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Hyaluronic Acid Production via Fermentation – Process Modeling and Techno-

Economic Assessment (TEA) using SuperPro Designer.

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 Hyaluronic Acid Production via
Fermentation
Process Modeling and Cost Analysis

Using SuperPro Designer®

by

Rafael da Gama, Nikiforos Misailidis, and Demetri Petrides

May 2021

This is the ReadMe file of a SuperPro Designer example that deals with process modeling, cost analysis
and optimization of Hyaluronic Acid Production via Fermentation. The flowsheets of the two cases
analyzed are appended to the bottom of this document. You may test-drive this model by downloading
the functional evaluation edition of SuperPro Designer from the downloads page of our website
(www.intelligen.com). The files of this example can be found in the Examples \ Pharmaceuticals \
HyaluronicAcid folder. The default installation path of the SuperPro Designer Examples folder follows
below.

C:\ Users \ Public \ Public Documents \ Intelligen \ SuperPro Designer \ v12 \ Process Library \ Examples

If you have any questions regarding this example and SuperPro Designer in general, please send an
email message to dpetrides@intelligen.com

INTELLIGEN, INC.
Simulation, Design, and Scheduling Tools
For the Process Manufacturing Industries
www.intelligen.com
Introduction

Hyaluronic acid (HA), also known as hyaluronan, is a linear polysaccharide made of alternating D-glucuronic
acid and N-acetyl-D-glucosamine residues joined by β-1,3 and β-1,4 glycosidic bonds (Figure 1). It was first
isolated from bovine vitreous humor, after which it is named: hyaloid is Greek for vitreous, and uronic acid
refers to the glucuronic acid monomer. HA is found in various vertebrates; in humans, in particular, it is
present in the extracellular matrix of connective, epithelial, and nervous tissues [1]. It is especially abundant
in the synovial fluid, cartilage, the skin, the vitreous humor of the eye, the vocal folds and the umbilical cord
[2]. Initially thought to be an inert space filler, HA is now recognized to perform a variety of important
functions: it regulates tissue viscosity and osmosis, providing lubrication and shock absorption to the joints;
and in the skin, it keeps cells hydrated and participates in signal transduction, wound healing, tissue
regeneration and healthy tissue turnover [2–4]

Figure 1: Molecular structure of hyaluronic acid, a linear polymer composed of disaccharide units. Each disaccharide
is made up of a D-glucuronic acid molecule (on the left) and an N-acetyl-D-glucosamine molecule (on the right).

HA’s properties strongly depend on its molecular weight (MW), which typically ranges from 10 3 to 107 Da
[5]. Due to its large MW and capacity to establish multiple hydrogen bonds with water molecules, HA is very
hygroscopic and exhibits unique rheological and adhesive properties. In fact, at concentrations as low as
0.2%, HA solutions present highly non-Newtonian, shear-thinning behavior, and at concentrations higher
than 1.5%, HA forms stable hydrogels [3]. Interestingly, high-MW molecules and low-MW molecules display
different, and sometimes even opposite biological functions: high-MW HA has lubrication, shock absorption,
space-filling, anti-inflammatory and anti-angiogenic properties, whereas low-MW HA is pro-inflammatory
and associated with tumor growth [1,2,6].

Given that HA has numerous beneficial properties and is generally biocompatible and non-immunogenic, it
has found many medical and cosmetic applications [1,7–9]. Nowadays, it is widely used in skincare
products and cosmetic surgery for its hydration, wound healing and space-filling properties [9–11]. In eye
surgery, HA facilitates surgical manipulations and replaces the vitreous humor lost during the procedure.
In the treatment of various arthritic disorders, HA restores the lubrication and shock absorption properties
of the joints [2,9,12,13]. It has also been used in several types of surgery to promote wound healing and
prevent organ adhesion, to treat vesicoureteral reflux, and for drug delivery purposes [3,14,15].

Other than vertebrates, certain bacteria, notably Lancefield A and C streptococci (e. g. Streptococcus equi
subsp. zooepidemicus, S. equisimilis, S. pyogenes) and coccobacillus Pasteurella multocida, are also
capable of producing HA as an extracellular capsule. Those microorganisms are pathogenic to humans
and/or livestock, and the HA capsule is thought to trick the host immune system, which does not recognize
it as foreign. In addition, the HA capsule appears to help these bacteria migrate from epithelial layers into
the tissue and protect them from reactive oxygen species (these microorganisms are catalase-negative
anaerobes) [3].

Until recently, HA was mostly produced by extraction from animal sources, especially rooster combs. This
production method has certain drawbacks, however: HA from natural sources is entangled with protein-
bound glycosaminoglycans, and therefore a relatively harsh and extensive purification process is required
to separate HA from other molecules. There is also a growing preference for non-animal sources of medical
products out of safety concerns, notably the risk of virus or prion contamination [1–3]. For those reasons,
the microbial production of HA has largely replaced HA extracted from animal sources [9,16]. In fact, most
HA is manufactured via streptococcal fermentation [8,9]; however, recombinant production of HA was
employed by Novozymes in the past [1], and is currently used by Hyalose LLC [17]. In both cases, Bacillus
was the recombinant platform.

The global market of HA (as raw material) was estimated at US$ 4.30 billion in 2015, and it is expected to
grow at a compound annual growth rate (CAGR) of 6.7% from 2018 to 2024 (Figure 2). The most significant
market segments are those of dermal fillers, osteoarthritis, and ophthalmology, and the current increase in
sales is primarily driven by the growing demand for anti-aging skin products and the need to treat arthritic
disorders in aging populations [18]. HA production is booming particularly in China, where sales are
expected to grow at a CAGR of 14.1% from 2018 to 2022, and to reach a volume of 613 metric tons (MT)
by the end of this period [19]. In 2018, Chinese sales of HA raw materials were estimated at 430 MT,
accounting for more than 80% of the global market [20]. By way of comparison, HA production in the early
2000s was estimated to be 10 – 20 MT/year for ophthalmic, cosmetic, and dietary applications, and less
than 1 MT/year for medical grade applications. HA prices in those segments were in the ranges of 1,000 –
2,000 $/kg and 40,000 – 60,000 $/kg, respectively [3]. This huge price difference is primarily due to the
extremely high purity required by medical applications, and, to a lesser extent, to the higher MW required
by those. Recently, the average price of HA was said to fall between 1,000 and 5,000 $/kg [7].
Figure 2: Global market of hyaluronic acid raw material, from 2013 to 2024. Each color indicates a major market
segment. Source: [18]

Process Description

This SuperPro Designer example includes two cases (A & B). The models were created in collaboration
with Prof. Maria Helena Santana from the State University of Campinas and Prof. Adriano Azzoni from the
University of Sao Paulo, Brazil, drawing from their experience with microbial production of HA and
purification of biomolecules. The original models were developed to perform a techno-economic analysis
of HA production, which was published in a peer-reviewed journal [21].

Both models share the same processing steps, except for the final portion of the downstream branch. In
case (A) we assume that a single HA product is manufactured, in the form of a dry powder, which is suitable
for topical formulations (e.g., skincare products). In case (B), on the other hand, 10% of the HA recovered
is diverted to the production of a highly pure, sterile, and high-MW HA that is suitable for injectable
applications (e.g., treatment of osteoarthritis). The upstream portion of both models is based on the
fermentation of an attenuated, non-hemolytic strain of Streptococcus.

For reporting and analysis purposes, cases (A) and (B) have been divided into four and five sections,
respectively:

• Upstream Section (cases A and B)

• Primary Recovery (cases A and B)
• Purification (cases A and B)
 • Injectable HA Manufacturing (only case B)
• Isopropanol Recycle (cases A and B)

Flowsheet sections in SuperPro are simply sets of related unit procedures (processing steps). In these
models, a distinct icon color was assigned to each process section. For information on how to specify
flowsheet sections and edit their properties, please use the Help tool (HelpIndexSection). The contents
of each process section are described in greater detail below. The flowsheets of the two cases are
appended to the bottom of this document.

Upstream Section

The Upstream Section includes the main fermentation step that produces HA (P-25 / FR-101), as well as a
seed train to generate the required inoculum volume for the production step. The seed train is composed
of three seed fermentation steps (P-18 / SFR-101; P-20 / SFR-102; P-22 / SFR-103), having an expansion
factor of 10×. The nominal volumes of the seed fermentors are 13.5 L; 135 L; and 1,345 L. The first seed
fermentor (SFR-101) is inoculated by a cell suspension with 30 g/L of dry biomass, while each subsequent
step is inoculated by the whole broth from the previous step, and the broth from the third seed fermentor
inoculates the production fermentor. The production fermentation procedure is carried out using two
fermentors (FR-101 and FR-102) that alternate between batches (i.e., operating in Stagger Mode; for more
details, please refer to the document section “Process Scheduling and Debottlenecking”), which have 15.9
m3 each. The Upstream Section also encompasses media preparation and aeration. The medium is
assumed to be the same for all fermentation steps; it is composed of 25 g/L of glucose, 60 g/L of yeast
extract and 11 g/L of salts. Each one of these components is dissolved in water and heat-sterilized using a
dedicated blending tank and heat sterilizer, and then distributed to all the fermentors. The solutions have
the following concentrations: yeast extract, 40% w/w; glucose, 50% w/w; salts, 15% w/w. Water is also
heat-sterilized and distributed to the fermentors, to complete the batch medium volume. All fermentation
steps are aerated with 1.0 VVM (volume of air per volume of liquid per minute) of sterile air; although S.
zooepidemicus is unable to perform oxidative phosphorylation, it can use oxygen to generate reducing
equivalents for HA synthesis [3]. The temperature is kept constant at 37 °C with chilled water, and the pH
is maintained at 7.0 with NaOH 20% w/w in all fermentation steps. The NaOH 20% solution is added in fed-
batch mode to all fermentors. With respect to media, however, the seed fermentation steps operate in batch
mode, while the main fermentation step operates in fed-batch mode (for information on how to implement
the fed-batch mode in SuperPro Designer, please consult the end of this document). In addition, the
fermentation time is assumed to be 12 h in the seed fermentation steps, and 24 h in the main fermentation
step. The seed and production fermentation steps were all modeled with the same mass stoichiometric
equation, given below:
100 Glucose + 15 O2 + 1 Salts + 165 Yeast Extract → 5.2 Acetic Acid + 18.6 Biomass + 80 Carbon
(1)
Dioxide + 9.7 Hyaluronan + 27.5 Lactic Acid + 140 Water

The extent of the fermentation reaction was set at 95% in the seed fermentation steps, and at 99% in the
main fermentation step. The heat released by fermentation was assumed to be 1800 kcal/kg of glucose
consumed in every step. The acetic acid, lactic acid and HA produced during fermentation are neutralized
by the NaOH solution that keeps the pH constant.

In addition, a cell lysis reaction was added to the main fermentation step to represent the spontaneous
death of bacterial cells towards the end of the fermentation process. The mass stoichiometry of this reaction
is given below:

100 Biomass → 20 Cell Debris + 50 Contaminating Proteins + 15 Impurities + 15 Nucleic Acids (2)

The extent of this reaction was set at 2%.

After the fermentation is deemed complete, a solution of HCl (20% w/w) is added to the broth to inactivate
the microorganism and re-convert the HA salt to the uncharged form of HA, which is less viscous [22,23].
The acid also converts the organic acid salts to their corresponding protonated forms. As a result, 13.3 m3
of fermentation broth is produced per batch, containing:

• 5.0 g/L of HA.

• organic acids (acetic acid and lactic acid).
• biomass, cell debris, bacterial proteins, nucleic acids, and small impurities (amino acids,
nucleotides, minerals, etc.).
• residual medium components (glucose, yeast extract, and medium salts).

Primary Recovery Section

The Primary Recovery Section comprises a clarification step and a crossflow filtration step. First, the
fermentation broth is cooled down (P-27 / HX-201) and sent to a disk-stack centrifuge (P-29 / DS-201) to
separate the bacterial cells from the HA molecules, which stay in the supernatant. It is assumed that 97%
of the cells are removed, and that the heavy stream has a wet solids concentration of 600 g/L (~18% w/w
on a dry basis). The supernatant then goes through a 0.22 µm dead-end filter (P-31 / DE-201) to remove
any residual cells. Next, the liquid is sent to a tangential flow filtration (TFF) system with a UF membrane
having a molecular weight cut-off (MWCO) of 100 kDa (P-32 / RT-201 and P-33 / DF-201). The solution is
initially concentrated 2× and then diafiltered with 8 volumes of deionized water (RO Water). The rejection
coefficient of the UF membrane for HA is assumed to be 0.995 in both operations, which leads to an overall
yield of approximately 96% in this unit procedure.
Purification Section

In both scenarios, the Purification Section includes Granular Activated Carbon (GAC) adsorption,
isopropanol precipitation, centrifugation, and tray-drying of HA. It is assumed that the GAC column (P-35 /
GAC-301) binds most of the contaminating proteins, nucleic acids, cell debris, glucose, and organic acids
that were not removed in the Primary Recovery Section, letting 99% of HA pass through. After that, HA is
converted to its salt form by addition of NaOH 20% w/w in a blending tank (P-36 / BT-301). Subsequently,
an isopropanol (IPA)-rich solution (87% w/w) is added to the same tank so that the final IPA concentration
is equal to 42% w/w, leading to the precipitation of the HA salt (as well as of some residual nucleic acids).
The HA precipitation yield was assumed to be 95%. The precipitate is then separated from the supernatant
using a decanter centrifuge (P-37 / DC-301), which leads to the recovery of 99% of the precipitated HA
under the form of a jelly cake containing 250 g/L of solids. This cake is then further dewatered using a
basket centrifuge (P-39 / BC-301); after centrifugation, the cake is washed with the same 87%-IPA solution
used before. The HA recovery in this step is 95%, and the resulting cake contains 53% w/w of HA salt.
Lastly, the product is dried under vacuum in a tray-dryer, to a final solids content of 95% w/w (P-40 /
TDR-301 in case (A); P-42 / TDR-301 in case (B). In case (A), a total of 61.7 kg of topical-grade product is
generated per batch, whereas in case (B), 55.6 kg of topical-grade product is generated. A slightly lower
amount is produced in the latter case because the final portion of the downstream branch is different in
case (B); a fraction of the HA cake (S-318) is diverted to produce injectable-grade HA.

Injectable HA Manufacturing Section

In case (B), 10% of the jelly cake is sent to a second TFF system that has a membrane MWCO of 1,000
kDa (P-45 / RT-401 and P-46 / DF-401). Firstly, the cake is dissolved in water for injection (WFI) to produce
a 5.0 g/L solution. Then, the solution is diafiltered with 6 volumes of WFI. In this diafiltration step, it is
assumed that the HA salt is composed of two major fractions: a high-molecular weight (HMW) fraction that
is larger than 1,000 kDa, and a low molecular weight (LMW) fraction that is smaller than 1,000 kDa. It was
assumed that 86.5% of the HA salt belongs to the HMW fraction, and 13.5% belongs to the LMW fraction.
The rejection coefficient of the UF membrane was assumed to be 0.90 for the HMW fraction, and 0.10 for
the LMW fraction. The retentate of the diafiltration is then re-concentrated to 5.0 g/L, filter-sterilized with a
0.22 µm dead-end filter (P-47 / DE-401) and freeze-dried to a final solids content of 95% w/w (P-49 /
FDR-401). In this case, 2.7 kg of injectable-grade sodium hyaluronate is produced (alongside with the
aforementioned 55.6 kg of topical-grade product).

Isopropanol Recycle Section

The light streams of the decanter and basket centrifugation steps, which contain 44% w/w and 74% w/w of
IPA, respectively, are sent to a continuous distillation column (P-46 / C-401 in case A; P-53 / C-501 in case
B) for solvent recovery and purification. The vapor-liquid equilibrium of IPA and water was predicted in
SuperPro Designer using an NRTL model (for details on how to implement rigorous vapor-liquid equilibrium
models in SuperPro, please consult the Isobutanol example in the Bio-Fuels folder). The distillate of the
column is recycled to the process and combined with fresh IPA to make up the volume required by the
Purification Section. The bottom phase of the distillation column, on the other hand, is used to pre-heat the
distillation feed through a shell and tube heat-exchanger, and then sent to wastewater treatment.

Process Scheduling and Debottlenecking

The overall batch time for this process (case A) is approximately 158 h (6.6 days). This is the time elapsed
from the start of a given batch (i.e., the preparation of glucose, yeast extract, etc.) to the end of that
particular batch (the generation of pure product). However, the batch cycle time is only 24 h because the
individual procedures in this process are much shorter than the overall batch time, and multiple (staggered)
equipment items are used in some parts of the process.

To visualize the process schedule, click Charts Equipment Occupancy Multiple Batches. This will
generate the Equipment Occupancy Chart (EOC), shown in Figure 3 for case A. The chart displays 6
consecutive batches of HA production, and the Upstream portion indicates that two production fermentors
are employed in this process: FR-101 and FR-102. In addition, it shows that they operate in Stagger Mode
(out of phase with each other). This configuration enables the plant to initiate a new batch every 24 h, even
though each fermentation procedure takes longer (37 h). Further details on specifying equipment in Stagger
Mode can be found in the Farnesene example, in the Bio-Materials folder.

The bottom portion of the chart displays downstream equipment. Most downstream equipment items exhibit
small gaps between batches, indicating that their idle time is low. Moreover, we can see that the activated
carbon column (GAC-301) bars have a distinctive pattern of repeated segments, reflecting the fact that the
column runs multiple adsorption cycles per batch. For more information on procedures that cycle multiple
times per batch, please refer to the Industrial Enzymes example in the Bio-Materials folder.
 CIP

Upstream

Primary Recovery

Purification

Figure 3: Equipment Occupancy Chart (EOC) for Hyaluronic Acid Production (Case A)
Figure 4: Operations Gantt Chart for Hyaluronic Acid Production (Case A)
Another view of the process schedule is provided by the Operations Gantt chart (in the MS Project style).
This chart displays detailed scheduling information for one or multiple batches. The Gantt chart for a single
batch is generated by selecting ChartsGantt ChartsOperations GC. Figure 4 displays a portion of that
Gantt chart, illustrating the scheduling of operations in the third seed fermentation and in the production
fermentation procedure. The dark blue and cyan bars represent the durations of procedures and operations,
respectively.

The Gantt chart enables users to visualize the execution of a batch process in detail. It also facilitates
editing of batch recipes. Double-clicking on any of its bars brings up the dialog of the corresponding entity
(e.g., operation, procedure, recipe, etc.). The simulation calculations can then be redone, and the chart can
be updated by clicking on the refresh button of the chart ( ).

Furthermore, SuperPro can export its scheduling data to MS Project by selecting FileExport to MS
Project XML File. Likewise, SuperPro can export its recipe data to SchedulePro by selecting FileExport
to SchedulePro’s Recipe DB. SchedulePro is a resource management, production planning and
scheduling tool available from Intelligen. Please consult the Help facility for information on these two
exporting options (HelpIndexExporting).

Material Requirements

Tables 1 and 2 display the material requirements in kg/yr, kg/batch, and kg/kg of MP for case A and B,
respectively (“MP” stands for main product, which is topical-grade sodium hyaluronate). These tables were
extracted from the RTF version of the respective Materials & Streams reports, which can be generated by
selecting Reports Materials & Streams from the main menu bar of SuperPro. The format of the report
can be specified through the dialog that is displayed when you select Reports Options from the main
menu bar. Table 1 and Table 2 are similar to each other in most respects; they show that acidic and basic
solutions are the materials used in the largest volumes (apart from air and water), due to the extensive
cleaning required by this type of process, and to the pH adjustment required by the fermentation steps and
the conversion between uncharged and salt forms of HA. Medium components, especially yeast extract
and glucose, are also required in large quantities. Even though a large volume of isopropanol is used to
precipitate HA in every batch, the demand for fresh isopropanol is relatively small, thanks to the efficient
recovery process embedded in the process design.

The only notable difference between the two tables is the use of WFI in case B, justified by safety reasons
when manufacturing injectable-grade products.
Table 1: Material Requirements for Hyaluronic Acid Production (Case A).

BULK MATERIALS (Entire Process)

Material kg/yr kg/batch kg/kg MP

Air 7,380,003 22,777.79 369.00
Biomass 10 0.03 0.00
CIP-Acid 947,736 2,925.11 47.39
CIP-Caustic 1,953,751 6,030.10 97.69
Glucose 225,626 696.38 11.28
HCl (20% w/w) 194,927 601.63 9.75
Isopropanol 126,331 389.91 6.32
NaOH (20% w/w) 197,620 609.94 9.88
NaOH (4% w/w) 136,046 419.89 6.80
RO Water 34,481,633 106,424.79 1,724.08
Salts 8,622 26.61 0.43
Water 3,465,965 10,697.42 173.30
Yeast Extract 372,352 1,149.23 18.62
TOTAL 49,490,622 152,748.83 2,474.53

Table 2: Material Requirements for Hyaluronic Production (Case B).

BULK MATERIALS (Entire Process)

Material kg/yr kg/batch kg/kg MP

Air 7,357,116 22,777.45 410.00
Biomass 10 0.03 0.00
CIP-Acid 1,057,824 3,275.00 58.95
CIP-Caustic 2,136,261 6,613.81 119.05
Glucose 224,926 696.36 12.53
HCl (20% w/w) 194,322 601.62 10.83
Isopropanol 176,315 545.87 9.83
NaOH (20% w/w) 197,007 609.93 10.98
NaOH (4% w/w) 135,624 419.89 7.56
RO Water 34,800,120 107,740.31 1,939.35
Salts 8,595 26.61 0.48
Water 3,455,217 10,697.27 192.55
WFI 4,288,370 13,276.69 238.98
Yeast Extract 371,197 1,149.22 20.69
TOTAL 54,402,906 168,430.05 3,031.79

Cost Analysis

SuperPro Designer performs thorough cost analysis, estimating capital costs (CAPEX) as well as operating
costs (OPEX), and generates the following three pertinent reports (through the Reports menu): the
Economic Evaluation Report (EER), the Cash Flow Analysis Report (CFR), and the Itemized Cost Report
(ICR). Table 3 and Table 4 display the Executive Summary of the Economic Evaluation Report for case
A and B, respectively. Notice that the Cost Basis Annual Rate is slightly different between the two cases:
20,000 kg of topical-grade HA is produced per year in case A, and 17,944 kg of topical-grade HA in case
B. This is because a fraction of the HA precipitate is diverted to produce injectable-grade HA in case B.

The total capital investment is estimated in approximately $42 million and $87 million, respectively. This
reflects the cost of additional equipment and more sophisticated buildings in case b. The latter is justified
by the stricter conditions required to produce injectable-grade products. To modify the buildings cost in
case B, the “Injectable HA Manufacturing” section was selected in the Sections & Branches Toolbar, and
the “Section Capital Cost Adjustments” dialog was accessed by clicking on the dollar sign button ( ).
More details on how to adjust capital costs can be found in the Industrial Enzymes example in the Bio-
Materials folder, or through the Help facility (under the topic “Capital Investment Dialog: DFC Tab”).

Likewise, the unit production cost per kg of topical-grade HA is substantially higher in case B (1665 $/kg)
than in case A (931 $/kg). The unit production cost is calculated by dividing the annual Operating Cost by
the Cost Basis Annual Rate. The Operating Cost is higher in case B because the “Injectable HA
Manufacturing” section entails additional facility-dependent costs, labor costs, consumable costs, raw
material costs, and utility costs. Besides, the Cost Basis Annual Rate is slightly lower in case B, as explained
earlier.

Table 3: Executive Summary for Hyaluronic Acid Production (Case A)

EXECUTIVE SUMMARY (2021 prices)

Total Capital Investment 41,819,000 $

Capital Investment Charged to This Project 41,819,000 $
Operating Cost 18,612,000 $/yr
Revenues 40,000,000 $/yr
Batch Size 61.73 kg MP
Cost Basis Annual Rate 20,000 kg MP/yr
Unit Production Cost 930.61 $/kg MP
Net Unit Production Cost 930.61 $/kg MP
Unit Production Revenue 2,000.00 $/kg MP
Gross Margin 53.47 %
Return On Investment 46.12 %
Payback Time 2.17 years
IRR (After Taxes) 35.71 %
NPV (at 5.0% Interest) 117,977,000 $
MP = Total Flow of Stream 'S-319'
Table 4: Executive Summary for Hyaluronic Acid Production (Case B)

EXECUTIVE SUMMARY (2021 prices)

Total Capital Investment 86,844,000 $

Capital Investment Charged to This Project 86,844,000 $
Operating Cost 29,880,000 $/yr
Main Revenue 35,888,000 $/yr
Other Revenues 43,565,570 $/yr
Total Revenues 79,454,000 $/yr
Batch Size 55.55 kg MP
Cost Basis Annual Rate 17,944 kg MP/yr
Unit Production Cost 1,665.16 $/kg MP
Net Unit Production Cost 1,665.16 $/kg MP
Unit Production Revenue 4,427.84 $/kg MP
Gross Margin 62.39 %
Return On Investment 50.64 %
Payback Time 1.97 years
IRR (After Taxes) 40.16 %
NPV (at 5.0% Interest) 281,615,000 $
MP = Total Flow of Stream 'S-322'

Total revenues, on the other hand, are much higher in case B ($79 million) than in case a ($40 million). The
reason is that injectable-grade HA has a much higher selling price than the topical-grade HA. Consequently,
case B presents better profitability metrics such as gross margin, return on investment, and net present
value (NPV), in spite of having higher capital and operating costs.

Figure 5 and Figure 6 show a breakdown of the operating costs in case A and B, respectively. These charts
were also extracted from the corresponding Economic Evaluation Report. They can be included in the EER
by selecting Reports Options and checking the “Include Charts” box on the lower right corner of the dialog.

In both cases, we observe that labor and facility-dependent costs predominate, accounting together for
approximately 70% of the total operating cost. This is typical of high-value, low-volume products such as
HA. It is noticeable, however, that the facility-dependent cost has a considerably larger weight in case B
(44%) than in case A (36%). This is due to the additional equipment costs associated with the “Injectable
HA Manufacturing” section. In fact, facility-dependent costs encompass maintenance and depreciation
costs, which are calculated based on equipment purchase costs.
Figure 5: Annual Operating Cost Breakdown for Hyaluronic Acid Production (Case A)

Figure 6: Annual Operating Cost Breakdown for Hyaluronic Acid Production (Case B)

The results of the economic analysis suggest that the proposed process design is adequate for the
production of HA in both scenarios. This can be credited to a relatively simple recovery and purification
process that attains high purification yields and high purity while avoiding the use of expensive
chromatographic steps. A key feature of the present design is the use of an ultrafiltration-diafiltration step
early in the downstream portion of the process using a 100-kDa membrane, as proposed by Corsa and
Carpanese [23]. The diafiltration mode enables the elimination of most impurities present in the extracellular
broth, such as organic acids, salts, sugars, and peptides, which are much smaller than the product of
interest. This step also eliminates molecules of HA smaller than 30 kDa, which are known to be
proinflammatory and therefore unacceptable in cosmetic or medical applications.

The results also suggest some strategies to improve the economics of HA production. The large facility-
dependent and labor costs, for instance, could be reduced if the plant were built in a country that has lower
facility and labor costs than the U.S. Increasing the fermentation titer and downstream yields would also be
useful for reducing HA cost. Fermentation titer, in turn, could be boosted by optimizing the microbial culture,
by genetically modifying native producers or by engineering recombinant systems. Recombinant production
of HA in a non-pathogenic host, such as C. glutamicum or B. subtilis, could also improve the process by
diminishing health risks to personnel and by reducing the size and complexity of the purification section.

In any case, many assumptions underlie this kind of analysis, such as the selling price and market demand
for topical-grade and injectable-grade HA; the costs of raw materials, consumables, labor, utilities, and
equipment; the actual fermentation and purification yields; etc. As a result, the actual economics of such a
plant may be substantially better or worse than the current projection. Indeed, a useful exercise is to perform
“what-if” analyses with SuperPro to determine the impact of various changes (such as a change in
fermentation yield). This allows one to understand the potential risks and rewards of a project under different
sets of assumptions. What-if scenarios can be evaluated individually (by simply changing parameter values
manually and re-running the simulation to see the results), or they can be automated through MS Excel.
For information on how to drive SuperPro through MS Excel, please consult the examples in the
“…EXAMPLES \ COM” folder. Related information is available in the Help Facility of the tool, which can be
accessed by selecting Help  COM Interface and Library.

Modeling Tips

Fed-Batch Mode

In certain chemical processes and many biotechnological processes, it is advantageous to add

reactants/media gradually or intermittently over a certain period, instead of having all of them inside the
vessel right at the beginning of the reaction/fermentation. In the case of chemical reactors, this is usually
done to improve reaction selectivity or to have a better control of the reactor temperature when dealing with
strongly exothermic reactions. In the case of bioprocesses, on the other hand, the fed-batch mode is usually
employed because high concentrations of substrates often have inhibitory effects on enzymes/cells. The
fed-batch mode may also be useful when a reactant/substrate has a low solubility limit in the reaction
mixture/culture medium.

In SuperPro Designer, the fed-batch mode is available to the following operations in Batch Vessel
Procedures:
 • Batch Stoichiometric Reaction
• Batch Kinetic Reaction
• Batch Stoichiometric Fermentation
• Batch Kinetic Fermentation

The fed-batch mode can be activated by accessing the operation dialog (right-click on the procedure, click
on Operation Data, and select the pertinent operation); clicking on the Fed Batch tab, and checking the box
“Consider Fed-Batch Supply of Reactants”, as shown in Figure 7.

Figure 7: Specification of Fed-Batch Mode in the Fermentation Operation of the Main Fermentation Procedure (P-25)
In that tab, the user must select the Feed Stream and provide other fed-batch specifications. In this
example, the fed-batch mode was specified in terms of Amount & Rxn Time, i.e., the total amount of material
present in the Feed Stream, and the Reaction Time over which the material is fed to the vessel.

Given that only one Feed Stream is allowed, and that in this example we wanted to add three solutions
(yeast extract 40%, glucose 50% and NaOH 20%) in fed-batch mode to the production fermentor, a mixing
procedure (P-24 / MX-101) was also added to the flowsheet. That procedure mixes the yeast extract
solution (S-111), the glucose solution (S-122), and the NaOH solution (S-159); its output stream (S-160)
was then specified as the Feed Stream in the Fermentation operation, as indicated in Figure 7.

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