein assay kit with a BSA standard.b A <30,000 Da MWCO membrane was used to enrich low molecular weight proteins.

supplierAAB
number of proteins identified in FBS799091
amount of total protein from 100 μL of FBS (μg)3,2003,9004,200
amount of LMWb proteins/peptides enriched from 100 μL of FBS (μg)343840
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Figure Figure 1. Growth of ARPE-19 cells varied with three serum lots from different manufacturers. (A) ARPE-19 cells grown in FBS-Ia at day 1 post-seeding. FBS-Ib showed a similar result. (B) ARPE-19 cells grown in FBS-II at day 1 post-seeding.

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In an effort to understand the different growth-promoting properties of these lots, we performed a proteomic analysis on the three different lots of FBS (Ia, Ib, and II). Also, to improve the sensitivity of the proteomic analysis and to examine lower molecular weight proteins and peptides, we repeated the proteomic analysis on a low molecular weight fraction (<30 KDa) prepared by a membrane-based ultra-centrifugation step (see Materials and Methods). Table 1 shows that based on the BCA protein assay this fractionation step depleted up to 99% of high abundance proteins, which dramatically decreased the complexity of fetal bovine serum. In addition, most growth factors and the corresponding binding proteins have a molecular weight less than the cutoff of the filter (30 KDa).

We also performed a more detailed proteomic analysis on the cell-culture media from cells grown in FBS II. Samples were collected at three different time-points (T0, control; T5, 5 days; T8, 8 days). The purpose of this analysis was to explore if the protein composition of the media could be used to monitor depletion of factors such as growth-promoting proteins, as well as the appearance of host cell proteins in the fermentation medium.

There are two common platforms for proteomic analysis, one is based on 2-D gel electrophoresis and the other is LC-MS, which is performed on a tryptic digest of the protein mixture (10). The advantage of the 2-D gel electrophoresis approach is that one can analyze expression patterns of intact proteins, but the method is time-consuming and also has a limited dynamic range. We therefore chose to use the LC-MS approach in our study of different FBS lots. As is discussed in the experimental section, we used conservative criteria for the matching of mass spectrometry fragmentation data with the theoretical spectra determined from a genomic database. The database used was based on bovine gene sequences that we derived from the nr database. The final database used in this study contained 3583 proteins including 30 proteins that were growth factor related proteins.

Analysis of Fetal Bovine Serum. Table 1 lists a relatively consistent total number of proteins identified from FBS-Ia, FBS-Ib, and FBS-II (79, 90, and 91 for the lots FBS-Ia, Ib, and II, respectively). The relatively low number of proteins identified in this assay is due to a number of factors such as conservative criteria used for the protein identification and the less well annotated nature of the bovine database relative to the human database. It can be seen that the number of protein identifications for the lot with superior growth properties was similar to the other lots (91 vs 90 and 79). Table 2 shows that the composition of the lots in terms of high abundance proteins, such as albumin, α-2-HS-glycoprotein, and α-1-antiproteinase, also could not be used to distinguish between growth properties of different lots. This is despite the fact that many of the proteins in this list have important growth-promoting and transport properties. For example, albumin is a well-defined component of human and other mammalian sera (11) and is the most abundant protein in all FBS lots (Table 2). It supports cell growth and protects materials from oxidation (12).

Table Table 2. Comparison of Abundant Proteins in FBS Identified by LC-MS
  approximate relative amountsa
IDproteinsFBS- IaFBS- IbFBS-II
  1. a Based on the number of unique peptide sequencing attempts, which is an approximate estimation of relative concentration (4).

ABBOSserum albumin676560
CNRCcone cGMP-specific 3′,5′-cyclic phosphodiesterase α-subunit251918
A1ATα-1-antiproteinase242927
PLMNplasminogen232022
PERLlactoperoxidase222416
KNL2kininogen, LMW II201612
NUAMNADH-ubiquinone oxido- reductase 75201916
A2HSα-2-HS-glycoprotein191816
A60166hemiferrin171713
THRBprothrombin171215
I45853apolipoprotein A-I16204
AMBPα-1-1-microglobulin and inter- α-trypsin inhibitor light chain13117
ANT3antithrombin-III121017
ITB1integrin β-1111719
APOHβ-2-glycoprotein I101312
A2APα-2-antiplasmin10149
HBBFhemoglobin beta fetal chain81011
APA2apolipoprotein A-II676
HABOhemoglobin α chain676
S66289α 1 antichymotrypsin5109

We therefore turned our attention to the analysis of growth factors and related proteins as indicators of the suitability of different lots. These proteins are at much lower levels than the components listed in Table 2, but as shown in Table 3 several could be detected in the LC-MS analyses. To assess variability, each trypsin digestion was performed three times. In the case of single peptide identifications we required that such identifications were made consistently in replicate analyses (typically triplicate), and the MS/MS spectra were manually interpreted for consistency with established fragmentation processes. As will be described later these identifications were confirmed with the analysis of the low MW fraction. On the basis of our LC-MS results, we found the most abundant factor was insulin-like growth factor-binding protein 2 (IGFBP2, Figure 2), and it was found in each FBS lot. In fact IGFBP2 and IGF II were the only growth factor related proteins identified in the lots that promoted less growth (FBS-Ia and FBS-Ib). In addition, transforming growth factor β 1 (TGF-β 1) and glial growth factor (GGF) were identified in FBS-II (Table 4). This initial analysis indicated that proteomics has potential to distinguish between different lots of growth media.

Table Table 3. Summary of Growth Factors and Related Binding Proteins Identified by LC-MS in Unfractioned FBS and the Corresponding LMW Fractions
   no. of unique peptides
approachprotein IDprotein referenceFBS-IaFBS-IbFBS-II
  1. a Not detected.

1 (unfractionated sample)TGF1transforming growth factor β 1aa2
 GGFglial growth factoraa1
 IGFBP2insulin-like growth factor-binding protein 2132
2 (LMW fraction)IGFIIinsulin-like growth factor-II334
 TGF1transforming growth factor β 1aa2
 GGFglial growth factoraa2
 CAA33746.1prepro-insulin-like growth factor Iaa2
 bFGFbasic fibroblast growth factoraa2
 IGFBP2insulin-like growth factor-binding protein 2335
 IGFBP4insulin-like growth factor-binding protein 4aa2
thumbnail image

Figure Figure 2. Example of MS/MS spectrum of identified peptide (R.GECWCVNPNTGK.L) from insulin-like growth factor-binding protein 2.

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The bovine database that we used in this study contained 30 proteins that were growth factor binding proteins, but the complexity of the media prevented the observation of more than three factors in the LC-MS analysis. We found, however, that 24 growth factors have molecular weights between 3 and 30 KDa, and this lead us to develop a depletion step using a molecular weight cutoff membrane (MWCO 30 KDa) to deplete high molecular weight proteins (13). The success of this approach was shown by the approximate doubling of the number of proteins identified in each of the FBS lots (data not shown). High molecular weight proteins such as albumin, plasminogen, and apolipoprotein A-I were reduced. The number of peptide sequencing events for albumin decreased from 60 to 15. In addition, this approach gave a significant increase in the number identifications of growth factors and other low level proteins (see Table 4 and the specific example of IGF II in Figure 3). Growth factors found in the original LC-MS serum analysis were identified again with the molecular weight (MW)-based filtration step but with increased sequence coverage. In FBS-II, IGFBP-2 was originally identified with two unique peptides in serum, and five unique peptides were successfully sequenced in the LMW fraction. The qualitative difference between FBS lot II and lots Ia and Ib was confirmed and strengthened in the analysis of the low MW pool. As shown in Table 3, five growth factor related proteins were identified only in FBS-II, such as prepro-insulin-like growth factor and IGFBP-4. For example, prepro-insulin-like growth factor is structurally and functionally related to insulin but has a much higher growth-promoting activity (14). Additional information on the importance of these factors will be described in the Discussion section.

Table Table 4. Analysis of Growth Factors and Related Binding Proteins Present in the Cell Culture Supernatanta
  no. of unique peptides
protein IDprotein referenceT0T5T8
  1. a Samples were taken over three time-points (T0, control; T5, 5 days; T8, 8 days.b Not detected.

IGFBP2insulin-like growth factor-binding protein 2211
TGF1transforming growth factor β 1211
bFGFbasic fibroblast growth factor111
GGFglial growth factor1bb
IBP4insulin-like growth factor-binding protein 41bb
CAA33746.1prepro-insulin-like growth factor I1b 
thumbnail image

Figure Figure 3. Example of MS/MS spectrum of identified peptide (-.AYRPSETLCGGELVDTLQFVCGDR.G) from insulin-like growth factor II.

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Analysis of Cell Culture Media Supernatant. In addition to the analysis of FBS as a raw material, we also analyzed the fermentation medium for consumption of key bovine components that are related to cell growth and spreading. Another potential benefit of such an analysis was the ability to detect the appearance of host cell proteins in the fermentation media, which may be used to monitor growth. In this study, proteomic analysis by LC-MS was used to monitor a cell culture medium that had been supplemented with 2% fetal bovine serum. Proteomic analysis was carried out on T0 (control), T5 (5 days), and T8 (8 days). The relative abundance changes of proteins were initially measured using differences in peptide sequencing events as described in the Materials and Methods section. In selected examples differential abundance changes were measured by comparing the peak area ratios in the extracted ion chromatogram. These ions were selected with a mass window of 2 Da and with a retention time window of 0.5 min. In this study the average coefficient of variance (CV) for variation in retention time from five different ions was 0.15% in a triplicate analysis. Figure 4 shows the base peak chromatograms of the tryptic digests of the supernatant collected at T0, T5, and T8 time points. As shown in Table 4, the number of FBS related growth factors decreased over the period of the fermentation, which provides further evidence that these components were associated with cell growth. Initially six growth factor related proteins were identified, three in T5 samples and three in the final time point. For example, one could expect that the abundance of IGFBP-2 would be decreased during cell culture because IGFBP-2 stimulates cell spreading and over time it would be expected to be utilized.

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Figure Figure 4. Example of extracted ion chromatogram of two peptides. (A) The peptide (R.EGAPGAEGSPGR.D) was derived from collagen α 1(I) over three time-points (T0, control; T5, 5 days; T8, 8 days). The peak area of this peptide was increased over time, indicating that the amount of collagen α 1(I) was increasing. (B) The peptide (R.VPCELVR.E) was derived from insulin-like growth factor binding protein 2 over 3 time-points (T0, control; T5, 5 days; T8, 8 days). The peak area of this peptide was decreased over time, indicating the amount of insulin-like growth factor binding protein 2 was decreasing.

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In addition, markers for cellular growth such as the collagen α 1(I) chain were increased in the culture medium overtime. The number of identified peptides of collagen α 1(I) was 0 in T0, 0 again in T5, and 10 in T8. Collagen α 1(I) chain is part of the extracellular matrix and is synthesized during cell growth. Collagen α 2 (I), thrombospondin-1, and actin are also part of extracellular matrix structural constituent and were similarly increased as shown in Table 5 (15).

Table Table 5. Presence of Structural Proteins from RPE Cells in the Cell Culture Supernatant at Different Time Pointsa
 no. of unique peptides
protein referenceT0T5T8
  1. aT0, control; T5, 5 days; T8, 8 days.b Not detected.

fibronectinb123
collagen α 2(I) chainb211
collagen α 1(I) chainbb10
keratin, type II cytoskeletal 1227
keratin, type I cytoskeletal 91b7
SPARC (secreted protein acidic and rich in cysteine)bb5
thrombospondin-1114
keratin, type I cytoskeletal 10113
actin, α cardiac112

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References and Notes

Recent developments in the biotechnology industry have resulted in the manufacture of ever-more-complex protein pharmaceuticals by a variety of different fermentation processes and novel cell-lines. In addition, the process development is often challenged to establish well-defined manufacturing processes that may be carried out at multiple manufacturing sites and even on an international basis. It is clear that in this context proteomics has much to offer to the biotechnology industry as a new approach to monitor a wide range of steps in the production processes. Recently we published a study in which proteomics was used to better understand the growth of E.coli on glucose in high density, fed-batch cultures and response to the overexpression of plasmid-encoded 6-phosphogluconolactonase (PGL) (1). Measurement of relative abundance changes in significant cellular proteins showed up-regulation of key enzymes in the citric acid cycle that was consistent with observed changes in the cell culture. The study also confirmed the importance of the pentose-phosphate shunt for high yield protein biosynthesis.

In a similar manner other issues related to a well-regulated manufacturing process can also profit from a proteomic analysis. The advantage of a proteomic analysis is that one can achieve a more global view of protein expression levels in a fermentation or cell culture system. In addition, however, proteomics can be used to examine the purity and effectiveness of raw materials used in the fermentation process. It has been noted that materials such as primatone are often poorly defined complex biological samples (16). In this context, one issue about the production of biologic pharmaceuticals that is particularly troubling is the inability for a process development scientist to effectively assay the effectiveness of different media supplements.

It is of importance to the biotechnology industry to better understand the composition of growth supplements such as fetal bovine serum. In addition to safety considerations caused by the presence of adventitious biological agents, the cost effectiveness of the fermentation components as well as the product yield and throughput are important economic considerations. With the sequencing of the human genome there is now a significant increase in the understanding of the biological complexity of mammalian extracts. We will therefore briefly discuss the biology of the growth factors reported in this study in the context of the bioprocess.

Growth factors are proteins that bind to receptors on the cell surface, with the primary result of activating cellular proliferation and/or differentiation (17). Many growth factors are quite versatile, stimulating cellular division in different cell types, whereas others are specific to a particular cell type. The insulin-like growth factors possess growth-promoting activity, and in vitro they are potent mitogens for cultured cells. In this study IGF-II was observed with several peptides after the ultrafiltration step, but we did not observe IGF-I, which is present at lower levels than IGF-II in fetal bovine serum (18).

The corresponding binding proteins (IGFBPs) hold a central position in IGF ligand−receptor interactions through influences on both the bioavailability and distribution of IGFs in the extracellular environment (19). These proteins belong to the superfamily of proteins that bind the insulin-like growth factors, IGF-I and IGF-II. The superfamily has six high affinity members, IGFBP-1, −2, −3, −4, −5, and −6, and four low affinity members, IGFBP-7, −8, −9, and −10 (20). In addition IGFBP-1 and IGFBP-2 can stimulate cell spreading through an action mediated by integrins (21). In this analysis IGFBP2 was found in all samples and with several peptides, which indicated relatively high abundance (see Table 4). Also the related protein IBGFBP4 was found in FBS-II after the ultrafiltration step. These members prolong the half-life of IGF-I and IGF-II by modulating interactions with cell surface receptors. In this manner one could either inhibit or stimulate the growth-promoting effects of the IGFs on cell culture and perhaps regulate cellular proliferation and migration.

The proteomic analysis also characterized several other growth factors that are important to cellular growth, as will be summarized in the following text. Transforming growth factor β 1 (TGF-β) is a multifunctional peptide that controls proliferation, differentiation, and other functions in a variety of cell types. Many cells synthesize TGF-β and have specific receptors for this peptide (22). TGF-β regulates the actions of many other peptide growth factors and determines a positive or negative direction for their effects. Glial growth factor (GGF) is one isoform of neuregulin 1 (NRG1), which was originally identified as a 4-kD glycoprotein that interacts with the NEU/ERBB2 receptor tyrosine kinase. The receptors for all NRG1 isoforms are the ERBB family of tyrosine kinase transmembrane receptors. Through interaction with ERBB receptors, NRG1 isoforms induce the growth and differentiation of epithelial, neuronal, glial, and other types of cells (23). We also identified basic fibroblast growth factor (FGF), which is one of the heparin-binding growth factors and is a potent mitogen for a variety of cell types in vitro. This protein binds heparin more strongly than does acidic fibroblast growth factor. Basic fibroblast growth factor is known to be important in cell culture because it may locally regulate cell growth and differentiation during angiogenesis (24).

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References and Notes

Using LC-MS/MS, we identified approximately 80 proteins from each fetal bovine serum lot by capillary HPLC-MS analysis of the tryptic digests. The employment of an ultrafiltration step to select proteins of less than 30 kDa greatly improved the dynamic range of the analysis and allowed the characterization of approximately 150 proteins. We found that the identity and relative amounts of high abundance proteins were consistent and did not discriminate between lots that exhibited good and poor growth properties. With the ability to characterize lower level proteins by our LC-MS system, we found that the serum lot with the highest growth rate (FBS-II) contained additional growth factors and related binding proteins that were not found in the other two serum lots (FBS-Ia and FBS-Ib). We also analyzed the change in relative amounts of growth factors and major host cell proteins in the cell culture supernatant. Based on differential analysis, the amount of several structural proteins such as collagen α 1(I) was increased over time, whereas the amount of growth factors such as IGFBP-2 decreased. This study has indicated the potential of proteomics for improved characterization of bioprocess manufacturing such as in the testing of raw materials and for monitoring of cell culture processes.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References and Notes

The authors want to thank GE Healthcare, ThermoFinnigan, and Berlex Biosciences for support of this work and Dr. Marina Hincapie for her helpful suggestions. Barnett Institute contribution number is 879.

References and Notes

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References and Notes
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