Usability of size-excluded fractions of soy protein hydrolysates for growth and viability of Chinese hamster ovary cells in protein-free suspension culture

To investigate the effect of size-excluded fraction of non-animal protein hydrolysate on growth, viability and longevity of Chinese hamster ovary (CHO) cells, several commercially available protein hydrolysates were evaluated as a feed supplement to chemically-defined protein-free suspension culture. Soy protein hydrolysates showed better supporting capability for cell growth and viability than the other types of hydrolysates. Maximal cell growth was not affected greatly by size exclusion of some soy hydrolysates such as bacto soytone and soy hydrolysates. CHO cells supplemented with size-excluded fractions of the two hydrolysates showed viable cell density and viability almost equal to those with their crude hydrolysates, although soy hydrolysates showed a little better performance. This suggested that the size-excluded hydrolysate fractions of some soy hydrolysate might be a potential culture medium additive to achieve better downstream operation in a large-scale production as well as enhanced productivity.     

适合棉铃虫细胞HzAm1生长的培养基筛选及低血清驯化

昆虫细胞-杆状病毒系统是昆虫杀虫剂生产和医用外源基因表达的有效工具。昆虫细胞的无血清或低血清培养是十分必要的。从三种商业化的培养基TC-100、GRACE和IPL-41中筛选出了最适合棉铃虫细胞HzAm1生长的基础培养基TC-100。以该培养基为基础,将血清用量从常用的10%降至1%,同时补加一定量的水解乳蛋白以及酵母提取物等,对棉铃虫细胞HzAm1进行驯化培养,效果良好。     

Influence of the rapeseed protein hydrolysis process on CHO cell growth

Different protein hydrolysates were prepared from enzymatic hydrolyses of a rapeseed isolate (>90% protein content) using different commercial enzymes of non-animal origin. The extent of hydrolysis was controlled to produce hydrolysates corresponding to various degrees of hydrolysis (DH) from 5 to 30. These hydrolysates were characterized according to their solubility and size peptide pattern. Different growth behaviours of Chinese Hamster Ovary cells were observed when these various hydrolysates were added in serum-free medium containing transferrin, albumin and insulin. Hydrolysates from low degree of hydrolysis generally did not exhibit significant positive effect on cell growth; conversely hydrolysates from extensive hydrolysis, corresponding to a major low molecular size peptides content, usually allowed an increase of the maximal cell density. However, depending on the enzyme used, the supplementation with hydrolysates corresponding to a high degree of hydrolysis and composed of at least 70% peptides with a molecular size under 1kDa, led to different maximal cell density values, indicating the importance of enzyme specificity and consequently the nature of the released peptides. This result showed that the positive influence of the rapeseed hydrolysates on cell growth was not only due to a nutritional support tied to the addition of small peptides but may be related to the presence of peptides exhibiting growth or survival factor effects. Furthermore, total substitution of proteins (transferrin, albumin and insulin) in the cell culture medium by some rapeseed hydrolysates appeared to be a promising alternative to improve the cell growth in protein-free media.     

Promoting effect of rapeseed proteins and peptides on Sf9 insect cell growth

The Baculovirus Expression Vector System has become widely used for the production of recombinant proteins for research and diagnostics. Serum-free culture media able to support high cell densities have been developed for the large scale culture of insect cells. While serum elimination aims at avoiding the risks associated with the introduction of an ill defined component of bovine origin, additives such as protein hydrolysates from animal sources are still used. An alternative could be the supplementation of culture media with protein hydrolysates derived from plants. In this study, we describe the replacement of lactalbumin hydrolysate with a laboratory produced hydrolysate of rapeseed proteins. Its effect on Sf9 cell growth kinetics, substrate consumption and by-product formation in low-serum or serum-free medium was evaluated. Cells were unable to grow in the presence of a rapeseed protein hydrolysate generated by PTN 3.0 Special^® enzyme and containing only 24% of peptides under 1 kDa in size. On the other hand, serum-free medium supplementation with a rapeseed protein hydrolysate obtained with Orientase 90N^® enzyme had a strong growth promoting effect, leading to a 60% increase in maximal cell density without affecting cell metabolism. This significant positive effect could be explained by the higher degree of hydrolysis of this digest, with 74% of peptides under 1 kDa in size.     

Development of serum-free medium supplemented with hydrolysates for the production of therapeutic antibodies in CHO cell cultures using design of experiments

An efficient method of formulating serum-free medium (SFM) for production of therapeutic antibodies by recombinant CHO (rCHO) cells was developed using two rCHO cell lines producing a therapeutic antibody. In this method, ten kinds of SFM were prepared by supplementing the basal SFM with statistically designed mixtures (total 5 g L^?1) of three non-animal-derived hydrolysates: yeastolate, soy hydrolysate, and wheat gluten hydrolysate. When the two rCHO cell lines were cultivated, the mixtures of soy hydrolysate and wheat gluten hydrolysate showed a positive effect on cell growth. On the other hand, the mixtures including a high portion of yeastolate significantly enhanced specific antibody productivity. To reconstitute the mixture ratios of the three hydrolysates for high growth and antibody production, the effect of each medium was analyzed by the statistical program Design-Expert®. The resulting medium gave a 1.9–3.3-fold increase in the maximum antibody concentration, compared to the basal SFM. Taken together, the supplementation of hydrolysates to the basal SFM with the help of statistical analysis is an efficient means of developing SFM for therapeutic antibody production by rCHO cells.     

Investigation of proteins and peptides from yeastolate and subsequent impurity testing of drug product

Hydrolysates play an important role in modern biological production. These mixtures are mostly undefined and contain a mixture of proteins, peptides, and amino acids along with other non–amino acid‐based components. Recently, there has been an interest in defining and sequencing proteins and peptides in these hydrolysates to subsequently develop an assay to ensure removal during product purification. This work investigates an ultrafiltrate of yeastolate to determine whether any protein is present. Size exclusion chromatography indicated a possible high molecular weight component (>10 kDa). This suspected high molecular weight fraction was collected and investigated. It was determined that this fraction consists of nucleic acids; and no protein was detected using sensitive modern techniques including HPLC, mass spectrometry, and SDS‐PAGE. Next, five unique, yeast‐specific peptides were identified, sequenced, and confirmed. Finally, an impurity assay for any residual yeast specific peptides was developed and the analytical metrics were determined including accuracy, precision, linearity, range, and limits of detection and quantitation. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Mosquito larvae soluble fractions induce DNA synthesis alterations on human mononuclear cells.

Mosquito larvae soluble fractions obtained by molecular exclusion chromatography altered the mitotic rate of several epithelial cell populations in hepatectomised mice, as well as the proliferation of human mononuclear cells (MNC), stimulating or inhibiting them depending on the fraction and dose applied. The effect was also thermolabile, suggesting a proteic nature of the compounds involved. Analysis of cell viability after culture indicated that the extract did not have lethal toxic effects. One fraction with a molecular weight ranging between 12-80 kDa caused only an inhibitory effect. In the present study, we performed further characterisation of this fraction by assaying the effect of new fractions obtained from this one, by the use of a column with a lower molecular weight exclusion range. Assays were performed on the proliferation of adult human MNCs. Our results showed that two out of four of the sub-fractions analysed, with a MW of about 70 and 17 kDa, caused a dose-dependent response, either inhibiting or stimulating MNC proliferation respectively.     

Development of a tissue cell culture bioassay for identifying mechanisms of inhibition of settlement of barnacle and tunicate larvae by surface‐colonizing marine bacteria

A marine bacterium D2 (CCUG 26757) isolated from a tunicate Ciona intestinalis specimen produced a low molecular weight component which inhibits barnacle and tunicate larvae and prevents their settlement on solid surfaces (Holmström et al., 1992). In order to perform chemical and structural analyses of the component independent of season, a bioassay, complementary to tests with invertebrate larvae was developed. This bioassay is based on tissue cell culture techniques and growth of the AGS cell line. It was previously shown that the toxic component is a stationary phase released product, which is heat stable and less than 500 Dalton in size (Holmström et al., 1992). Furthermore, it is not a peptide or a protein and metaperiodate treatment increases its toxicity to larvae indicating that it binds to or contains carbohydrate moieties. In this study, these results were confirmed by using the anchorage dependent human gastric adenocarcinoma (AGS) cell line as a bioassay. Fractionation of the D2 supernatant on a Sephadex G‐200 column and addition of different size fractions to the AGS tissue culture cells, showed that both a low and a high molecular weight fraction inhibited cell growth. Exposure of Balanus amphitrite and C. intestinalis larvae to the same fractions, showed that the low molecular weight fraction that inhibited growth of the cells corresponds to the component that inhibited larvae of both organisms. The high molecular weight fraction was found to inhibit larvae of B. amphitrite.     

A cytotoxic substance produced by a wild culture of Lactobacillus casei D-34 against tumour cells

A wild lactic culture isolated from dahi (fermented milk) sample and characterised as L. casei D-34 was found to be significantly cytotoxic (34-36%) against three tumour cell lines, HeLa, HEp-2 and HFS-9. The cytotoxic substance (CS) was found to be in the culture supernatants, protein in nature, with a molecular weight ranging from 17,000-20,000. The crude culture supernatant was partially purified by dialysis and ion exchange chromatography as anionic, cationic and neutral fractions. Among the fractions, except for the anionic fraction, others were found to be highly cytotoxic against all three tumour cell lines. The cationic, neutral and pooled (anionic:cationic:neutral in 1:1:1 ratio) fractions showed 50, 70, 70% cytotoxicity against Hep-2 cells, 70, 88, 94% against HFS-9 cells and 50, 89, 90% against HeLa cells respectively. Pooled fraction was found to exhibit higher percent of cytotoxicity compared to individual fractions indicating a synergistic effect. (3H)-thymidine incorporation studies revealed that CS and its fractions inhibited DNA synthesis in tumour cells. The CS was stable towards heat and pH changes.     

Study of Glial Fibrillary Acidic Protein in a Human Glioma Cell Line Grown in Culture and as a Solid Tumor

Abstract: A continuous human glioma cell line grown in culture and as a solid tumor was analyzed for glial fibrillary acidic (GFA) protein. This material provided a rich source for GFA protein that could also be manipulated and controlled. Immunoperoxidase staining at the light and electron microscopic levels revealed that the cell culture and tumor specimens were strongly positive for GFA protein. When aqueous soluble fractions of the cell culture and tumor were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, electroblotted onto nitrocellulose and stained immunochemically, they contained exclusively low molecular weight (41–43 K-dalton) GFA peptides. SDS (0.15%)-soluble fractions contained either low molecular weight only (culture) or a mixture of peptides ranging from 41 to 49K daltons. SDS (1%) extracts of either cell culture or tumor contained only 49K dalton GFA protein. Two-dimensional gel separation revealed that the GFA protein extracted from either the culture or tumor with 1% SDS resolved to two or three spots at pH 5.8. Low molecular weight GFA peptides (<49K daltons) in aqueous and 0.15% SDS-soluble extracts became increasingly more acidic with decreasing molecular weight. The extremely rapid degradation seen suggests that this cell line may be a valuable system for further study of intermediate filament protein turnover.     

Characterization of yeastolate fractions that promote insect cell growth and recombinant protein production

Yeastolate is effective in promoting growth of insect cell and enhancing production of recombinant protein, thus it is a key component in formulating serum-free medium for insect cell culture. However, yeastolate is a complex mixture and identification of the constituents responsible for cell growth promotion has not yet been achieved. This study used sequential ethanol precipitation to fractionate yeastolate ultrafiltrate (YUF) into six fractions (F1–F6). Fractions were characterized and evaluated for their growth promoting activities. Fraction F1 was obtained by 65% ethanol precipitation. When supplemented to IPL-41 medium at a concentration of 1 g L⁻¹, fraction F1 showed 71% Sf-9 cell growth improvement and 22% β-galactosidase production enhancement over YUF (at 1 g L⁻¹ in IPL-41 medium). However, the superiority of F1 over YUF on promoting cell growth gradually diminished as its concentration in IPL-41 medium increased. At 4 g L⁻¹, the relative activity of F1 was 93% whereas YUF was 100% at the same concentration. At 1 g L⁻¹, four other fractions (F2–F5) precipitated with higher ethanol concentrations and F6, the final supernatant, showed growth promoting activities ranging from 32 to 80% as compared to YUF (100%). Interestingly, a synergistic effect on promoting cell growth was observed when F6 was supplemented in IPL-41 medium in presence of high concentrations of F1 (>3 g L⁻¹). The results suggest that ethanol precipitation was a practical method to fractionate growth-promoting components from YUF, but more than one components contributed to the optimum growth of Sf-9 cells. Further fractionation, isolation and identification of individual active components would be needed to better understand the role of these components on the cell metabolism.     

Isolation from lactalbumin hydrolysate of a high molecular weight mitogenic factor.

A new mitogenic factor has been isolated from tissue culture grade lactalbumin hydrolysate. Incubation of postconfluent Swiss 3T3 cells in serum-free medium containing lactalbumin hydrolysate resulted in enhanced synthesis and release of plasminogen activator. Sephadex G-100 gel filtration of concentrated, dialyzed lactalbumin hydrolysate revealed two fractions, LH-FI and LH-FII. LH-FI elutes at the void volume, indicative of high molecular weight, and contains all plasminogen activator stimulatory activity of the original lactalbumin hydrolysate, whereas LH-FII has no activity. In addition, LH-FI also induces DNA synthesis in Swiss 3T3 cells in a dose-dependent fashion, 1 to 2 microgram/ml being equivalent to 10% fetal calf serum. Again, LH-FII is without effect. Induction of DNA synthesis in LH-FI or serum-stimulated quiescent sparse cells followed essentially identical kinetics at least through the first 20 h. Furthermore, LH-FI also enhances 3T3 cell growth. Preliminary results of Sepharose 4B filtration of LH-FI reveal the presence of five subfractions each with plasminogen activator stimulatory as well as mitogenic activity.     

Submerged monoxenic culture medium development for Heterorhabditis bacteriophora and its symbiotic bacterium Photorhabdus luminescens: protein sources

Most medium formulations for improving culture of entomopathogenic nematodes (EPN) based on protein sources have used enriched media like animal feed such as dried egg yolk, lactalbumin, and liver extract, among other ingredients. Most results, however, showed unstable yields and longer production time. Many of the results do not show the detailed parameters of fermentation. Soy flour, cotton seed flour, corn gluten meal, casein powder, soytone, peptone, casein hydrolysates, and lactalbumin hydrolysate as protein sources were tested to determine the source to support optimal symbiotic bacteria and nematode growth. The protein hydrolysates selected did not improve bacterial cell mass compared with the yeast extract control, but soy flour was the best, showing 75.1% recovery and producing more bacterial cell number (1.4×10?/ml) than all other sources. The highest yield (1.85×10? IJs/ml), yield coefficient (1.67×10? IJs/g medium), and productivity (1.32×10? IJs/l/day) were also achieved at enriched medium with soybean protein.     

Plant protein hydrolysates: preparation of defined peptide fractions promoting growth and production in animal cells cultures

A new approach was applied with the aim at producing plant protein hydrolysates less heterogeneous and less contaminated with nonpeptide substances than are the presently available digests. A significant reduction of nonprotein contaminants was achieved by extraction of the plant material, soy flour or wheat flour, with acetone prior to isolation of the protein. Enzymes of nonanimal origin, papain or Pronase, were used for protein hydrolysis. The components of the hydrolysates were resolved by low-pressure liquid chromatography. Separation of peptide fractions and of remaining nonpeptide contaminants was achieved using small-pore size-exclusion chromatography matrices, Sephadex G-15 or Biogel P-2. Individual peptide fractions, both from soy protein and from wheat gluten, varied substantially in their growth-promoting and production-enhancing activities when tested on a mouse hybridoma culture in protein-free medium. The highest enhancement of viable cell density in batch cultures was 180% of control, and the highest enhancement of final immunoglobulin concentration was more than 230% of control. The existence of marked differences in activity of individual peptide fractions leads to a suggestion that the hydrolysates may provide peptides exerting specific positive effects on cultured animal cells.     

Design of an efficient medium for insect cell growth and recombinant protein production

We report the development of a new serum-free medium based on the use of factorial experiments. At first, a variety of hydrolysates were screened using a fractional factorial approach with High-Five cells. From this experiment yeastolate ultrafiltrate was found to have, by far, the most important effect on cell growth. Furthermore, Primatone RL was found to remarkably prolong the stationary phase of Sf-9 and High-Five cell cultures. The optimal concentrations for yeastolate and Primatone were determined to be 0.6 and 0.5%, respectively, on the basis of a complete factorial experiment. This new medium, called YPR, supported good growth of both Sf-9 and High-Five cells in batch cultures, with maximal densities of 5.4 and 6.1 x 10(6) cells/ml, respectively. In addition, both cell lines achieved good growth in bioreactor batch culture and had a prolonged stationary phase of 3-4 d in YPR medium compared to Insect-XPRESS medium. The ability of the new medium to support recombinant protein expression was also tested by infecting Sf-9 or High-Five cells at high density (2 x 10(6) cells/ml) with a baculovirus expressing secreted placental alkaline phosphatase (SEAP). The maximum total SEAP concentration after 7 d was about 43 lU/ml (58 mg/L) and 28 lU/ml (39 mg/L) for High-Five and Sf-9 cells, respectively.     

Purification of a nuclear trans-acting factor involved in the regulated transcription of a human immunoglobulin heavy chain gene

The expression of the rearranged human immunoglobulin gamma 1 heavy chain gene (HIG1) was shown to be induced through its enhancer by the positive regulatory trans-acting factor(s) that was contained only in cells of B lineage. The trans-acting factors were purified from mouse myeloma NS1 cells, and HIG1-inducing activity was found mainly in fractions of molecular weight 53-127 kd and in a fraction eluted from a heparin-Sepharose column with 0.5 M KCI. This semipurified fraction contained proteins binding to the conserved octamer sequence, ATGCAAAT, in the promoter region, as well as to sequences in the enhancer region. The 0.5 M KCI eluates from a heparin-Sepharose column were applied to a DNA affinity column of synthetic oligonucleotides of the octamer sequence and the sequence TATTTTAGGAAGCAAA in the HpaII-BgIII region of the HIG1 gene enhancer. The protein eluted from the enhancer sequence-specific DNA affinity column showed a strong inducing activity for the HIG1 gene, and the molecular weight of a predominant protein was 96 kd.     

Growth promotion by yeastolate and related components on insect cells

In this study, we tested a number of defined and undefined compounds in comparison with yeastolate in Sf-9 insect cell culture including a group of B vitamins and a series of purines and pyrimidines. Yeastolate promoted the cell growth appreciably, increasing the maximum cell density by more than 60%. Vitamins and purines, which are considered as the major constituents of yeastolate, are not responsible for its growth-promoting effect.     

Growth inhibitors in plasma derived human serum

Summary It was reported previously that plasma derived human serum (PDS) inhibited the growth of cells established from malignant human breast tissues and the MCF-7 cell line but did not inhibit the growth of cells from nonmalignant mammary tissues, including the HBL-100 cell line. Plasma derived human serum was fractionated in the current study by molecular sieve chromatography on Sephadex G-100 in an effort to characterize the factor(s) responsible for inhibition. Plasma derived human serum contained several growth inhibitory fractions, which were designated G-1, G-2, G-3, and G-4. The G-1 was associated with the lipoproteins and immunoglobulins of serum. The lipid portion of G-1 inhibited the growth of both MCF-7 and HBL-100 cells, whereas the protein fraction contained a low activity factor directed against MCF-7 cells only. The G-2 also inhibited MCF-7 cell growth at a low specific activity and was separated in the serum albumin fraction. The MCF-7 inhibitory activity in the G-3 fractions from individual donors fluctuated with the level of activity in the starting sera. The cell specific G-3 components were purified further by Sephadex G-100 superfine chromatography and gel electrophoresis. A tentative molecular weight of 50,000 was assigned to the G-3 inhibitor. The G-4 fraction consisted of small molecular weight materials migrating in advance of the column volume, which inhibited the growth of both cell lines. This investigation was supported by Grant PDT-140 from the American Cancer Society, Inc., and PHS Grant CA30284 awarded by the National Cancer Institute, Bethesda, Maryland.     

Partial characterization of mosquito larvae extract inducing dna synthesis alterations on human mononuclear cells

A crude mosquito larvae and dialysed extract alters the mitotic rate of several epithelial cell populations in normal young and adult hepatectomized mice. A crude extract also showed a biphasic effect on the proliferation of human mononuclear cells (MNCs), either stimulating or inhibiting them depending on the dose applied. In the present paper, we assayed the effect of the dialysed mosquito larvae extract and two different protein fractions on human MNCs. Analysis of cell viability after culture indicated that the extract did not have toxic effects. Our results show a dual response of the MNCs to the dialysed, as well as to the protein fraction, with the highest molecular weight inhibiting or stimulating proliferation, depending on the dose applied. The protein fraction with the lowest molecular weight (range between 12-80 kDa) showed only an inhibitory effect on cell proliferation.     

The bioactivity and fractionation of peptide hydrolysates in cultures of CHO cells

Peptide hydrolysate supplements in mammalian cell cultures provide enhanced growth and productivity. The objective of this study was to compare the bioactivity of ten different commercially available hydrolysates from plant, microbial, and animal sources. The peptide hydrolysates were tested as supplements to cultures of Chinese hamster ovary (CHO) cells that produce human beta interferon (β‐IFN). A soy hydrolysate was shown to support high cell growth but not protein productivity compared to an animal component hydrolysate (Primatone RL). On the other hand, a yeast hydrolysate showed lower cell growth, but comparable productivity of the recombinant protein. Glycosylation analysis showed that the glycan profile of β‐IFN produced in yeast hydrolysate supplemented cultures was equivalent to that from Primatone RL‐supplemented cultures. Fractionation of the yeast hydrolysate and Primatone RL produced a similar protein‐assayed pattern except for one extra peak at around 1 kDa in the Primatone RL profile. A fraction taken at a molecular weight range of 1.5–1.7 kDa showed the highest growth promoting activity in both samples. However, four other fractions in yeast hydrolysate and two in Primatone RL at lower molecular weights showed some growth promoting activity. In conclusion, the yeast hydrolysates provided a good alternative to the animal sourced Primatone RL for high productivity of β‐IFN from CHO cells with equivalent glycosylation. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:584–593, 2014