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For many decades bovine serum has been used as an essential component for the growth of animal cells in culture. However, the combined disadvantages of variability in composition, cost but particularly the potential for contamination with viruses or prions has been the driver for the substitution of serum with a more defined and animal-component free media. For some cell lines substitution with just a few simple ingredients can provide an effective liquid media for growth. However, for a number of cell lines finding a suitable serum-free formulation for growth has been very challenging. Because of the complexity of these formulations statistically designed methods have been adopted to ensure a rational approach to media design. This, as well as the increasing availability of microbially-produced recombinant forms of animal proteins has been significant in the development of animal-component free and chemically defined media. Sometimes chemically-defined media have poorer characteristics for growth promotion than the serum-based formulations that they replace. However, incremental steps of improvement are possible by the addition of key ingredients or by adaptation of the cells to newly formatted serum-free media.
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Chapter 8
Serum and Protein Free Media
Michael Butler
Abstract For many decades bovine serum has been used as an essential compo-
nent for the growth of animal cells in culture. However, the combined disadvan-
tages of variability in composition, cost but particularly the potential for
contamination with viruses or prions has been the driver for the substitution of
serum with a more defined and animal-component free media. For some cell lines
substitution with just a few simple ingredients can provide an effective liquid media
for growth. However, for a number of cell lines finding a suitable serum-free
formulation for growth has been very challenging. Because of the complexity of
these formulations statistically designed methods have been adopted to ensure a
rational approach to media design. This, as well as the increasing availability of
microbially-produced recombinant forms of animal proteins has been significant in
the development of animal-component free and chemically defined media. Some-
times chemically-defined media have poorer characteristics for growth promotion
than the serum-based formulations that they replace. However, incremental steps of
improvement are possible by the addition of key ingredients or by adaptation of the
cells to newly formatted serum-free media.
Keywords Serum • Basal media • Chemically-defined media • Peptide
hydrolysates
8.1 Introduction
Various biological fluids including serum, tissue extracts and homogenized chicken
embryos have been used over the past 100 years to grow animal cells in vitro. It was
in the 1950s that serious efforts were made to adopt systematic approaches to
determine the nutrients required for mammalian cell growth. One approach was
an attempt to analyze the contents of biological fluids. This was only partially
successful because of the multiple components present at micromolar concentra-
tions (Morgan et al. 1950).
M. Butler (*)
Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada
e-mail: butler@umanitoba.ca
©Springer International Publishing Switzerland 2015
M. Al-Rubeai (ed.), Animal Cell Culture, Cell Engineering 9,
DOI 10.1007/978-3-319-10320-4_8
223
A second approach was to determine the minimal chemically-defined compo-
nents that were essential for growth. This approach led to the development of
Eagles Minimal Essential Media (EMEM) (Eagle 1959). This consisted of
13 amino acids, 8 vitamins, 6 ionic species and supplemented with dialyzed
serum to provide the undefined components required for growth. Although
serum-supplemented EMEM was suitable for the growth of a number of cell
lines, other cells required more complex formulations. By increasing the compo-
nent concentrations of EMEM, basal media formulations such as Dulbeccos
modification of Eagles medium (DMEM) were developed for the growth of other
cell lines, to higher cell densities and for the propagation of viruses (Dulbecco and
Freeman 1959). Enrichment of media with an enhanced range of nutritional com-
ponents allowed clonal cell growth of selected cell lines, largely by the early work
of Richard Ham who gave his name to the widely-used Hams F-12 medium (Ham
1965). Sato had the ingenious idea of blending basal media formulations and
developed the now commonly used 1:1 v/v DMEM/F-12 mix that has become
widely used for the growth of multiple cell lines to high density (Jayme et al. 1997).
However, despite the inclusion of up to 70 defined components, these chemically-
defined media are still designated as basal media because they require supplemen-
tation with serum (typically 10 %) to sustain the growth of most cell lines,
This chapter focuses on the use of techniques to replace serum and the devel-
opment of serum-free formulations.
8.2 The Advantages and Disadvantages of Serum
Serum, as the supernatant from clotted blood of bovine or equine sources has been
found to provide high growth-promoting activity for a range of mammalian cell
lines. This is a rich source of often unidentified components such as attachment
factors, micronutrients, trace elements, water insoluble nutrients, growth factors,
hormones, proteases, and protective elements (antitoxins, antioxidants,
antiproteases), not provided by the basal medium and that promote rapid cell
growth. Furthermore, the high albumin content of serum ensures that the cells are
well-protected from potentially adverse conditions such as pH fluctuations or shear
forces, which may occur particularly in large-scale cultures. However, supplemen-
tation of culture media with serum has many inherent disadvantages.
(a) Batch-to-batch variation in composition. The composition of serum is variable
and undefined, which leads to inconsistent growth and productivity. Each
batch of serum can vary in composition depending upon the diet and environ-
mental conditions of the donor animals. This variation can cause significant
differences in the growth-promoting characteristics of the serum, and ulti-
mately causes significant differences in productivity of the cell-culture
process.
224 M. Butler
(b) A high protein content that hinders product purification. The cells grown in a
bioreactor secrete the product of interest (normally a protein) into the culture
medium. If the culture medium contains serum, its protein concentration is
already high. This causes difficulties in purification of the final product.
Culture medium with a serum content of 10 % v/v has a high protein concen-
tration that approaches 10 g/L. In comparison, the concentration of a recom-
binant protein secreted by the cells may only reach 0.1 g/L. This poses a
problem in the purification process of the targeted protein within a large mix of
serum protein. Furthermore, if the target is a specific monoclonal antibody, it
may well be mixed with any other non-specific antibodies present in the serum
and these are very difficult to separate.
(c) The potential for product contamination. The threat of contamination arises
from unwanted viruses and mycoplasma that may be present in serum as well
as the vectors of bovine spongiform encephalopathy (BSE, or “Mad Cow
Disease”). Although there have been no proven cases of such contamination
getting into the final product, no one wants to take the chance of manufactur-
ing biopharmaceuticals that may have had contact with unknown agents of
disease (Merten 2002). Prions as agents of BSE are of particular concern
because of the difficulty of their removal. The incidences of new variant
CJD (the human version of BSE) that have occurred in Europe are a great
source of concern for the vaccine industry that has used serum and for which
there is up to now no ideal substitute. Because of the concern over the potential
human consequences of the presence of these contaminants in therapeutic
products, most regulatory authorities have demanded the use of serum-free
processes for biopharmaceutical manufacture.
(d) Cost and availability. Fetal bovine serum (FBS) is often regarding as the best
available serum supplement for supporting cell growth. However, the cost is
often prohibitive at $500–1,000 per litre and this can account for up to 95 % of
the overall cost of the media. The most desirable source of FBS is from
countries that have had minimal incidences of BSE such as New Zealand.
However, there has from time to time been a global shortage of this serum and
this can cause a problem in the continuity of a bioprocess.
(e) Ethical concerns. There is an increasing concern for the ethical treatment of
animals that includes standard procedures needed to obtain serum from a
bovine fetus.
8.3 Serum-Free Media
The challenge of formulating a serum-free medium is to identify and substitute all
the components in serum that provide growth support for cells. Some cells are quite
fastidious in their growth requirements and these requirements vary considerably
from one cell line to another. Therefore, it has not been possible to design a single
universal serum-free formulation to act as a serum substitute suitable for the growth
8 Serum and Protein Free Media 225
of all cell lines. In fact even different clones of the same cell line may require
different formulations for optimal growth. As a result of this there are a multitude of
different serum-free media formulations available, each normally directed for the
growth of a specific cell line. Some formulations have been published. However,
many are proprietary and are sold in liquid or powder form without an available list
of components.
A serum-free formulation, by definition, means the absence of serum. However,
within this definition there are various possibilities and classifications. Figure 8.1
shows the evolution ‘of different types of media from those supplemented with
serum to serum-free media with various characteristics. A chemically-defined
medium is one in which all the components are known through well-defined
molecular characteristics and may include proteins. A protein-free medium is one
in which there is an absence of large proteins, although this may not be chemically-
defined. An animal-component-free medium is one without components derived
from animal sources but again this may not be chemically-defined. Each of these
classifications is associated with potential advantages for growing mammalian cells
in culture. Undoubtedly the most desirable medium is one which can be classified in
each of these sub-types. That is: chemically-defined, protein-free and animal
component-free. However, it is often the case that performance of the medium
decreases with increased definition (Hodge 2005).
8.4 Basal Media
There are several rational approaches for obtaining a suitable serum-free formula-
tion for the growth and productivity of a specific cell line. The general objective is
to obtain a mixture of supplements that can substitute for the serum in a growth
Fig. 8.1 A schematic showing the evolution of different media types
226 M. Butler
media. However, one of the early considerations should be the basal media. Many
of these media formulations were developed in the 1950s and 1960s and contain
components of complex mixture of carbohydrate, amino acids, salts, vitamins,
hormones, and growth factors. The most widely known are:-
Eagles basal medium (BME). This was originally designed for the growth of
mouse-L and HeLa cells. There are several versions of BME which have been
used for the growth of a wide range of cell lines. The medium needs to be
changed at least every other day to support continued cell growth (Eagle 1955).
Eagles minimum essential medium (EMEM). EMEM has a higher concentration
of amino acids than BME. It contains a balanced salt solution (Earles), alter-
natively Hankssalt solution can be used. This was developed as an improve-
ment of BME for the optimal growth of a wide variety of clonal cultures (Eagle
1959).
Glasgows modification of Eagles medium (GMEM). This is a modification of
BME and contains 2X the concentrations of the amino acids and vitamins with
extra glucose and bicarbonate. This media was originally developed for the
growth of BHK21 cells (Stoker and Macpherson 1964).
Dulbeccos modification of Eagles medium (DMEM). This has four-times the
BME concentration of amino acids and vitamins as well as addition
non-essential amino acids and trace elements. A glucose concentration as high
as 25 mM can be used in this medium for the growth of several cell types. The
medium was first reported for the culture of embryonic mouse cells but has been
used for a wide variety of applications for various cells, including primary
mouse and chicken cells, and in virus production (Vogt and Dulbecco 1960).
RPMI 1640. This was developed as a modification of McCoy 5A medium for the
long-term culture of peripheral blood lymphocytes. It is now recognized as a
general-purpose medium particularly for lymphocyte and hybridoma cultures
[5].
These basal formulations contain published lists of up to 60 components and are
available as sterilized liquids from media vendors. The basal media are normally
supplemented with 10 % serum in order to promote maximum growth of specific
cell lines. The serum may supplement the concentration of components already
contained in the basal media. Therefore, one of the early steps in serum-free media
development should be to optimize the basal medium. This is often done by
blending existing formulations. For example, one choice which has proved popular
is a 1:1 v/v mixture of DMEM and RPMI1640. DMEM provides high concentra-
tions of amino acids and vitamins whereas RPMI1640 provides a long list of
micronutrients to support cell growth.
8 Serum and Protein Free Media 227
8.5 Approaches for the Development of Serum-Free Media
8.5.1 Top-Down Approach for Serum Replacement
There are two basic approaches to developing serum-free media formulations. The
first approach (top-down) might involve selecting an existing formulation for a
similar cell line, supplemented with serum to obtain a reasonable level of growth.
The content of serum could then be gradually reduced to cause a decrease in the cell
growth to say 50 % of that originally obtained. At this point selected components
could be added to the medium to restore the original level of cell growth.
The top-down approach is often easier to pursue since a working serum-free
formulation can often be developed more quickly. Cell lines that belong to the same
group, such as epithelial or transformed, often require the same growth factors for
growth. Therefore a formulation that works for one epithelial cell line may work for
another with minimal modifications to certain growth factors or hormones. For this
reason serum-free formulations can be designed faster by this approach.
This method involves a systematic approach for the gradual replacement of
serum by substitution with essential nutrients or growth factors. In this procedure
the concentration of serum in gradually reduced to a level which around 50 % of
maximal growth as measured by the cell yields after 3 or 4 days. This procedure
may involve the adaptation of cells to gradually reduced levels of serum. Typically
adaptation can be successful in reducing the serum concentration from 10 % to 2 %.
At the lower serum level the growth rate will decrease until appropriate supple-
ments are provided in the medium or until the cells adapt. Cellular adaptation can
involve the synthesis of essential components by the cells.
The drawback to the top-down approach is that many components in the
formulation may be unnecessary, and often inhibitory for growth. This can often
result in the “capping” of the optimal performance of the medium (i.e.: the
maximum growth may not be achieved) as improvements are hindered by the
presence of unwanted compounds.
8.5.2 Bottom-Up Approach for Serum Replacement
The bottom-up approach involves the addition of potentially growth promoting
components systematically to the media in an attempt to obtain incremental
increases in cell growth. Although more labour-intensive and time-consuming,
this approach can lead to higher quality media. Only the components that are
required for growth are included in the formulation, allowing for greater control
of optimizing the medium. Thus media developed in this way tend to have higher
growth rates and are more easily improved since inhibitory compounds are less
likely to be present.
228 M. Butler
There is likely to be a wide concentration range over which any nutrient
supplement is not limiting. Using Hams approach (Ham and McKeehan 1979)
the optimum concentration for the nutrient can be set at the mid-point of the broad
optimum plateau of the growth-response curve (Fig. 8.2). This enables the concen-
tration to be bracketed between a maximum and minimum value. This reduces the
likelihood that the component will become growth limiting through the adjustments
of other medium components. In the response curve shown in Fig. 8.2 the mid-point
of the optimum concentration is 10
5
M. The concentration range of a supplement
can be established initially by testing a broad range at tenfold increases e.g.: 0.1,
1, 10 and 100 of a nominal starting concentration. This can be followed by
focus on a narrower range to obtain the optimum concentration for growth.
8.6 A Statistical Approach to Serum-Free Media
Development
Because of the multiple potential components involved in the development of
serum-free media it is almost essential to utilize an experimental design suitable
for evaluating the relative importance of each factor. One of the most popular
factorial “Design-of-experiments” (DOE) is the Plackett-Burman statistical
approach, which was designed to be simpler than a full factorial experiment. This
enables the evaluation of components to affect growth or any other measurable
property in a small a manageable number of experiments. This screening method
that can evaluate systematically a complex set of supplements for the promotion or
inhibition of cell growth. A large number of components can be studied at once to
determine which combination of factors is important for a serum-free formulation.
This statistical approach has been utilized in the design of various serum-free
formulations. Castro et al. obtained a medium for CHO cells that resulted in 45 %
higher productivity than previous formulations (Castro et al. 1992). From this study
glycine, phenylalanine, tyrosine and BSA were shown to improve the specific
0
50
100
3.0E-07 1.0E-06 1.0E-04 3.0E-02
% growth
Moles/litre
Fig. 8.2 The determination of the optimal concentration of a component
8 Serum and Protein Free Media 229
growth rate whereas other amino acids, such as methionine, proline and histidine
enhanced the production of interferon-gamma. Other potential components, such
as, insulin, arginine, aspartate, and serine produced an inhibitory effect on both cell
growth and interferon-gamma production. Using a similar approach Lee
et al. improved medium for CHO cell growth and erythropoietin production (Lee
et al. 1999). This medium was based on IMDM. Supplements of glutamate, serine,
methionine, phosphatidylcholine, hydrocortisone and Pluronic F68 were all identi-
fied as positive determinants for cell growth. The final optimized medium resulted
in a 79 % higher product yield compared to the original serum-supplemented
media.
A Plackett-Burman simple matrix is shown in Table 8.1 for the analysis of four
potential media components. In this, 16 separate cultures would be established –
normally in a multi-well plate. Each component is added at a high (+) or low ()
concentration. These values are chosen arbitrarily, although prior knowledge of the
typical concentration effects of each component would be valuable. Culture 1 in the
matrix contains all four components at their high concentrations whereas culture
16 has all four components at their low concentration. All other cultures are
established with a mixture of high and low concentrations of the four components.
Statistical analysis of the results would typically be performed of the cell growth
following 3 or 4 days. However this approach is not limited to cell growth and can
also be valuable in determining the effect of components on any culture parameter
which might include productivity or glycosylation profiles. The primary goal of the
method is to identify the primary components that influence the response – in this
case the growth of cells. This is done by analysis of variance (ANOVA) and main
Table 8.1 A typical Plackett-
Burman matrix Variables (components)
Medium A B C D
1 ++++
2 +++
3++
4+
5+++
6++
7+
8++
9+
10 +
11 + + +
12 + +
13 ++
14 + ++
15 + +
16 
+/represents high/low concentration of component
230 M. Butler
effect plots. A main effect is identified when different concentration of a media
affects cell growth. This type of analysis is best performed using statistical software
such as that offered by SAS.
The steps involved in a typical Plackett-Burman experiment are outlined below:
(a) Select the standard basal medium or combinations analyzed for their ability to
enhance growth.
(b) Establish two concentrations for each component to be tested : a high (+) and a
low ().
(c) Grow the cells in the serum-supplemented basal medium with serum (5–10 %)
in a well-plate culture. Allow the cells to initiate growth (~24 h).
(d) Collect the cell pool from the serum-medium and inoculate into each of the
16 wells established according to the matrix in Table 8.1, each with the same
basal medium plus combinations of media components (no serum).
(e) Include a positive control (serum-supplemented medium) and a negative
control (medium with no supplements)
(f) Count the viable cells (by MTT assay or Trypan-Blue exclusion) in each well
after a suitable time period (4 days).
(g) Calculate the variances from all of the effects from the single factors using
suitable statistical software (e.g.: SAS). This should identify the components
that were tested that have a significant effect on growth. The best combination
of components can then be included in the serum-free formulation under
development.
(h) Initiate an adaptation procedure for the cells to the new serum-free medium.
8.7 Mitogenic Components Needed to Replace Serum
There are specific groups of compounds that have been found to promote cell
growth and serve well in serum-free formulations. In this section some examples
of these are discussed but this is far from an exhaustive list.
8.7.1 Peptide Hydrolysates
These are obtained by the enzymatic hydrolysis of proteins derived from animal,
plant or microbial sources and contain a variable composition of small peptides,
which have proved valuable for many years as food supplements (Howard and
Udenigwe 2013). Their use as cell culture media components has been widespread,
although the batch-to-batch variability of their composition can be a problem. Meat
hydrolysates such as Primatone RL have been shown to be particularly effective in
promoting cell growth (Schlaeger 1996) but clearly this would be unacceptable in
the current drive for the industry standard animal component-free media.
8 Serum and Protein Free Media 231
Yeast, soy, wheat, cotton and pea hydrolysates have been particularly widely
used as individual components or as part of a defined cocktail to maximize the
growth of a particular cell line.
Attempts to identify and isolate the active components of hydrolysates have
proved particularly difficult because of their complexity and variability of compo-
sition (Michiels et al. 2011; Pasupuleti and Demain 2010; Hsueh and Moskowitz
1973). Analysis of a soy hydrolysate by HPLC and mass spectrometry identified
410 compounds of which 253 were assigned as peptides (Gupta et al. 2014). A
statistical analysis of the correlation of these compounds and antibody production
suggested that enhanced productivity occurred by the complex network of compo-
nents rather than a single component. This type of analysis has proved difficult
because of the variability of hydrolysate preparations from one batch to another.
This has lead to the development of a predictive model as a screening tool for high
performance hydrolysate lots (Luo and Pierce 2012). Without necessarily defining
the composition of the hydrolysate, NMR fingerprinting can identify a pattern
which could predict process consistency and product quality.
In order to meet the more stringent conditions of consistency of hydrolysates
needed for culture media a novel hydrolysate production process can be employed
to meet higher standards of control (Siemensma et al. 2010). The basis of the
method developed by Sheffield is the use of a rationally designed animal
component-free (ACF) enzyme cocktail that includes both proteases and
non-proteolytic hydrolases. These enzymes can release peptides as well as primary
components of the polymerized non-protein portion of the raw material. The
enzyme cocktail ensures the release of not only the growth-promoting peptides
and amino acids, but also key carbohydrates, lipids, minerals, and vitamins that may
well play a part in the bioactivity of the final product. The use of ultrafiltration with
typically a 10 kDa cut-off filter can add to the consistent quality of the final batch of
the peptide hydrolysate.
8.7.2 Insulin and Insulin-Like Growth Factor
Insulin is a small polypeptide (5.7 kDa) that is known to have multiple effects on
cell physiology, including membrane transport, glucose metabolism, and biosyn-
thesis of nucleic acids and fatty acids (Komolov and Fedotov 1978). It is normally
present in serum and so must be added as a supplement in serum-free media.
Whereas many bioactive components are required specifically for certain cell
lines, insulin appears to be a universal requirement for the growth of cells in culture.
The stimulation of DNA synthesis by insulin is particularly important for cell
growth and maintenance of the normal mitotic cycle (Komolov and Fedotov
1978; Simms et al. 1980). Although, insulin is an animal protein it is available
commercially by manufacture from genetically-engineered microbial systems that
enables it to be incorporated into an animal component-free medium formulation.
232 M. Butler
The concentration of insulin in culture media is usually around 5 ug/ml which is
approximately 10
3
greater than serum. However this has been shown to be the
minimal level required for significant growth of many cell lines (Chang et al. 1980;
Florini and Roberts 1979; Simms et al. 1980). It is possible that the high insulin
requirement is linked to cell metabolism in the presence of the high glucose
concentrations added to most media. However, a more likely explanation is the
instability of insulin under non-physiological conditions. It has been shown that
90 % of insulin can be reduced in 1 h at 37 C (Hayashi et al. 1978), which may be
due to the high redox potential related to the presence of such components as
cysteine in the media. Replacement of cysteine by cystine decreases the reduction
of insulin significantly.
Insulin-like growth factors (IGF) are naturally occurring peptides that have high
sequence similarity to insulin and also have similar metabolic actions as insulin.
However they are more potent mitogens and are required in media at lower
concentrations to promote the same metabolic effects (Morris and Schmid 2000).
Long R
3
IGF-1 is a modified recombinant form of IGF which has been found
valuable as a component in serum-free media. The modifications that include an
amino acid substitution at position 3 as well as a 13 amino acids extension prevents
inactivation of the molecule by specific IGF binding factors that may be secreted by
mammalian cells. The modified form of IGF has been shown to 200 more potent
and 3 more stable than insulin, both advantageous properties for media
components.
8.7.3 Epithelial Growth Factor (EGF)
This is one of many naturally occurring growth factors with a potent mitogenic
activity. It activates a signalling pathway by dimerization of a receptor (EGFR)
embedded in the cell membrane and activation of a tyrosine kinase. During
transformation cells tend to lose their sensitivity to EGF (Gopas et al. 1992).
Therefore the factor is normally only included in the media of non-transformed
and usually anchorage-dependent cells such as epithelial or fibroblast cells. Fortu-
nately, recombinant forms of the factor are available from microbial sources
including a recombinant Long EGF similar to the Long R
3
IGF-1 described above
and with which it may have synergistic activity (Simmons et al. 1995).
8.8 Transferrin: A Carrier Protein
This is a standard protein (~80 kDa) component of serum that is well-characterized
for its importance in the transport of iron under physiological conditions. It is
available as a single component from animal sources or as a microbially derived
recombinant protein. Virtually every cell line has shown a response to the presence
8 Serum and Protein Free Media 233
of transferrin but the extent of the response is dependent on the availability of other
forms of iron in the media. It was shown for mouse melanoma cells that the
presence of transferrin enhanced cell growth 15 in the absence of iron but only
by 2 in the presence of iron as FeSO
4
(Mather and Sato 1979). The value of
transferrin has been shown to include the ability to detoxify potentially toxic trace
elements in media (Barnes and Sato 1980), which is particularly important for low
protein media. Some reports have shown that it is possible to replace the iron
transport function of transferring by other forms of chelated iron such as ferric
citrate or tropolone chelated iron (Metcalfe and Froud 1994.).
8.9 Attachment Factors
These factors are often termed extracellular matrix proteins and are essential for
anchorage-dependent cells to allow rapid attachment to an available surface prior to
growth. These protein factors such as fibronectin and laminin may be available in
serum and may also be synthesised by many cell lines. However, they may be
provided in the media or on a surface coated with one of the factors such as
fibronectin. This can increase the speed of attachment of cells to the substratum,
which may well preserve the viability of the cell population (Orly and Sato 1979).
Conclusion
The driver for the removal of serum as a component of a bioprocess for the
production of biopharmaceuticals has been the recognized danger of viral and
prion contamination in the final product. Regulatory approval would now be
very difficult for a process that included animal-derived components partic-
ularly serum if there were an alternative. The difficulty in complying with this
requirement is in the identification of media components that can provide the
same growth promoting capacity as serum. Because of the extensive range of
growth promoting factors in serum it has proved to be a universal supplement
to basal media for growth promotion of almost any cell line. An equivalent
non-animal sourced universal supplement has not been found. Rather,
non-animal component media formulations have been designed for specific
cell lines. The design of such formulations is not trivial given the long list of
potential components and broad range of concentrations of each that could be
tested. Plant and microbial sourced protein hydrolysates have proved useful
components but their use is marred by variability of composition. The
availability of recombinant forms of natural growth factors or major serum
proteins has been particularly valuable as these can be isolated from micro-
bial cultures and therefore designated non-animal-components. The difficulty
of formulating serum-free media can be eased by statistical design-of-
(continued)
234 M. Butler
experiment procedures, although this may well require an extensive number
of reiterative cycles before a working formulation can be found. At the
present there are a number of serum-free media formulations available for
suspension cell lines but relatively fewer for anchorage-dependent cells. This
reflects the more exacting requirements of anchorage-dependent cells for
attachment and growth factors. Although many serum-free media types are
now available commercially their formulations are usually not in the public
domain. Rather, the composition of the media is proprietary to the supplier.
This is a relatively new phenomenon that did not occur in the early days of
investigation of the nutritional requirements of cells and the development of
basal media. However the secrecy surrounding the composition of serum-free
formulations reflects the commercial reality which has developed with the
range of high value products that can now be produced from mammalian cells
in culture.
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236 M. Butler
... In order to achieve optimized large-scale vaccine production in a fixed-bed bioreactor, two factors, serum in the medium and cell inoculum density, also need to be seriously considered. As for an important component for mammalian cell culture, animal serum provides nutrients, hormones and growth factors for cell growth, which is widely used for virus production (Butler 2015). However, addition of serum also results in complication of the downstream purification process and increases risk of virus contamination, especially for production of biopharmaceuticals (Butler 2015). ...
... As for an important component for mammalian cell culture, animal serum provides nutrients, hormones and growth factors for cell growth, which is widely used for virus production (Butler 2015). However, addition of serum also results in complication of the downstream purification process and increases risk of virus contamination, especially for production of biopharmaceuticals (Butler 2015). In some special cases, serum in media even needs to be removed in the upstream process. ...
... Presence of serum leads to loss of trypsin activity and thus it must be removed before virus infection (Le Ru et al. 2010). Therefore, use of serumfree medium began to draw attention in recent years in pharmaceutical industry, which overcomes drawbacks of using serum-containing media (Butler 2015;Genzel and Reichl 2009;Huang et al. 2015). Cells grown in serum-free media show increased sensitivity to shear force due to impaired adhesive ability on macrocarriers without serum (Ozturk and Palsson 1991), but this issue typically can be solved through addition of shear protectant Pluronic F-68 (Wu 1999). ...
Article
Full-text available
Fixed-bed bioreactors packed with macrocarriers show great potential to be used for vaccine process development and large-scale production due to distinguishing features of low shear force, high cell adhering surface area, and easy replacement of culture media in situ. As an initial step of utilizing this type of bioreactors for Pseudorabies virus production (PRV) by African green monkey kidney (Vero) cells, we developed a tube-fixed-bed bioreactor in the previous study, which represents a scale-down model for further process optimization. By using this scale-down model, here we evaluated impacts of two strategies (use of serum-free medium and low cell inoculum density) on PRV production, which have benefits of simplifying downstream process and reducing risk of contamination. We first compared Vero cell cultures with different media, bioreactors and inoculum densities, and conclude that cell growth with serum-free medium is comparable to that with serum-containing medium in tube-fixed-bed bioreactor, and low inoculum density supports cell growth only in this bioreactor. Next, we applied serum-free medium and low inoculum cell density for PRV production. By optimization of time of infection (TOI), multiplicity of infection (MOI) and the harvesting strategy, we obtained total amount of virus particles ~ 9 log10 TCID50 at 5 days post-infection (dpi) in the tube-fixed-bed bioreactor. This process was then scaled up by 25-fold to a Xcell 1-L fixed-bed bioreactor, which yields totally virus particles of 10.5 log10 TCID50, corresponding to ~ 3 × 105 doses of vaccine. The process studied in this work holds promise to be developed as a generic platform for the production of vaccines for animal and human health.
... Two culture media and their mixes were used and the best culture media formulations were selected based on the levels of expression in cell culture supernatant, using PFHM-II as a control. This approach does not consider a factorial "Design-of-Experiments" (DOE), such as the Plackett-Burman statistical method, to evaluate components that affect growth or any other measurable property (Butler 2015;Mandenius and Brundin 2008). Although, ...
... Indeed, media blending is an alternative method that rapidly generates new media by mixing existing formulations (Hunter et al. 2019). Moreover, once a reduced number of mixed media is determined, further experiments related to DOE can be achieved as a screening method to assess systematically a complex set of supplements or components for the promotion or inhibition of cell growth and productivity (Butler 2015;Mandenius and Brundin 2008). In addition, through metabolite profiling, it is possible to develop optimal culture medium and understand the use of medium components for a rational improvement of bioprocesses (Sellick et al. 2011a(Sellick et al. , 2011b. ...
Article
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Infliximab is a mouse/human chimeric IgG1 monoclonal antibody which recognizes the proinflammatory cytokine, tumor necrosis factor α (TNFα), and inhibits receptor interactions, thereby decreasing inflammation and autoimmune response in patients. This monoclonal antibody has been successfully used to treat rheumatoid arthritis, ankylosing spondylitis, and psoriatic arthritis. However, the high treatment cost limits patient access to this biotherapy. One alternative to this problem is the use of biosimilars. In this work, we describe the stable expression and physicochemical characterization of an anti-TNFα antibody. While infliximab is produced in recombinant murine SP2/0 cells, our anti-TNFα IgG antibody was expressed in recombinant murine NS0 myeloma cells. The best anti-TNFα antibody-expressing clone was selected from three clone candidates based on the stability of IgG expression levels, specific productivity as well as TNFα-binding activity compared to commercial infliximab. Our results indicate that the selected cell clone, culture medium, and fermentation mode allowed for the production of an anti-TNFα antibody with similar characteristics to the reference commercially available product. An optimization of the selected culture medium by metabolomics may increase the volumetric productivity of the process to satisfy the demand for this product. Further experiments should be performed to evaluate the biological properties of this anti-TNFα antibody. Key points • An anti-TNFα antibody was produced in NS0 cells using perfusion culture. • A proprietary chemically defined culture medium was used to replace commercially available protein-free medium. • The purified anti-TNFα antibody was comparable to the reference marketed product.
... Promising, low-cost alternatives to albumins are animalfree protein hydrolysates. They are inexpensive and have previously shown to successfully promote A promising attribute of hydrolysates is that they contain bioactive compounds such as isoflavones, saponins, bioactive peptides and phytosterols, which can potentially act as FBS/albumin or growth-factor replacements as they are associated with in vitro and in vivo antioxidative, immune-regulating, anticancer, antihypertensive, hypocholesterolemic, antiosteoporotic and antiobesity effects [44][45][46][47][48][49][50][51][52]. ...
... cell growth[44]. Protein hydrolysates are from animal, plant or microbial protein origin and are obtained through enzymatic, acidic or heat hydrolysation, yielding complex mixes of nutrients and growthfactor-like compounds (single amino acids and peptides)[44][45][46]. For cultured meat purposes, plant, microbial and also insect-derived protein could be considered. ...
Article
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Cultured meat is a bearer of hope for sustainable meat production, aiming at ensuring global food security while preserving environmental resources. Being a nascent industry, there are still many hurdles to overcome, some of which are cost reduction and ensuring a sustainable supply chain. This review focuses on the current state of the industry, identifies medium-related costs as one of the main cost drivers for scalable cultured meat production and discusses the latest findings on potential cost-effective nutrient replacements for cell propagation and differentiation, based on hydrolysates. Opportunities for medium recyclability with a focus on algae are also explored.
... If proper adapted -which can be a challenge, since MSCs might initially not expand sufficiently in such media -, the growth performance of many primary MSC types can be as effective as in serum or HS/hPL-containing media. To allow the successful expansion in such media, additional additives, special coatings/treatments of the growth surface and special medium compositions as well as appropriate adjusting strategies are regularly required [37,38]. Consequently, either sophisticated protocols need to be established to allow scaled expansion in such media, or primary MSCs need to be expanded to critical cell numbers in another medium than used for the conditioning of supernatant for the EV production. ...
... Consequently, either sophisticated protocols need to be established to allow scaled expansion in such media, or primary MSCs need to be expanded to critical cell numbers in another medium than used for the conditioning of supernatant for the EV production. Although it is a common way in the production of biotherapeutics to separate the production in a cellular growth and production phase [37], both of which use different media that must be well adapted to each other, it complicates USP. Furthermore, with a strategy using different expansion and harvesting media, CM from early passages are lost for the EV production. ...
Article
Extracellular vesicles (EVs) especially of mesenchymal stem/stomal cells (MSCs) are increasingly considered as biotherapeutic agents for a variety of different diseases. For translating them effectively into the clinics, scalable production processes fulfilling good manufacturing practice (GMP) are needed. Like for other biotherapeutic agents, the manufacturing of EV products can be subdivided in the upstream and downstream processing and the subsequent quality control, each of them containing several unit operations. During upstream processing (USP), cells are isolated, stored (cell banking) and expanded; furthermore, EV-containing conditioned media are produced. During downstream processing (DSP), conditioned media (CM) are processed to obtain concentrated and purified EV products. CM are either stored until DSP or are directly processed. As first unit operation in DSP, clarification removes remaining cells, debris and other larger impurities. The key operations of each EV DSP is volume-reduction combined with purification of the concentrated EVs. Most of the EV preparation methods used in conventional research labs including differential centrifugation procedures are limited in their scalability. Consequently, it is a major challenge in the therapeutic EV field to identify appropriate EV concentration and purification methods allowing scale up. As EVs share several features with enveloped viruses, that are used for more than two decades in the clinics now, several principles can be adopted to EV manufacturing. Here, we introduce and discuss volume reducing and purification methods frequently used for viruses and analyze their value for the manufacturing of EV-based therapeutics.
... However, due to the presence of ill-defined components in serum, batch-to batch variation and the potential presence of microbial contamination, the use of serum has been largely disbanded in the commercial production of therapeutics (Dimasi 2011;Urbano and Urbano 2007;Doucet et al. 2005). Although serum-supplemented media can support the growth of a range of cell lines, serum-free formulations are usually specific for certain cell types (Butler 2015). ...
... The composition of serum-free media is a key factor in the ability to produce biologicals such as monoclonal antibodies with high productivity in a bioprocess (Butler 2015). The nutrients and growth-promoting factors in media can support cell growth to a high cell density and maintain cell viability over a prolonged period. ...
Article
Full-text available
Four independent mAb-producing CHO cell lines were grown in media supplemented with one of seven protein hydrolysates of animal and plant origin. This generated a 7x4 matrix of replicate cultures which was analysed for viable cell density and mAb productivity. In all cultures, a consistent growth rate was shown in batch culture up to 4 to 5 days. Differences between cultures appeared in the decline phase which was followed up to 7 days beyond the start of the cultures. There was a marginal but significant overall increase (x1.1) in the integral viable cell density (IVCD) in the presence of hydrolysate but a more substantial increase in the cell-specific mAb (qMab) productivity (x1.5). There were individual differences between hydrolysates in terms of enhancement of mAb productivity, the highest being a 166% increase of mAb titre (to 117 mg/L) in batch cultures of CHO-EG2 supplemented with UPcotton hydrolysate. The effect of one of the most active hydrolysates (HP7504) on antibody glycosylation was investigated. This showed no change in the predominant seven glycans produced but a significant increase in the galactosylation and sialylation of some but not all the antibodies. Overall, the animal hydrolysate, Primatone and two cotton-derived hydrolysates provided the most substantial benefit for enhanced productivity. The cotton-based hydrolysates can be viewed as valuable supplements for animal-derived component-free (ADCF) media and as a source for the investigation of chemically defined bioactive components. Key points • Protein hydrolysates enhanced both IVCD & qMab; the effect on qMab being consistently greater. • Cotton-based hydrolysates showed high bioactivity and potential for use in serum-free media. • Enhanced galactosylation and sialylation was shown for some of the Mabs tested.
... Media formulations can contain 60-100 components, which change in concentration during a batch culture [57]. Glucose and glutamine are key nutrients utilized for energy metabolism during cell growth. ...
Article
Full-text available
The insect cell expression system has previously been proposed as the preferred biosecurity strategy for production of any vaccine, particularly for future influenza pandemic vaccines. The development and regulatory risk for new vaccine candidates is shortened as the platform is already in use for the manufacturing of the FDA-licensed seasonal recombinant influenza vaccine Flublok®. Large-scale production capacity is in place and could be used to produce other antigens as well. However, as demonstrated by the 2019 SARS-CoV-2 pandemic the insect cell expression system has limitations that need to be addressed to ensure that recombinant antigens will indeed play a role in combating future pandemics. The greatest challenge may be the ability to produce an adequate quantity of purified antigen in an accelerated manner. This review summarizes recent innovations in technology areas important for enhancing recombinant-protein production levels and shortening development timelines. Opportunities for increasing product concentrations through vector development, cell line engineering, or bioprocessing and for shortening timelines through standardization of manufacturing processes will be presented.
... For cultured meat too, animals are involved, as stem cells of animals are needed to grow meat. Furthermore, uncertainties persist on precise processes; it is yet unclear whether bovine muscle tissue, for example, can profitably be grown without foetal calf serum, which originates from animals (Butler, 2015). The identification of alternative sustainable, affordable, and animal-free growth media and their large-scale production is seen as one of the major challenges for cultured meat production (Stephens et al., 2018). ...
Chapter
Full-text available
The industrial production and excessive consumption of animal meat is increasingly related to environmental, ethical, and health issues. Meat alternatives have emerged as a promising solution to these issues. These foodstuffs that are intended to liken animal-based meat are frequently made from plant-based proteins, but may also be grown from animal tissue in laboratories. With an increasing interest in meat-free and meat-reduced diets, this chapter contributes to the book in two ways: It reviews the growing markets and trends as well as the sustainability potential of meat alternatives, and investigates how these trends may constitute opportunities and threats to the meat industry. Assuming that all ‘meats’ are mutually substitutable, it is argued that ‘non-animal meats’ can be considered a further step in the industrialisation of agriculture; hereby, the social, symbolic, and financial capital of the global meat industry enables it to continue ‘producing meat’ and to use these trends for further growth.
Chapter
In vitro cultured cells possess discrete properties in comparison to the normal cells, and therefore, they require a suitable environmental system for their growth. The nature of different environmental factors significantly affects the cells in culture conditions, most commonly the kind of substrate and/or phase in which cells are growing, the constituents of the culture medium such as nutrients present in the medium, the physicochemical properties of the medium, supplementation of hormones, and growth factors to the medium and the temperature of culture incubation. Thus, different cells can be grown in selectively different culture conditions, and interestingly the cultured cells have certain distinguishing features from cells growing in in vivo conditions. Also, subculturing of these cultured cells depends on several factors such as the cell density, exhaustion of the media, subculture schedule and serum. Most importantly, the cells growing in culture conditions follow a characteristic growth pattern. Subculturing several times may cause morphological changes, where the actual cultured cells lose their shapes and sizes and may cause misguided results. So, the selection of culture media for the cultured cells is a crucial step in cell maintenance as well as keeping the actual morphology of the cultured cells intact.
Chapter
For more than three decades, mammalian cells have been the host par excellence for the recombinant protein production for therapeutic purposes in humans. Due to the high cost of media and other supplies used for cell growth, initially this expression platform was only used for the production of proteins of pharmaceutical importance including antibodies. However, large biotechnological companies that used this platform continued research to improve its technical and economic feasibility. The main qualitative improvement was obtained when individual cells could be cultured in a liquid medium similar to bacteria and yeast cultures. Another important innovation for growing cells in suspension was the improvement in chemically defined media that does not contain macromolecules; they were cheaper to culture as any other microbial media. These scientific milestones have reduced the cost of mammalian cell culture and their use in obtaining proteins for veterinary use. The ease of working with mammalian cell culture has permitted the use of this expression platform to produce active pharmaceutic ingredients for veterinary vaccines. In this chapter, the protocol to obtain recombinant mammalian cell lines will be described.
Article
In vitro methods that can replace animal testing in the identification of skin sensitisers are now a reality. However, as cell culture and related techniques usually rely on animal-derived products, these methods may be failing to address the complete replacement of animals in safety assessment. The objective of this study was to identify the animal-derived products that are used as part of in vitro methods for skin sensitisation testing. Thus, a systematic review of 156 articles featuring 83 different in vitro methods was carried out and, from this review, the use of several animal-derived products from different species was identified, with the use of fetal bovine serum being cited in most of the methods (78%). The use of sera from other animals, monoclonal antibodies and animal proteins were also variously mentioned. While non-animal alternatives are available and methods free of animal-derived products are emerging, most of the current methods reported used at least one animal-derived product, which raises ethical and technical concerns. Therefore, to deliver technically and ethically better in vitro methods for the safety assessment of chemicals, more effort should be made to replace products of animal origin in existing methods and to avoid their use in the development of new method protocols.
Chapter
Full-text available
In the light of the growing demand for high quality plant-derived hydrolysates (i.e., HyPep™ and UltraPep™ series), Sheffield Bio-Science has developed a new hydrolysate platform that addresses the need for animal-free cell culture medium supplements while also minimizing variability concerns. The platform is based upon a novel approach to enzymatic digestion and more refined processing. At the heart of the platform is a rationally designed animal component-free (ACF) enzyme cocktail that includes both proteases and non-proteolytic enzymes (hydrolases) whose activities can also liberate primary components of the polymerized non-protein portion of the raw material. This enzyme system is added during a highly optimized process step that targets specific enzyme-substrate reactions to expand the range of beneficial nutritional factors made available to cells in culture. Such factors are fundamental to improving the bio-performance of the culture system, as they provide not merely growth-promoting peptides and amino acids, but also key carbohydrates, lipids, minerals, and vitamins that improve both rate and quality of protein expression, and serve to improve culture life due to osmo-protectant and anti-apoptotic properties. Also of significant note is that, compared to typical hydrolysates, the production process is greatly reduced and requires fewer steps, intrinsically yielding a better-controlled and therefore more reproducible product. Finally, the more sophisticated approach to enzymatic digestion renders hydrolysates more amenable to sterile filtration, allowing hydrolysate end users to experience streamlined media preparation and bioreactor supplementation activities. Current and future development activities will evolve from a better understanding of the complex interactions within a handful of key biochemical pathways that impact the growth and productivity of industrially relevant organisms. Presented in this chapter are some examples of the efforts that have been made so far to elucidate the mechanisms for the often dramatic benefits that hydrolysates can impart on cell culture processes. Given the variety of roles that hydrolysates likely play in each cell type, close collaboration between protein hydrolysate manufacturers and biopharmaceutical developers will continue to be critical to expanding the industry’s knowledge and retaining hydrolysates as a tool for enhancing media formulations. KeywordsAnimal cell culture-Serum-Serum free-Monoclonal antibodies-Protein hydrolysates
Chapter
Iron is an essential component in media for hybridoma and myeloma cell lines and is often delivered to the cell using the protein transferrin. In the development of defined protein-free media for large scale suspension culture, alternative iron delivery systems were tested. A number of alternatives supported growth in static culture. In agitated cultures, however, acceptable growth comparable to that observed with transferrin was only obtained when 2-hydroxy-2,4,6-cycloheptarin-1-one (tropolone) was used as the transferrin substitute. Interestingly, although high levels of ammonium ferric citrate (FAC) supported growth in static culture, little growth was obtained when this was used in agitated cultures. Growth of a mouse hybridoma cell line was comparable when tropolone was used in place of transferrin in a 30 litre fed batch airlift fermenter. Tropolone has also been used as a transferrin replacement in medium for recombinant murine myelomas using the glutamine synthetase expression system; Growth and productivity were unaffected.
Article
Media and feed development have been responsible for the largest share of improvement in cell culture productivity. New assay methods and high-throughput tools can be expected to extend this trend.
Article
The polyoma(PY) virus or parotid tumor agent (1,2) -- a DNA-containing virus (3,4) -- is characterized by a duality of action: it produces neoplasias of various types in different species of rodents (5), and causes cell degeneration in mouse embryo tissue cultures (6). In the experiments to be reported here, it was possible to obtain in cellular cultures in vitro the oncogenic effect of the virus; this afforded the possibility of studying the relationship between the oncogenic and cytocidal effect of the virus. The results so far obtained reveal a situation novel in animal viruses and suggest the existence of a host-virus interaction with characteristics reminiscent of temperate bacteriophage.
Book
The protein hydrolysates industry is growing rapidly yet there is no single book that describes the challenges and opportunities for manufacturers and end users, techniques used in manufacturing, characterization and screening of protein hydrolysates, their applications in a wide variety of industries in biotechnology. One of the misconceptions in using protein hydrolysates in fermentations is that the end user believes and uses it as a mere nitrogen source. However, the functionality of the product obtained is not necessarily due to protein hydrolysates alone because it may not be a pure peptide or a combination of peptides and may contain carbohydrates, lipids, micronutrients, etc., present in the raw material used or sometimes the manufacturers deliberately add to the process to bring unique functionality. Only a handful of manufacturers dictate this market that tend to keep manufacturing process proprietary making it harder to understand. This book will close the gap by unfolding information on latest developments.
Article
Hyperlipidaemia is an important risk factor for developing cardiovascular disease, a leading global health issue. While pharmaceutical interventions have proved efficacious in acute conditions, many hypolipidaemic drugs are known to induce adverse side effects. Due to a strong positive link between functional food components and human health, emerging research has explored the application of natural food-based strategies in disease management. One of such strategies involves the use of food proteins as precursors of peptides with a wide variety of beneficial health functions. Some plant, animal and marine-derived protein hydrolysates and peptides have shown promising hypolipidaemic properties when evaluated in vitro, in cultured mammalian cells and animal models. The products exert their functions via bile acid-binding and disruption of cholesterol micelles in the gastrointestinal tract, and by altering hepatic and adipocytic enzyme activity and gene expression of lipogenic proteins, which can modulate aberrant physiological lipid profiles. The activity of the protein hydrolysates and peptides depends on their physicochemical properties including hydrophobicity of amino acid residues but there is knowledge gap on detailed structure-function relationships and efficacy in hyperlipidaemic human subjects. Based on the prospects, commercial functional food products containing hypolipidaemic peptides have been developed for enhancement of cardiovascular health.
Article
While bacteria and yeast are used for the production of rather simple proteins, mammalian cells are generally required for the production of more complex proteins (i.e. post-translational modifications and, in particular, complex glycosylations). In order to boost cell growth and protein production, cultivation media are commonly supplemented with plant peptones, which are hydrolysed plant proteins. Therefore, plant peptones consist mainly of an undefined mixture of peptides, but also contain carbohydrates, phenolic compounds, salts, etc. To increase biosafety, the current trend is the use of chemically defined media. The straightforward idea that only a small number of compounds from peptones are biologically active is attractive, but their identification remains challenging. We first characterised global chemical families of compounds present in a soy peptone. The main constituents of this batch were peptides (60%) and carbohydrates (20%). The addition of this peptone or derived fractions to the culture medium was then tested on Chinese hamster ovary cells, CHO-320 line, expressing γ-interferon (γ-IFN), cultivated in BDM. Its presence did not increase the time-integral of the viable cell density, it increased slightly that of the γ-IFN concentration, but significantly the product/cell yield. Upon fractionation of this peptone by anion exchange chromatography and gel filtration, beneficial and detrimental fractions were obtained. The antioxidant activity of detrimental fractions was higher than that of beneficial fractions. Whereas the chemical nature of antioxidants contained in the detrimental fractions remains unknown, it may be assumed that they could inhibit the proliferation of CHO cells. Although the beneficial compounds were not identified, a fraction exhibiting the same effect as the peptone, but less complex, was obtained.
Article
Plant-derived hydrolysates are widely used in mammalian cell culture media to increase yields of recombinant proteins and monoclonal antibodies (mAbs). However, these chemically varied and undefined raw materials can have negative impact on yield and/or product quality in large-scale cell culture processes. Traditional methods that rely on fractionation of hydrolysates yielded little success in improving hydrolysate quality. We took a holistic approach to develop an efficient and reliable method to screen intact soy hydrolysate lots for commercial recombinant mAb manufacturing. Combined high-resolution (1) H nuclear magnetic resonance (NMR) spectroscopy and partial least squares (PLS) analysis led to a prediction model between product titer and NMR fingerprinting of soy hydrolysate with cross-validated correlation coefficient R(2) of 0.87 and root-mean-squared-error of cross-validation RMSECV% of 11.2%. This approach screens for high performance hydrolysate lots, therefore ensuring process consistency and product quality in the mAb manufacturing process. Furthermore, PLS analysis was successful in discerning multiple markers (DL-lactate, soy saccharides, citrate and succinate) among hydrolysate components that positively and negatively correlate with titer. Interestingly, these markers correlate to the metabolic characteristics of some strains of taxonomically diverse lactic acid bacteria (LAB). Thus our findings indicate that LAB strains may exist during hydrolysate manufacturing steps and their biochemical activities may attribute to the titer enhancement effect of soy hydrolysates.