Factors Causing Compositional Changes in Soy Protein Hydrolysates

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Factors Causing Compositional Changes in Soy Protein Hydrolysates and the Effects on Cell Culture Functionality Abhishek J. Gupta, Harry Gruppen, Dominick Maes, Jan-Willem Boots, and Peter A. Wierenga J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 12 Oct 2013 Downloaded from http://pubs.acs.org on October 13, 2013

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Journal of Agricultural and Food Chemistry

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Factors Causing Compositional Changes in Soy Protein Hydrolysates and the

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Effects on Cell Culture Functionality

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Abhishek J. Gupta

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Wierenga 1*

1, 2

, Harry Gruppen 1, Dominick Maes 2, Jan-Willem Boots 2, Peter A.

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1

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The Netherlands

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2

Wageningen University, Laboratory of Food Chemistry, P.O. Box 17, 6700 AA Wageningen,

FrieslandCampina Domo, P.O. Box 1551, 3800 BN Amersfoort, The Netherlands

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11

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13

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* Corresponding author:

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Dr. ir. Peter A. Wierenga

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Wageningen University, Laboratory of Food Chemistry, P.O. Box 17, 6700 AA Wageningen,

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The Netherlands

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Email: [email protected]; Tel: +31 317 483 786

19 1

ACS Paragon Plus Environment


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Abstract

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Soy protein hydrolysates significantly enhance cell growth and recombinant protein production in

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cell cultures. The extent of this enhancement in cell growth and IgG production is known to vary

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from batch-to-batch. This can be due to differences in the abundance of different classes of

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compounds (e.g. peptide content), the quality of these compounds (e.g. glycated peptides), or the

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presence of specific compounds (e.g. furosine). These quantitative and qualitative differences

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between batches of hydrolysates result from variation in the seed composition and seed/ meal

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processing. Although a considerable amount of literature is available which describes these

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factors, this knowledge has not been combined in an overview yet. The aim of this review is to

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identify the most dominant factors that affect hydrolysate composition and functionality. While

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there is a limited influence of variation in the seed composition, the overview shows that the

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qualitative changes in hydrolysate composition result in the formation of minor compounds (e.g.

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Maillard reaction products). In pure systems, these compounds have a profound effect on the cell

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culture functionality. This suggests that presence of these compounds in soy protein hydrolysates

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may affect hydrolysate functionality as well. This influence on the functionality can be of direct

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or indirect nature. For instance, some minor compounds (e.g. maillard reaction products) are

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cytotoxic; while other compounds (e.g. phytates) suppress protein hydrolysis during hydrolysate

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production resulting in altered peptide composition, and thus, affect the functionality.

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Keywords

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Meal Processing. Soybeans.

Enzymatic hydrolysis. Compositional changes. Maillard reaction. Racemization.

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Introduction

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The performance of a mammalian cell culture system is primarily determined by the product

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yield (production of recombinant proteins, such as immunoglobulins (IgG) and interferon γ) and

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product quality (glycosylation of recombinant proteins).1 In many cell culture studies, viable cell

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growth is used as a second parameter to describe the cell culture performance.1, 2 This is because

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the recombinant protein production can be affected by cell growth. In some studies, recombinant

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protein production has been reported to increase with an increase in cell growth,3 while in other

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studies, recombinant protein production increased following a suppression in the cell growth.4

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To improve the performance of mammalian cell culture processes, fetal calf serum has been used

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as a supplement for several decades. This supplement provides growth factors, proteins, lipids,

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attachment factors, minerals, hormones, and several trace elements that are important for

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promoting cell growth and enhancing recombinant protein production.1,

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fetal calf serum has become restricted due to the risk of transmissible diseases like bovine

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spongiform encephalopathy. As a result, substantial research has been performed to identify

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alternatives for fetal calf serum. Currently, two alternatives have been described in literature: 1)

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to use chemically defined medium,6-9 or 2) Supplement the basal chemically defined medium

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with plant protein hydrolysates.10-13 The latter approach has become a common practice in the

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biopharma industry. In table 1, an overview of the influence of soy protein hydrolysate

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supplementation to chemically defined medium on cell growth and recombinant protein

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production is provided. In this overview, the cell growth and recombinant protein production for

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chemically defined medium is set to 100%. The cell growth and IgG production in hydrolysate-

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supplemented chemically defined medium ranged from 90-178% and 95-300%, respectively as

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compared to that in the chemically defined medium (100%) (Table 1).

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However, the use of

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Commercially, plant protein hydrolysates from several sources such as soy, cotton, rice, wheat

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gluten, pea, and rapeseed proteins are available for cell culture applications.14-16 The cell growth

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of the Chinese hamster ovary (CHO, CHO DG44/ dhfr-/-) cells cultivated in HyQ® CDM4-CHO

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medium supplemented with 16 hydrolysates obtained from different sources varied from 106% to

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144% relative to that in the chemically defined medium (100%).2 Although, the enhancement in

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cell growth in culture supplemented with soy protein hydrolysate was the highest, the reason why

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soy protein hydrolysates performed better than other hydrolysates were not explained.

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Several cell culture studies have been performed, in which various cell lines (e.g. CHO 320, ME

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750, and WuT3),11,

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IMDM:Ham’s F12:NCTC, and 2:1:1 (v/v) of DMEM:F12:RPMI)7, 9, 10 supplemented with soy

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protein hydrolysates10,

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comparison of results obtained in these studies is limited, because these chemically defined

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media and hydrolysates have different chemical compositions and different cell lines have

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diverse nutritional requirements.1 This comparison is further complicated by the fact that

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although, the chemically defined media to which hydrolysates are supplemented are called

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‘chemically defined’, an exact definition, or composition of such a medium is not reported in the

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scientific literature. This lack of comparability limits the understanding of the role of soy protein

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hydrolysates in enhancing cell culture functionality. Moreover, this results in wide variability in

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the functionality of soy protein hydrolysates in cell culture (Table 1). For several studies, while

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the cell growth was low in hydrolysate supplemented cultures as compared to the chemically

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defined media, the recombinant protein production was always higher in the former than the

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latter. The beneficial effects of soy protein hydrolysate are attributed to its complex composition,

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wherein it contains a large variety of different classes of compounds (e.g. peptides and

12, 17, 18

chemically defined media (e.g. RPMI 1640, 5:5:1 (v/v) of

12, 13, 17

produced using different processes have been tested. The

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carbohydrates). Typically, soy protein hydrolysates (60% peptides/ amino acids and 20%

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carbohydrates)17 are supplemented to chemically defined medium at 0.1-1.0% (w/v). A typical

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chemically defined medium like Iscove’s modified Dulbecco’s medium has an amino acid and

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glucose content of 1.2 g/ L and 4.5 g/ L, respectively.19 The hydrolysate supplementation to

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chemically defined medium significantly increases the protein content (50-500%) and

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carbohydrate content (4-44%) of the supplemented medium. As a result, these compounds

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considerably enhance the cell culture functionality of chemically defined medium. Firstly, these

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compounds provide carbon and nitrogen to cells resulting in enhanced cell growth and/ or

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recombinant protein production;16, 20 Secondly, there might be certain specific (key) compounds

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in hydrolysates that specifically enhance cell growth, recombinant protein production, or both.13,

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17

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annotated peptide) were identified as key compounds in soy protein hydrolysates that correlated

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positively with CHO (CRL-11397) cell growth.13 Lactate, ferulate, syringic acid, galactarate,

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adenine, and X-198 (an un-annotated peptide) were shown to be the key compounds that

In previous work, phenyllactate, lactate, trigonelline, chiro-inositol, and X-190 (an un-

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correlated positively with IgG production.13,

21, 22

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concentration of the key compounds was linked to the batch-to-batch variability in the cell

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culture performance of soy protein hydrolysates. A comparison of supplementation of thirty

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batches of soy protein hydrolysate to chemically defined media to culture CHO (CRL-11397)

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cells showed that the cell growth and IgG production varied from 148-438% and 117-283%

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relative to the chemically defined medium (100%), respectively.13 For 29 batches of another soy

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protein hydrolysate, the recombinant protein production ranged from 52-164% of the average

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recombinant protein production of all hydrolysates.23

In these studies, the variation in the

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The extent of variability in the cell culture performance between different batches of a

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hydrolysate also depends on the cell lines used and the test concentration of hydrolysates. The

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IgG production in CHO cells ranged from 103-138% and that in avian cells ranged from 93-

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115% as measured for 16 batches of a soy protein hydrolysate.24 When the same set of

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hydrolysates were supplemented at 0.5% (w/v) to CHO cells, the IgG production ranged from

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103-138%, while it was 83-138% when hydrolysates were supplemented at 1.5% (w/v)

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concentration, respectively.24

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In addition to cell line and hydrolysate concentration, batch-to-batch variation in the hydrolysate

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functionality is influenced by variability in the gross composition and qualitative changes that

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occur in the chemical composition of hydrolysates. The gross composition includes the total

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content and composition of each class of compound present in the hydrolysate, for instance, total

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peptide content and content of individual peptides. The qualitative changes refer to the chemical

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modification reactions (e.g. cross-linking, and glycation of proteins) that occur in hydrolysates.

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These reactions result in the formation of minor compounds, such as furosine, lysinoalanine

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(LAL), peptide-phytate complexes, and peptide-polyphenol complexes. Conventionally, these

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compounds are not included in the gross compositional analysis of hydrolysates. Therefore,

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qualitative changes could result in large differences in the hydrolysate functionality, while the

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gross composition of different batches of hydrolysate stays identical. Both gross and qualitative

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compositional changes occur in soy protein hydrolysates due to variations in processing and raw

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material (seed and/ or meal) composition. While there is a lot of literature available on

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compositional (gross and qualitative) changes in soybean and its derived products, this

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knowledge, in relation to cell culture functionality, has not been combined in an overview yet.

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In this review, we first discuss the effects of hydrolysate composition- both gross and qualitative

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on cell growth and recombinant protein production in cell culture applications. Subsequently, it is

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discussed how variations in the hydrolysate composition may be induced by variations in the raw

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material and processing.

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Chemical composition of soy protein hydrolysates

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The major compounds present in soy protein hydrolysates are peptides, carbohydrates, and

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minerals. The typical gross composition of a commercial soy protein hydrolysate produced from

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defatted soybean meal is 60 ± 5% (w/w) peptides/ amino acids, 20 ± 5% (w/w) carbohydrates and

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10% (w/w) minerals†.17 While the total carbohydrate and mineral content of soy protein

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hydrolysate is known, the monosaccharide and mineral composition is not yet reported. For 30

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batches of another commercial soy protein hydrolysate ‘Proyield Soy SE50MAF-UF’ produced

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from defatted soybean meal, the protein content varied in a narrow range of 56-58% (w/w).13

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This suggests that the batch-to-batch variability in the gross composition of a specific hydrolysate

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produced by a particular manufacturer is relatively small (< 5%).

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In addition to peptides, carbohydrates, and minerals, several compounds like phytates ( 10 kDa) and less-

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extensively hydrolyzed (18% peptides < 1 kDa; 26% peptides > 10 kDa) rapeseed protein

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hydrolysates supplemented to chemically defined media showed cell growth of 133% and 85%,

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respectively.30 Similar observations were reported when rapeseed protein hydrolysates made with

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Esperase 7.5L®, Neutrase 0.8L®, and Orientase 90N® were tested in cell culture. Esperase is a

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serine protease from Bacillus lentus, Neutrase is a neutral metallo-protaese from Bacillus

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amyloliquefaciens, and Orientase is a serine protease from Bacillus subtilis.30 All of the enzymes

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had low specificities.30 The activity of the enzymes was standardized using a hemoglobin

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standard allowing comparison of different enzymes. The hydrolysates had similar molecular size

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distribution of peptides but the cell growth observed was not similar. For e.g. the hydrolysate 10


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made with Esperase enhanced the cell growth 1.5 times higher than hydrolysates made with

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Neutrase.30 This showed that hydrolysis with different enzymes resulted in formation of different

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peptides which affected the functionality of hydrolysates.

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(b)

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carbohydrates differ in the rates at which they are consumed by cells and hence, the maximum

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achievable cell growth differs.31,

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concentration were tested in a media formulation used for CHO-TF-70R cells. While the average

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cell concentration after 140 hours of culturing in media containing glucose or mannose was 1.2 *

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106 cells/ mL, it was 0.7 * 106 cells/ mL in media with galactose or fructose.31 Furthermore, the

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consumption rate of glucose or mannose was much faster than galactose or fructose. After 150

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hours of culturing, 12.5 mM of glucose/ mannose and 2.5mM of galactose/ fructose was

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consumed by CHO-TF-70R cells.31 In a similar study, Barngrover et al. (1985) studied the

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influence of fructose and galactose concentration on cell growth of Vero African green monkey

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cells. After 6 days culture, the cell growth was 27.5, 23.8, 11.3, and 16.3 * 105 cells/ mL in 5, 10,

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15, and 20 mM of galactose in L-15 (Leibovitz) medium.33 On the other hand, the cell growth

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was 23.8, 27.5, 23.8, and 16.8 * 105 cells/ mL in 5, 10, 15, and 20 mM of fructose concentration

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in L-15 (Leibovitz) medium.33 Therefore, a strategic selection of carbohydrates and concentration

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in cell culture can be used to enhance the recombinant protein production.34 While the maximum

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cell growth and tissue plasminogen activator production in 20 mM glucose medium was 1.6 * 106

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cells/ mL and 3.9 µg/ mL, it was 1.9 * 106 cells/ mL and 4.5 µg/ mL in 5 mM glucose and 20 mM

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galactose medium.34

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(c)

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has been investigated in few studies. In HeLa35 and chick embryo cell culture36 racemized D-

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amino acids did not inhibit or enhance cell growth. Similarly, Naylor et al. (1976) tested

Carbohydrates: Carbohydrates are a source of carbon that supports cell growth. Different

32

Glucose, mannose, galactose, and fructose at 20mM

Racemized amino acids/ peptides: The influence of racemized amino acids on cell growth

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racemized amino acids in several mammalian cell lines (e.g. LM (TK-), A9, HTC+, 6TG-11,

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CHO, and B16) and reported that racemized amino acids did not affect cell growth.37

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(d)

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phytates,40 and MRP41, 42 have been shown to chelate cations. Cations have an important role in

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mammalian cell culture, for e.g. magnesium controls the activities of many glycolytic enzymes in

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the Krebs cycle,43 and calcium and zinc are essential for cell growth and differentiation, apoptosis

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and recombinant protein production.44, 45 For instance, the supplementation of phytic acid (0.27

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mM to 2.7 mM) resulted in decreased BALB/c mouse 3T3 fibroblast cell growth (93% to 45%)

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as compared to the non-supplemented control (100%).46

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(e)

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amino acids (e.g. lysine), as the MRP cannot be metabolized in cell cultures. Moreover, Maillard

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reaction also results in formation of new compounds, such as pyrazines and hydroxymethyl

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furfural, which affect cell cultures. Pyrazine and its derivatives at 1% (w/v) concentration

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induced genotoxicity in the range of 0.4-29% in CHO cells.47 More than 1% (w/v) pyrazine

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concentration was toxic and completely inhibited cell growth. Another MRP, hydroxymethyl

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furfural, at 0.1% (w/v) concentration exhibited similar genotoxic and growth inhibition effects in

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chick-embryo fibroblasts.48

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(f)

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investigated by supplementing growth medium with sunflower meal hydrolysates containing 1%

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and 6% (w/w) of polyphenols. While the cell growth and recombinant protein production for the

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former hydrolysate was 9 g biomass/ L and 180 units of streptokinase/ mL-1 hour-1, it was only 2

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g biomass/ L and 40 units of streptokinase/ mL-1 hour-1 for the latter, respectively.49 This clearly

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showed that high levels of polyphenol in hydrolysates inhibited cell growth and productivity.

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Although a large but similar extent of variability is reported for polyphenol content in sunflower

Chelating compounds (LAL, phytates, and MRP): In human and animal studies, LAL,38, 39

Maillard reaction products (MRP): The Maillard reaction results in loss of essential

Peptide-polyphenol complexes: The influence of peptide-polyphenol complexes was

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(1.4-6.1%)49-52 and soybean meal (0.02-9%)53-55, the polyphenol composition between the two

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meals differ greatly. While the sunflower meal primarily contains chlorogenic acid, caffeic and

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quinic acids, soybean meals are rich in isoflavones such as daidzein, glycitein, and genistein. In

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chemically defined cell cultures, influence of soy isoflavones on the cell culture functionality has

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also been investigated. The supplementation with genistein at > 10µM concentration (2.7 µg/mL)

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to the cell culture media resulted in drastic reduction of CHO cell viability, and apoptosis and

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genotoxicity was induced.56 In addition to cytotoxicity, genotoxic effects on cell cultures due to

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polyphenols are reported. A 40-fold increase in chlorogenic acid concentration from 0.01 mg/ mL

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to 0.4 mg/ mL resulted in 40-fold increase in genotoxicity in CHO cells.57 These effects increased

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further in the presence of metal ions like manganese and copper.57

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In addition to a direct effect, the above-mentioned minor compounds influence the cell culture

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functionality by affecting hydrolysate composition. These compounds interfere with the

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production of peptides during enzymatic hydrolysis in hydrolysate production (Figure 1).

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Production of soy protein hydrolysates

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Soy protein hydrolysates for cell culture applications are produced by enzymatic hydrolysis of

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defatted soybean meal, soy protein concentrate, or protein isolate, followed by enzyme

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inactivation, coarse-filtration, ultra-filtration, and spray drying (Figure 2).58 The industrial

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enzyme preparations that are used for hydrolysis may contain carbohydrase side activities. Such

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side activities in Neutrase (0.025% on protein basis) resulted in release of 14% neutral sugars

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from the water unextractable solids fraction obtained from toasted soybean meal.59 The

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hydrolysis reaction is stopped by inactivating the enzyme using a heat treatment. After enzyme

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inactivation, the solution is coarse-filtered to remove large and insoluble impurities, mainly

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polysaccharides, non-hydrolyzed proteins, and aggregated peptides. Coarse filtration is usually

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performed using filter aids, which are available in wide range of particle sizes (median particle

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size from 1.5-26 µm).60 The coarse filtration can be substantially hindered by polysaccharides

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and protein-lysophospholipids complexes.61 After coarse filtration, the hydrolysate solution is

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ultra-filtered, typically using a 10 kDa membrane, to remove large molecular weight compounds,

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such

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lipopolysaccharides, which are a major component of the cell wall membrane of most gram

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negative bacteria. The presence of endotoxin in hydrolysate adversely affects its functionality. In

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B-9 cells, presence of 20 ng/ mL endotoxins resulted in 30% reduced production of a

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recombinant human protein called Mullerian inhibiting substance.62 After ultra-filtration, the

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hydrolysate is composed of only water-soluble compounds (including the minor compounds

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described in previous sections). Finally, the hydrolysate solution is concentrated and spray dried.

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Conventionally, commercial soy protein hydrolysates are characterized for molecular weight

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distribution of peptides. Other analyses may include determination of protein, ash, moisture, and

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amino acids.63

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Compounds affecting enzymatic hydrolysis of the soybean meal

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The enzymatic hydrolysis of the soybean meal is affected by the minor compounds following

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three mechanisms: firstly, due to processing treatments, the substrate protein may get modified

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(e.g. MRP and cross-linked proteins). Secondly, compounds present in the soybean meal (e.g.

310

protease inhibitors) may inhibit the enzyme used for hydrolysis. Thirdly, some compounds (e.g.

311

polyphenols and phytates) can both modify the substrate and inhibit the enzyme (Figure 1).

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Since systematic data on soy protein hydrolysis in relation to these mechanisms are not available,

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references that describe effects on other types of proteins are included (Table 2). In addition, data

as

partially

hydrolyzed

peptides

and

endotoxins.58

Endotoxins

are

complex

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on in vitro protein digestibility is not available and therefore, results from in vivo animal nutrition

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studies are used to illustrate the effects of these mechanisms on protein digestibility. These

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effects are then used as an indication to state that similar effects can be expected in in vitro

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protein hydrolysis during hydrolysate production.

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Specific substrate modification

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(a)

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been shown to occur in amino acids and peptides. In these studies, combinations of extreme

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experimental conditions (pH 2-12; temperature = 25 °C-130 °C; heating time = 1-24 hours) have

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been used to induce racemization.64, 65 However, these processing conditions never occur during

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hydrolysate production. The extent of racemization has not been investigated under

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representative conditions (neutral pH; temperature = 50 °C-100 °C; heating time = 0.5-1 hour).

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This is important because, the occurrence of even very small extent of racemization in proteins

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and/or peptides greatly affects its hydrolysis.66 In vitro hydrolysis of synthetic racemized and

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non-racemized tripeptides (Ala-L-Glu-Ala, Ala-D-Glu-Ala, Ala-L-Asp-Ala, Ala- D-Asp-Ala,

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Val-L-Asp-Val, Val-D-Asp-Val, Ala-L-Phe-Leu, Ala-D-Phe-Leu, Ala-Met-Ala.HCl, Ala-D-Met-

329

Ala.HCl, Val-Met-Phe.HCl, and Val-D-Met-Phe.HCl) using intestinal peptidases was

330

investigated. While, tripeptides containing L-amino acid residues were completely hydrolyzed,

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the tripeptides containing D-amino acid residues were not hydrolyzed at all.67, 68 In another study,

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peptic hydrolysis of benzyloxycarbonyl-L-Ala-L-Phe-L-Tyr, benzyloxycarbonyl-L-Ala-L-Phe-L-

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Leu-L-Ala, benzyloxycarbonyl-L-Ala-L-Gly-L-Phe-L-Tyr and their corresponding D-isomers

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was studied. While the rate of hydrolysis was 22, 95, and 584 nmoles/minute/mg pepsin for L-

335

isomers, it was 1, 7, and 4 nmoles/minute/mg pepsin for D-isomers, respectively.69

Racemization: In several studies, racemization, i.e. conversion of L-form to D-form, has

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(b)

Lysinoalanine (LAL) formation: The processing treatments used during oil extraction

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from soybeans, production of the meal and hydrolysates can lead to formation of compounds

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such as LAL, lanthionine, dehydroalanine, and β-aminoalanine. The cross-linking reaction and

339

formation of these compounds is well described in literature.70, 71 Soy proteins (Defatted soy flour

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precipitated at pH 4.5, washed with water and freeze dried) were heat treated at 100 °C-120 °C,

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pH 6.5 for 1-3 hours. The LAL content in these heat processed soy proteins was

Factors Causing Compositional Changes in Soy Protein Hydrolysates

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