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Blood Plasma Fractionation – Production of Albumin and IgG – Process Modeling

and Techno-Economic Assessment (TEA) using SuperPro Designer.

Preprint · August 2022

DOI: 10.13140/RG.2.2.31310.33606

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 Blood Plasma Fractionation
Production of Albumin and IgG
Modeling and Evaluation using
SuperPro Designer®
by

Rafael da Gama, Nikiforos Misailidis and Demetri Petrides

August 2022

This is the ReadMe file of a SuperPro Designer example that analyzes the production of therapeutic
proteins from blood plasma. Plasma contains numerous proteins, such as albumin, factor VIII, IgG, etc.,
that exert important physiological functions. The plasma proteins can be separated, purified, and used to
treat a variety of health conditions. The present example includes two SuperPro Designer models, one
focusing on the production of Albumin and the other on the production of IgG. The flowsheets of the two
models are appended to the bottom of this document. You may test-drive the two models by downloading
the functional trial edition of SuperPro Designer from the downloads page of our website
(www.intelligen.com). All the files of this example can be found in the Examples \ Pharmaceuticals \
BloodPlasmaFractionation folder. The default installation path of the SuperPro Designer Examples folder
follows below.

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

If you have any questions regarding this example or 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

Plasma is defined as the liquid fraction of blood, that is, blood without red cells, white cells, and platelets. It
makes up approximately 55% of blood volume and contains numerous proteins that exert important
physiological functions such as albumin, clotting factors, globulins, and hormones [1]. Many of these
proteins can be separated, purified, and utilized as therapeutic agents to treat a variety of health conditions.
The process of separating and purifying blood plasma components is referred to as plasma fractionation.
Currently, more than 25 therapeutic proteins are industrially produced from plasma [2]. They include:

- coagulation factors such as factor V, VII, VIII, X, XI, XIII, the prothrombin complex (a complex
fraction that includes factors II, IX and X), fibrinogen and von Willebrand factor. Most of these
proteins are used intravenously to make up for a protein that the patient lacks due to a congenital
deficiency. Fibrinogen is combined with thrombin to form a surgical clot called fibrin glue that is
employed for tissue sealing or healing
- protease inhibitors and anticoagulant concentrates including antithrombin, alpha 1-antitrypsin
and C1-esterase inhibitor [3]
- immunoglobulin G (IgG), which is a type of antibody. When IgG is recovered from a large donor
pool, it provides protection against a variety of antigens present in the environment of the donor
population and is said to be polyvalent IgG. It is used to treat people with immune deficiencies and
other immunological disorders (such as autoimmune diseases). It is also possible to produce IgG
from donors that have high concentrations of neutralizing antibodies against specific antigens (e.g.,
hepatitis, tetanus, rabies), typically resulting from vaccination. This is called hyperimmune IgG [3,4].
- albumin, which is by far the most abundant protein in human plasma, accounting for approximately
80% of the total plasma protein mass. The primary physiological function of albumin is to regulate
the osmotic pressure of blood and balance fluid distribution throughout the body (the colloid osmotic
pressure of plasma, also known as oncotic pressure, is largely due to albumin). Moreover, albumin
can bind a variety of molecules and ions, functioning as a carrier, storage or detoxification protein
depending on the particular species. Clinically, albumin is mainly used for blood volume expansion
in patients suffering from blood volume deficiency (hypovolemia) [3,4].

There are two basic ways to collect blood plasma: whole blood may be collected from donors and, after
that, centrifuged to separate the solid components (red cells, white cells, and platelets) from plasma; or
blood may be collected and plasma separated from the solids at the same time, so that cells and platelets
are immediately returned to the donor and only plasma is actually recovered [3,4]. The latter process is
called plasmapheresis and accounts for most of the plasma collected nowadays (roughly 80%) [2]. In both
cases, plasma is collected in bags containing an anticoagulant solution, usually made of citrate, that
prevents spontaneous clotting by plasma proteins. However, the collection method affects the composition
of plasma as well as the composition of the anticoagulant solution [2–4].

2
The history of plasma fractionation is closely related to that of World War II. Blood transfusion was
introduced in the battlefield in World War I; despite the risks related to blood type mismatch, whole blood
transfusions were performed then. During World War II, however, E. J. Cohn and coworkers at the Harvard
Medical School were charged by the American armed forces with developing a substitute for human
plasma. After a botched attempt with bovine albumin, Cohn and his team focused on the production of
human albumin. The first lot of human albumin suitable for clinical use was produced in 1941, and in 1943
– 1944 Cohn and his colleagues published the plasma fractionation methods they had developed. These
methods were based on a series of selective protein precipitation steps using ethanol under different
conditions of temperature, pH, and ionic strength. They not only led to the production of albumin at
considerable purity, but also to the separation of several protein fractions having different compositions,
properties, and applications. Peter Kistler and Hans Nitschmann, who worked at the central laboratory of
the Swiss Red Cross, published a variation of Cohn’s protocol a few years later which became popular in
Europe [4]. The ethanol fractionation methods of Cohn and Kistler-Nitschmann were widely adopted by the
industry and remain the bedrock of most plasma fractionation processes to this day, despite the increasing
utilization of chromatographical steps to complement or replace ethanol precipitation steps in modern
processes [3].

Manufacturing of Plasma Proteins for Therapeutic Use

Although albumin was the main driver of plasma fractionation in the early days of the plasma protein
industry, the introduction of factor VIII for the treatment of hemophilia made factor VIII concentrates the key
driving force for the plasma industry in the 1960s. With the development of recombinant factor VIII and a
greater understanding of the immunological role of IgG in the 1980s, intravenous IgG (IVIG) became the
main product of plasma fractionation processes and remains so to this date [5]. In any case, the production
of multiple proteins from the same volume of plasma can drastically improve the economics of plasma
fractionation, given that plasma is a very expensive raw material. In fact, the cost of plasma proteins
depends on their relative market demands: while the first liter of plasma processed leads to many proteins
that can be sold, the last liter is only used to produce the protein that is in highest demand [4]. This is called
“last liter economics”, and its effect can be seen in Figure 1, which displays the total revenues to produce
IgG alone and along with other plasma proteins.

3
Figure 1: Revenues from plasma fractionation considering the production of IgG alone or IgG along with
other proteins. GM = gross margin; HSA = Human Serum Albumin; F VIII = Factor VIII; F IX = Factor IX.
Source: [4].

In general, the fractionation process starts with thawing plasma packs (2000-4000 L) followed by
precipitation and centrifugation of the material at low temperature; this is called cryo-precipitation. The
resulting solid phase, named cryoprecipitate, is rich in factor VIII, fibrinogen and von Willebrand factor. The
aqueous phase, named cryo-poor plasma or cryo-supernatant, is then subjected to a series of ethanol
precipitation steps under different conditions of pH, temperature, and ionic strength. The precipitates are
usually separated by depth filtration or centrifugation. In the traditional Cohn process, the first precipitate
obtained by ethanol precipitation is named Fraction I; it is rich in fibrinogen. Another ethanol precipitation
step (II+III in the Cohn nomenclature) separates most globulins from albumin; the globulins form the
precipitate, while albumin remains in the supernatant. Additional ethanol precipitation steps can be used to
purify the albumin-rich supernatant as well as the IgG-rich precipitate. The introduction of
chromatographical steps at various points allows the separation and recovery of many other proteins, such
as alpha 1-antitrypsin, antithrombin, the prothrombin complex, factor VII, protein C, factor IX, etc. [3–5].
This is illustrated in Figure 2, which shows the complexity of a typical plasma fractionation process.

4
Figure 2: Typical plasma fractionation scheme [3].

5
Different types of chromatography may be used to separate, recover, and purify plasma proteins, such as
ion-exchange, affinity, and gel filtration chromatography. Ion-exchange chromatography is the type of
chromatography most employed in the industry due to the high capacity of ion-exchange resins and the
ease of controlling their selectivity by adjusting pH and conductivity. Affinity chromatography is another type
of chromatography extensively used in plasma fractionation processes; it may be based on group-specific
ligands such as immobilized heparin or lysine, or on highly specific ligands such as immobilized monoclonal
antibodies (immunoaffinity) [3,4].

In addition to chromatography, precipitation with agents other than ethanol are used in plasma fractionation,
whether to concentrate a desired protein or to remove impurities. Compounds such as ammonium sulfate,
polyethylene glycol and caprylic acid (a.k.a. octanoic acid) are employed for that purpose. Adsorption steps
using aluminum hydroxide or fumed silica are applied in specific processes to remove undesired molecules
too. Crossflow ultrafiltration is also widely employed in plasma fractionation to concentrate plasma proteins,
remove small molecule impurities, such as viral inactivation agents and exchange protein buffers (e.g., to
condition the protein for a subsequent chromatography step or to formulate it) [4].

Although precipitation and chromatography steps can reduce viral loads in plasma fractionation processes,
viral clearance may not be reproducible or sufficient to guarantee the safety of plasma proteins. For that
reason, dedicated viral removal and inactivation steps have become integral to plasma fractionation
processes. At least two viral removal & inactivation processes are typically used; one focuses on enveloped
viruses, which are generally more pathogenic but more labile, and another one addresses non-enveloped
viruses [3,4].

Viral removal & inactivation steps may be divided into three major types: heat inactivation, chemical
inactivation and nanofiltration:

- Heat inactivation may be performed on the bulk solution, on the final container or on freeze-dried
material. Dry heat is generally used, although moist heat (vapor treatment) may be applied to
freeze-dried products. Terminal pasteurization (i.e., dry heat applied to the final container) is
applied in most albumin production processes, for instance. Heat inactivation methods can however
damage the proteins of interest, and therefore cannot be applied indiscriminately.
- Chemical inactivation methods include the so-called solvent-detergent (S/D) treatment, which
employs tri-n-butyl-phosphate and a non-ionic detergent (Triton X-100 or Polysorbate-80); low pH
incubation, typically at pH 4; and caprylic acid precipitation. Chemical methods are usually effective
to inactivate enveloped viruses but not non-enveloped viruses.
- Nanofiltration employs multilayered membranes with 15 – 75-nm pores that can retain even small
viruses. Nanofiltration removes both enveloped and non-enveloped viruses and is often used to
complement chemical inactivation steps. It can be performed in dead-end (normal flow) or in
crossflow mode [3,4].

6
Process Description

Two SuperPro Designer files are included in this example:

• Plasma_Albumin.spf
• Plasma_IgG.spf

Both processes start from 2000 L of fresh frozen plasma (FFP) and their initial portion is the same,
consisting of two precipitation steps with ethanol. The supernatant of the second precipitation is rich in
albumin, and its purification is modeled in the Plasma_Albumin.spf file. The precipitate, on the other hand,
is rich in IgG, and its purification is modeled in the Plasma_IgG.spf file. The two processes have not been
modeled in the same file because, in practice, they are treated as separate processes with different batch
cycle times. Splitting these processes into two files also makes them simpler and easier to understand. For
the sake of simplicity, the recovery and purification of other plasma components such as fibrin glue or factor
VIII have been omitted.

In the albumin process, the supernatant of the second precipitation is subjected to additional ethanol
precipitation steps for the removal of impurities; diafiltered in a crossflow filter; subjected to anion-exchange
chromatography in negative mode; and diafiltered again for formulation. Approximately 56 kg of albumin
protein are produced per batch, corresponding to about 18 MT/yr.

In the IgG process, the precipitate of the second precipitation is treated with caprylic acid; diafiltered in a
crossflow filter; incubated at low pH; subjected to anion-exchange chromatography in negative mode; and
diafiltered again for formulation. Approximately 12 kg of IgG protein are produced per batch, corresponding
to about 4 MT/yr.

For reporting and analysis purposes, each process has been divided into multiple sections:

• Albumin process:
o Ethanol Precipitation (dark red)
o Ion-Exchange Chromatography (olive green)
o Formulation (violet)
• IgG Process:
o Ethanol Precipitation (dark red)
o Caprylic Acid Treatment (green)
o Low pH Treatment (blue)
o Ion-Exchange Chromatography (olive green)
o Formulation (violet)

7
Flowsheet sections in SuperPro are simply sets of related unit procedures (processing steps). For
information on how to specify flowsheet sections and edit their properties, please use the Help tool (Help
Index Section). Each process is described in greater detail next.

Albumin Process

Ethanol Precipitation

The process starts with thawing of 2000 L of fresh frozen plasma (FFP) at 0 °C (P-01 / T-101). FFP consists
of a pool of plasma bags containing 10% of anticoagulant solution (citrate). The average composition of
blood plasma considered in this example is provided in Table 1. During thawing, a small fraction of proteins
precipitate; this phenomenon is called cryoprecipitation. The precipitate (“cryoprecipitate”) is separated
from the supernatant (“cryo-supernatant” or “cryo-poor plasma”) by centrifugation in a bowl centrifuge (P-
02 / BC-101). A significant portion of fibrinogen (around 60%) ends up in the cryoprecipitate.

Table 1: Blood plasma composition (major components only)

Component Concentration (g/L)

Albumin 43.5
Fibrinogen 3.0
Alpha-Globulins 8.5
Beta-Globulins 9.0
Gamma-Globulins: IgG 9.0
Gamma-Globulins: non-IgG 3.0
TOTAL PROTEIN 76.0

The cryo-poor plasma is then subjected to the first ethanol precipitation step (P-10 / TPFF-101). The total
protein concentration is first adjusted with water for injection (WFI); a concentrated acetate buffer (4.8 M;
pH = 4) is added to adjust the pH to 7.2; and ethanol is added so that the final ethanol concentration is 8%
v/v. After that, the mixture is incubated at -3 °C for 4 h, which leads to the precipitation of the fibrinogen that
had not been removed by cryo-precipitation. This is modeled by a simple stoichiometric reaction in
SuperPro:

1 Fibrinogen → 1 Fibrinogen (s)

A conversion of 100% was assumed. Similar reactions were included for other proteins (albumin, IgG, etc.)
assuming a conversion of 2% to represent protein losses. All precipitation steps in this example were
modeled in a similar manner.

After precipitation is deemed complete, diatomaceous earth is added to the mixture to aid the subsequent
filtration step. The target concentration of filter aid is 1.5% w/w. The mixture is then filtered using a plate &

8
frame filter (P-12 / PFF-101), which retains the protein precipitates along with the filter aid. After filtration,
the resulting cake is washed with an ethanol solution (stream S-36), dried and collected. This cake
corresponds to Fraction I according to the nomenclature of Cohn, and it is rich in fibrinogen. The filtrate and
the cake wash are collected in another tank and subjected to a second precipitation step (P-13 / TPFF-102)
at a higher concentration of ethanol (19% v/v), a lower pH (5.85) and a lower temperature (-5 °C). Under
these conditions, most of the gamma-globulins and a significant portion of the alpha-globulins and beta-
globulins precipitate, while albumin remains in solution. This step corresponds to Precipitation A in the
Kistler-Nitschmann process. Filter aid is again added to the mixture after precipitation, and the slurry is
filtered in another plate & frame filter (P-15 / PFF-102). The resulting cake (Precipitate A) is washed, dried,
and collected for IgG production. The filtrate and cake wash, on the other hand, are collected in another
tank and subjected to a third precipitation step (P-16 / TPFF-103) at a higher concentration of ethanol (40%)
and a lower temperature (-8 °C), which removes most of the alpha- and beta-globulins present. The mixture
is again filtered using a plate & frame filter (P-18 / PFF-103); the resulting cake is washed, dried, and
collected; and the filtrate and cake wash are collected in another tank for a fourth precipitation step (P-19 /
TPFF-104). This corresponds to “Precipitation C” in the Kistler-Nitschmann process, and it occurs at the
same ethanol concentration and temperature as the previous precipitation, but at a lower pH (4.8). Under
these conditions, albumin precipitates. Another filtration is performed to recover the precipitate (P-24 / PFF-
104), and the resulting cake is washed, dried, and collected in another tank (P-28 / TPFF-105). The filtrate
from this precipitation is discarded. The albumin precipitate is then resolubilized with WFI (P-28 / TPFF-
105) and subjected to a fifth filtration step (P-29 / PFF-105) with the sole purpose of removing the filter aid
from the protein.

Ion-Exchange Chromatography

In this section, the albumin solution is subjected to ion-exchange chromatography in negative mode. Prior
to loading the column, however, a buffer exchange is performed using a crossflow filtration system in
diafiltration mode (P-33 / TDF-201 and P-34 / DF-201). Almost all the albumin and protein impurities are
retained by the membrane (a Rejection Coefficient of 0.995 is assumed for all proteins); the average filtrate
flux is assumed to be 40 L/m2/h; and the diafiltration is conducted with 10 volumes of acetate buffer (20
mM, pH = 4.6). After diafiltration, the pH of the solution is equal to 4.6, which corresponds to the isoelectric
point (pI) of albumin. The protein concentration is then adjusted to 10 g/L with WFI, and the solution is
loaded onto a weak anion-exchange column (P-45 / C-201) with a resin binding capacity of 100 g/L. The
column binds the protein impurities but does not bind albumin significantly; the retention is 95% for the
protein impurities and 5% for albumin. This occurs because the pH of the solution is equal to the pI of
albumin, which means that the overall electric charge of albumin is zero. As a result, the collected
flowthrough consists of a highly pure albumin solution. Its pH is adjusted to 7.0 with sodium hydroxide in
the collection tank (P-46 / MT-202). After loading, the column is stripped with a concentrated salt solution
(2M NaCl) and cleaned for the next cycle.

9
Formulation

In this section, the albumin solution is concentrated and subjected to a buffer exchange step using another
crossflow filtration system (P-50 / TDF-301 and P-51 / DF-301). The solution is first concentrated to 25 g/L,
then diafiltered with 10 volumes of buffer (90 mM NaCl), and subsequently concentrated to 220 g/L. The
membrane rejection coefficients for the proteins are assumed to be 1.0 in the concentration step and 0.995
in the diafiltration step. The average filtrate flux is assumed to be 20 L/m 2/h. Next, sodium caprylate and
WFI are added to the solution so that the final albumin concentration is 200 g/L and the final sodium
caprylate concentration is 32 mM. Lastly, the albumin solution goes through sterile filtration (P-52 / DE-302)
and is filled into 20-L storage bags (P-53 / DCS-301). A total of 280 L of albumin 20% (w/v) is produced per
batch (i.e., 56 kg of albumin protein per batch).

IgG Process

Ethanol Precipitation

The ethanol precipitation section of the IgG process is identical to that of the albumin process, except that
it is shorter, ending with the filtration of precipitate A (P-15 / PFF-102). The resulting filtration cake, rich in
gamma-globulins, is washed, dried, and sent for treatment with caprylic acid.

Caprylic Acid Treatment

Treatment with caprylic acid (a.k.a. octanoic acid) serves a double purpose: it inactivates enveloped viruses
and brings about the precipitation of alpha- and beta-globulins. First, the Precipitate A cake is re-solubilized
by adding 5 volumes of acetate buffer (0.2 M, pH = 4.8) and incubating for 4 h (P-20 / TPFF-201). Then,
caprylic acid is added to the vessel to a final concentration of approximately 0.17 mol/L, which leads to the
precipitation of 95% of the alpha- and beta-globulins. The precipitate is then removed using a plate & frame
filter (P-22 / PFF-201); the filtration cake is washed, dried, and discarded, while the filtrate and cake wash
are collected in a tank (P-26 / TDF-301).

Low pH Treatment

In this section, the protein mixture is incubated under low pH to inactivate viruses (enveloped viruses as
well as some non-enveloped viruses). First, the protein mixture is concentrated and diafiltered using a
crossflow filtration system (P-27 / DF-301) that retains IgG (as well as other plasma proteins). Upon
concentration, the protein titer is raised to 30 g/L. Diafiltration is performed with 10 volumes of acetate buffer
(0.2 M, pH = 4.1), and the average filtrate flux is 40 L/m2/h. The membrane rejection coefficients for the
proteins are assumed to be 1.0 in the concentration step and 0.995 in the diafiltration step. Next, the solution
is diluted to a protein concentration of 20 g/L and sent to a mixing tank (P-28 / TPFF-301) where the pH is
adjusted to 4 and a non-ionic detergent (polysorbate-80) is added to a final concentration of 18 μM. The
mixture is then heated to 37 °C and incubated for 9 h for extensive viral inactivation. Next, the pH is raised

10
to 6.5 with the aid of sodium hydroxide, in preparation for the next processing step (ion-exchange
chromatography). During this process, a substantial portion (60%) of the non-IgG gamma-globulins
precipitate. Filter aid is added to the mixture and then a plate & frame filtration step is performed (P-29 /
PFF-301) to remove the precipitated proteins. The resulting cake is washed, dried, and discarded, while
the filtrate and cake wash are collected in a tank (P-30 / T-401).

Ion-Exchange Chromatography

In this section, the protein solution is subjected to anion-exchange chromatography in negative mode, so
that impurities are bound by the column while the molecule of interest (IgG) essentially passes through
intact. This is achieved by loading the solution at pH 6.5, which corresponds to the isoelectric point of IgG.
Before loading the column, the mixture is diluted with WFI to a protein concentration of 10 g/L (P-30 / T-
401) and filtered through a dead-end microfilter (P-31 / MF-401) to avoid clogging the column. A weak-
anion exchange column is used (P-47 / C-401), with a resin binding capacity of 100 g/L. It was assumed
that 95% of the alpha-, beta- and non-IgG gamma-globulins bind to the resin, while only 5% of the IgG is
retained. The resin also binds the residual caprylic acid added earlier. The flowthrough is collected in a tank
(P-48 / T-402); it consists of a highly pure solution of IgG. During collection, the pH of the solution is adjusted
to 4.8 with the aid of 0.2M HCl. After the loading step, the column is stripped with a concentrated salt
solution (2M NaCl) and cleaned for the next cycle.

Formulation

The IgG solution first goes through a 20-nm filter (P-55 / MF-501) to remove enveloped and non-enveloped
viruses. After that, it is buffer exchanged using a crossflow filtration system (P-56 / TDF-501 and P-57 / DF-
501): first, the IgG solution is concentrated to 25 g/L; then, diafiltered with 10 volumes of acetate buffer (0.2
M, pH = 4.8); and, lastly, concentrated to 120 g/L. The membrane rejection coefficients for the proteins are
assumed to be 1.0 in the concentration steps and 0.995 in the diafiltration step. The average filtrate flux is
assumed to be 20 L/m2/h. Next, glycine and WFI are added to the solution, so that the final IgG
concentration is 100 g/L and the final glycine concentration is 0.25 M (glycine functions as a stabilizer). The
solution goes through a final sterile filtration step (P-58 / MF-502) and is filled into 20-L bags (P-59 / DCS-
501). A total of 124 L of IgG 10% (w/v) are produced per batch (i.e., 12 kg of IgG protein per batch).

11
Process Scheduling and Cycle Time Analysis

The overall Batch Time is 120.6 h for the albumin process and 81.5 h for the IgG process. This is the time
elapsed from the start of a given batch (i.e., thawing of fresh frozen plasma) to the end of that batch (sterile
filtration and storage of formulated albumin or IgG). However, the time between consecutive batches, called
Recipe Cycle Time (RCT), is 24 h in both processes. This is possible because most unit procedures in both
processes are shorter than one day, except for a few storage procedures, for which multiple (staggered)
tanks are employed. The user can find the overall Batch Time and the minimum RCT for the process in the
Recipe Scheduling Information dialog (Tasks Recipe Scheduling Information…). In this dialog, the user
may also specify the RCT that he or she desires, as long as it is larger than or equal to the minimum RCT.
The minimum RCT calculated by SuperPro Designer is 23.9 h for the albumin process, and 22.2 h for the
IgG process.

To visualize the process schedule, the user may click on Charts Equipment Occupancy Multiple
Batches. This will generate the Equipment Occupancy Chart (EOC), presented in Figure 3 for the albumin
process and in Figure 4 for the IgG process. The EOC displays the utilization of each equipment item over
time. A total of 6 consecutive batches are shown for each process, with each batch marked by a different
color. The process sections of each process are also indicated in the charts.

Notice that, in the albumin process, a few processing steps alternate between different equipment items
between batches; for example, batches #1, #3, and #5 utilize storage tank HT-101, whereas batches #2.
#4 and #6 utilize storage tank HT-101b. These tanks are said to operate in Staggered Mode. This
configuration enables the plant to initiate a new batch every 24 h, even though the storage step associated
with HT-101 takes longer than one day. Detailed information on how to specify equipment in Staggered
Mode can be found in the Farnesene example located in the Examples / Bio-Materials / Farnesene folder.

Another view of the process schedule is provided by the Operations Gantt chart (in the MS Project style).
That 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 5 displays a portion of
the Gantt chart for the albumin process, showing the scheduling of the first operations in the Ethanol
Precipitation section. The golden bar indicates the duration of the entire recipe, while 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 Designer can export its scheduling data to MS Project by selecting File Export
to MS Project XML File. Likewise, SuperPro Designer can export its recipe data to SchedulePro by

12
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).

13
 CIP Skids

Ethanol Precipitation

Ion-Exchange
Chromatography

Formulation

Figure 3: Equipment Occupancy Chart (EOC) for six consecutive batches of the albumin process.

14
 CIP Skids

Ethanol Precipitation

Caprylic Acid Treatment

Low pH
Treatment

Ion-Exchange
Chromatography

Formulation

Figure 4: Equipment Occupancy Chart (EOC) for six consecutive batches of the IgG process.

15
Figure 5: Operations Gantt Chart for the albumin process (portion of a single batch).

16
Material Balances

Table 2 and Table 3 display overall process data such as batch size, annual production rate and number
of batches per year for the albumin and IgG processes, respectively. These tables were extracted from the
RTF version of the corresponding 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.
The batch size is approximately 56 kg for albumin and 12 kg for IgG, which is reasonable considering that
the concentration of albumin in plasma is approximately 5 times that of IgG. The total number of batches
per year is almost identical for both processes: 325 for albumin and 327 for IgG; this was expected given
that the same recipe cycle time was specified for both processes, as discussed in the Process Scheduling
section. As a result, the annual production volume is approximately 18 MT for albumin and 4 MT for IgG.

Table 2: Overall process data for the albumin process.

OVERALL PROCESS DATA

Annual Operating Time 329.13 day

Unit Production Ref. Rate 18,187.89 kg MP/yr
Batch Size 55.96 kg MP
Recipe Batch Time 120.64 h
Recipe Cycle Time 24.00 h
Number of Batches per Year 325.00
MP = Flow of Component 'Albumin' in Stream 'S-159'

Table 3: Overall process data for the IgG process.

OVERALL PROCESS DATA

Annual Operating Time 329.43 day

Unit Production Ref. Rate 4,039.13 kg MP/yr
Batch Size 12.35 kg MP
Recipe Batch Time 81.46 h
Recipe Cycle Time 24.00 h
Number of Batches per Year 327.00
MP = Flow of Component 'IgG' in Stream 'S-161'

Table 4 and Table 5 were also extracted from the Materials & Streams report for albumin and IgG,
respectively. They list the raw material requirements for each process in kg/yr, kg/batch, and kg/kg of MP.
WFI, diafiltration buffers and CIP solutions are the materials used in largest quantities in both processes.
The consumption of fresh frozen plasma (FFP) is 36 kg/kg of product protein for albumin and 164 kg/kg of
product protein for IgG, reflecting the relative concentrations of those proteins in blood plasma.

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Table 4: Material requirements for the albumin process.

BULK MATERIALS (Entire Process)

Material kg/yr kg/batch kg/kg MP

Acetic Acid 1 M 10,589 32.58 0.58
AEX Cleaning Bf 165,584 509.49 9.10
AEX Eq/Wash Bfr 11,505,806 35,402.48 632.61
AEX Storage Bfr 76,479 235.32 4.20
AEX Strip Bfr 201,416 619.74 11.07
Buffer pH 4 10,678 32.86 0.59
Buffer pH 4.8 5,828 17.93 0.32
CIP-Acid 1,922,994 5,916.90 105.73
CIP-Caustic 3,208,132 9,871.18 176.39
Diatomite 81,515 250.81 4.48
Ethanol 95% 814,430 2,505.94 44.78
FFP 660,063 2,030.96 36.29
NaCl 90 mM 8,009,446 24,644.45 440.37
NaOH (1 M) 33 0.10 0.00
Sod. Caprylate 484 1.49 0.03
WFI 40,924,756 125,922.33 2,250.11
TOTAL 67,598,233 207,994.56 3,716.66

Table 5: Material requirements for the IgG process.

BULK MATERIALS (Entire Process)

Material kg/yr kg/batch kg/kg MP

AEX Cleaning Bf 122,411 374.34 30.31
AEX Cond Buffer 80,849 247.24 20.02
AEX Eq Buffer 120,460 368.38 29.82
AEX Regen Bfr 127,144 388.82 31.48
AEX Storage Bfr 76,233 233.13 18.87
Buffer pH 4 9,414 28.79 2.33
Buffer pH 4.1 2,446,072 7,480.34 605.59
Buffer pH 4.8 2,551,574 7,802.98 631.71
Caprylic Acid 13,306 40.69 3.29
CIP-Acid 1,577,836 4,825.19 390.64
CIP-Caustic 2,632,305 8,049.86 651.70
Diatomite 35,648 109.02 8.83
Ethanol 95% 326,001 996.95 80.71
FFP 664,125 2,030.96 164.42
Glycine 758 2.32 0.19
HCl (0.2 M) 40 0.12 0.01
NaOH (0.2 M) 166 0.51 0.04
Polysorbate 80 7 0.02 0.00
WFI 31,885,136 97,508.06 7,894.07
TOTAL 42,669,486 130,487.72 10,564.04

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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 6 and Table 7 display the Executive Summary extracted from the EER for albumin and IgG,
respectively.

Table 6: Executive summary for the albumin process.

EXECUTIVE SUMMARY (2022 prices)

Total Capital Investment 114,776,000 $

Capital Investment Charged to This Project 114,776,000 $
Operating Cost 81,078,000 $/yr
Revenues 91,322,000 $/yr
Batch Size 55.96 kg MP
Cost Basis Annual Rate 18,188 kg MP/yr
Unit Production Cost 4,457.82 $/kg MP
Net Unit Production Cost 4,457.82 $/kg MP
Unit Production Revenue 5,021.05 $/kg MP
Gross Margin 11.22 %
Return On Investment 13.72 %
Payback Time 7.29 years
IRR (After Taxes) 10.04 %
NPV (at 7.0% Interest) 18,404,000 $
MP = Flow of Component 'Albumin' in Stream 'S-159'

Table 7: Executive summary for the IgG process.

EXECUTIVE SUMMARY (2022 prices)

Total Capital Investment 90,735,000 $

Capital Investment Charged to This Project 90,735,000 $
Operating Cost 130,877,000 $/yr
Revenues 242,348,000 $/yr
Batch Size 12.35 kg MP
Cost Basis Annual Rate 4,039 kg MP/yr
Unit Production Cost 32,402.33 $/kg MP
Net Unit Production Cost 32,402.33 $/kg MP
Unit Production Revenue 60,000.00 $/kg MP
Gross Margin 46.00 %
Return On Investment 98.67 %
Payback Time 1.01 years
IRR (After Taxes) 73.73 %
NPV (at 7.0% Interest) 563,527,000 $
MP = Flow of Component 'IgG' in Stream 'S-161'

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The total capital investment (TCI) is estimated to be $115 million for albumin and $91 million for IgG. The
TCI includes equipment purchase and installation costs; costs related to plant construction such as
buildings, piping, and instrumentation; startup and validation costs; and the working capital required for this
project. Plant construction costs are estimated through multipliers in SuperPro Designer, and these have
been modified in this example to represent a facility that manufactures biopharmaceuticals. For more
information on capital cost estimations, please refer to the Industrial Enzymes example in the Examples
/ Bio-Materials / Industrial Enzymes folder or consult the Help tool (Help Search  Capital Investment
Dialog: DFC Tab).

In reality, the same facility would manufacture both albumin and IgG (and possibly other products such as
fibrinogen, factor VIII, etc.), so that a significant portion of the capital costs would be shared by both
processes. In particular, several equipment items in the Ethanol Precipitation section would be shared by
both processes, thus reducing the total capital investment.

Table 6 and Table 7 also display the unit production cost calculated for each process: approximately $4.5/g
of product albumin protein, and $32/g of IgG protein. Assuming a selling price of $5/g of albumin and $60/g
of IgG, the gross margin would be roughly 11% for albumin and 46% for IgG. As expected, IgG would be
the main source of profit for the overall plasma fractionation manufacturing facility.

To evaluate albumin and IgG production costs, it is necessary to account for the fact that many operating
expenses are shared by both products: a significant portion of the facility-dependent cost, labor, utilities,
waste treatment and, most importantly, raw materials in the Ethanol Precipitation section are shared by
both processes. In particular, the cost of blood plasma is quite large ($150/L), and it must be allocated to
all the different products manufactured in a plasma fractionation plant. Considering that for one liter of
plasma the revenues from IgG are about 3 times the revenues from albumin, the cost of plasma was set to
25% of its actual value in the albumin process, and to 75% in the IgG process. The cost of other key raw
materials employed in the Ethanol Precipitation section such as WFI, ethanol and diatomaceous earth were
allocated to both processes in a similar fashion.

Figure 6 and Figure 7 display breakdowns of the AOC for albumin and IgG, respectively; these were also
taken from the Economic Evaluation Report. Raw materials account for 50% of the production cost of
albumin, followed by the facility-dependent cost (19%) and consumables cost (16%). The contribution of
raw materials is even larger for IgG, corresponding to 75% of its production cost; the second largest
component is the facility-dependent cost, accounting for only 10%. A larger contribution of raw materials
was expected for IgG due to the higher allocation of plasma cost described in the previous paragraph.

20
Figure 6: Annual operating cost breakdown for the albumin process.

Figure 7: Annual operating cost breakdown for the IgG process.

In order to better understand the contribution of raw materials to manufacturing costs, Table 8 and Table 9
were extracted from the Economic Evaluation Report for albumin and IgG, respectively. These tables
provide breakdowns of raw material costs for each process. Fresh frozen plasma (FFP) corresponds to
62% of the total raw material cost for albumin; ethanol and WFI come next, accounting for 12% and 10%,
respectively. In the case of IgG, plasma represents 76% of the raw materials cost, followed by WFI and
ethanol, which constitute 10% and 6%, respectively. Even though the unit cost of plasma in the albumin
process ($37.50/L) was considered to be one-third of that in the IgG process ($112.5/L), the manufacturing
process for albumin is somewhat simpler and consumes lower amounts of other materials, which explains
why the percentage contributions of plasma are not so distant between the two processes. In fact, the

21
annual cost of raw materials is $40 million for albumin (of which $25 million corresponds to plasma) and
$98 million for IgG (of which $75 million corresponds to plasma).

Table 8: Breakdown of material costs for the albumin process.

MATERIALS COST - PROCESS SUMMARY

Unit Cost Annual Annual Cost

Bulk Material %
($) Amount ($)
Acetic Acid 1 M 0.43 10,589 kg 4,578 0.01
AEX Cleaning Bf 0.87 165,584 kg 144,469 0.36
AEX Eq/Wash Bfr 0.13 11,505,806 kg 1,440,634 3.59
AEX Storage Bfr 1.32 76,479 kg 100,749 0.25
AEX Strip Bfr 0.74 201,416 kg 149,353 0.37
Buffer pH 4 3.11 10,678 kg 33,203 0.08
Buffer pH 4.8 3.19 5,828 kg 18,562 0.05
CIP-Acid 0.50 1,922,994 kg 957,651 2.39
CIP-Caustic 0.49 3,208,132 kg 1,572,113 3.92
Diatomite 12.50 81,515 kg 1,018,933 2.54
Ethanol 95% 5.88 814,430 kg 4,786,705 11.93
FFP 37.50 660,063 kg 24,752,372 61.67
NaCl 90 mM 0.13 8,009,446 kg 1,049,590 2.62
NaOH (1 M) 0.77 33 kg 25 0.00
Sod. Caprylate 30.00 484 kg 14,508 0.04
WFI 0.10 40,924,756 kg 4,092,476 10.20
TOTAL 40,135,921 100.00

22
Table 9: Breakdown of material costs for the IgG process.

MATERIALS COST - PROCESS SUMMARY

Unit Cost Annual Annual Cost

Bulk Material %
($) Amount ($)
AEX Cleaning Bf 0.68 122,411 kg 83,260 0.08
AEX Cond Buffer 0.79 80,849 kg 63,502 0.06
AEX Eq Buffer 0.32 120,460 kg 39,087 0.04
AEX Regen Bfr 0.92 127,144 kg 116,943 0.12
AEX Storage Bfr 3.95 76,233 kg 301,274 0.31
Buffer pH 4 3.25 9,414 kg 30,606 0.03
Buffer pH 4.1 0.45 2,446,072 kg 1,107,205 1.13
Buffer pH 4.8 0.62 2,551,574 kg 1,569,725 1.60
Caprylic Acid 30.00 12,847 L(STP) 385,423 0.39
CIP-Acid 0.69 1,577,836 kg 1,095,018 1.12
CIP-Caustic 0.69 2,632,305 kg 1,806,077 1.84
Diatomite 37.50 35,648 kg 1,336,804 1.36
Ethanol 95% 17.63 326,001 kg 5,748,091 5.87
FFP 112.50 664,125 kg 74,714,081 76.24
Glycine 50.00 758 kg 37,901 0.04
HCl (0.2 M) 0.37 40 kg 15 0.00
NaOH (0.2 M) 0.45 166 kg 75 0.00
Polysorbate 80 100.00 7 kg 749 0.00
WFI 0.30 31,885,136 kg 9,565,541 9.76
TOTAL 98,001,377 100.00

The results of the present example suggest that the proposed processes for production of albumin and IgG
from plasma would make for a profitable investment, even though albumin production, considered
individually, would not be viable. This analysis corroborates the discussion on “last liter economics”
presented in the Introduction: the production of multiple products from plasma is economically
advantageous because the revenues from less profitable products such as albumin help to offset the large
cost of plasma.

It should be noted, however, that many variables affect the attractiveness of this kind of project, such as
the selling prices of IgG and albumin; the purchasing price of key raw materials such as plasma and their
allocation to different products; recovery yields in each processing step; etc. As a result, the actual
economics of such an investment may be substantially better or worse than the current projections. A useful
exercise is to perform sensitivity analyses with SuperPro Designer to determine the impact of these
changes; this allows the user to understand the potential risks and rewards of a project under different sets
of assumptions. Different 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 Designer through MS Excel and automate sensitivity analysis,
please consult the examples in the Examples / COM folder. Related information is available in the Help

23
facility of the tool, which can be accessed by selecting Help  COM Interface and Library. Math
optimization and Monte Carlo simulation can be performed in a similar manner.

References

[1] American Red Cross. Plasma Information n.d. https://www.redcrossblood.org/donate-

blood/dlp/plasma-information.html.

[2] Burnouf T. An overview of plasma fractionation. Ann Blood 2018;3:33–33.

doi:10.21037/aob.2018.05.03.

[3] Burnouf T. Modern Plasma Fractionation. Transfus Med Rev 2007;21:101–17.

doi:10.1016/j.tmrv.2006.11.001.

[4] Bertolini J, Goss N, Curling J, editors. Production of Plasma Proteins for Therapeutic Use. Hoboken,
NJ, USA: Wiley; 2013. doi:10.1002/pmic.201370099.

[5] Lundblad RL. Biotechnology of plasma proteins. 2012. doi:10.1201/b12368.

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