Before the fermentation can begin, the substrate must be sterilized to avoid contamination of the fermentation broth. For this procedure a batch sterilization has been chosen. To ensure sterility the feed is heated to 121 [°C], the pressure set at 2 [bar] and the residence time at 30 [min]. Reference: Michael L. Shuler and Fikret Kargi. Bioprocess Engineering - Basic Concepts; second edition; Pearson Education International; 2002 This was simulated in Aspen Plus using the BK10 method, and only the compounds available in the database were considered. The simulation showed a volume of the autoclave of 26.6 [L] and a heat duty of 48,483 [kJ/batch]. The substrate consist of Water (H₂O), Glucose, Ammonium chloride (NH₄Cl), Monopotassium phosphate (KH₂PO₄), Epsom salt (MgS·7 H₂O), Calcium chloride dihydrate (CaCl₂·2H₂O), Ironsulphate (FeSO₄), L-arganine·HCl, tracemetals and Ampicilin, the composition can be seen in table 1. The amount and composition of the substrate was based on a research artic le.. Reference: L. Yee and H. W. Blanch. Recombinant Trypsin Production in High Cell Density Fed-Batch Cultures in Escherichia coli. Biotechnology and Bioengineering. 1993; 41: 781-790

Table 1: Composition and conditions for A1

	S1: Feed 1	S2: Feed 1
Total mass [kg]	24.04	24.04
Temperature [°C]	21	121
Pressure [bar]	1	2
WT% H2O	87.53	87.53
WT% Glucose	2.00	2.00
WT% NH4Cl	0.13	0.13
WT% KH2PO4	1.50	1.50
WT% MgS·7 H2O	0.01	0.01
WT% CaCl2·2H2O	0.01	0.01
WT% FeSO4	0.01	0.01
WT% L-arginine·HCI	0.80	0.80
WT% TraceMetals	8.00	8.00
WT% Ampicilin	0.01	0.01

To avoid a flash occurring in the fermenter and ensure the survivel of cells, the substrate has to be cooled to 37 .[°C]. Reference: L. Yee and H. W. Blanch. Recombinant Trypsin Production in High Cell Density Fed-Batch Cultures in Escherichia coli. Biotechnology and Bioengineering. 1993; 41: 781-790 It has been chosen to grow the cells at this temperature, though growth at lower temperatures have been shown to decrease the amount of inclusion bodies formed. It has to be investigated at a later point if lower temperatures and the following prolongation of cycle time, could lead to an increase in production. Reference: Jeffrey Fu, David B. Wilson and Michael L. Shuler. Continuous, High Level Production and Excretion of a Plasmid-Encoded Protein by Escherichia coli in a Two-Stage Chemostat. Biotechnology and Bioengineering, 1993; 41: 937-946 As in A1 a simulation was run in Aspen Plus, the results showed the use of cooling water to be 5,474 [L/batch], and a heat duty of 15,196,014 [kJ/batch].

Table 2: Composition and conditions for E1

	S1: Feed 1	S2: Feed 1
Total mass [kg]	24.04	24.04
Temperature [°C]	21	121
Pressure [bar]	1	2
WT% H2O	87.53	87.53
WT% Glucose	2.00	2.00
WT% NH4Cl	0.13	0.13
WT% KH2PO4	1.50	1.50
WT% MgS·7 H2O	0.01	0.01
WT% CaCl2·2H2O	0.01	0.01
WT% FeSO4	0.01	0.01
WT% L-arginine·HCI	0.80	0.80
WT% TraceMetals	8.00	8.00
WT% Ampicilin	0.01	0.01

Fermentation is carried out under two conditions. In this first phase growth is carried out as a batch until glucose is depleted, and will take approximately 13 [h]. After which, cells will be grown to high densities in a fed-batch. One hindrance for accumulation of high biomass concentrations is the formation of inhibitors as acetate. By restricting the carbon source this formation can be reduced greatly, which can be viewed in figure 5. The figure describes the experimental results presented in the research paper used for estimates. The inoculum is scale linearly as in the case of the substrate. Therefore the same cell concentration is kept and the time will not be affected. The fed-batch and induction will be discussed further below. Reference: L. Yee and H. W. Blanch. Recombinant Trypsin Production in High Cell Density Fed-Batch Cultures in Escherichia coli. Biotechnology and Bioengineering. 1993; 41: 781-790

Table 3: Composition and conditions for E1.1

	S3: Feed 1	S4: innoculum	R1: first phase
Total mass [kg]	24.04	1.72	25.76
Temperature [°C]	37	21	37
Pressure [bar]	1	1	1
WT% Dry cells		0.03	0.70
WT% H2O	87.53	87.50	88.84
WT% Glucose		2.00	2.00
WT% NH4Cl	0.13	0.13	0.13
WT% KH2PO4	1.50	1.50	1.50
WT% MgS·7 H2O	0.01	0.01	0.01
WT% CaCl2·2H2O	0.01	0.01	0.01
WT% FeSO4	0.01	0.01	0.01
WT% L-arginine·HCI	0.80	0.80	0.80
WT% TraceMetals	8.00	8.00	7.99
WT% Ampicilin	0.01	0.01	0.005

The substrate used for the fed-batch phase of the fermentation is sterilized in A2. The same type of sterilization and conditions as in the A1 are chosen. Simulation in Aspen Plus yielded a volume of 16.2 [L] and a heat duty of 7.457 [kJ/batch]. The composition of the substrate can be viewed in table 4. It should be noted that in order to avoid dilution of the cell concentration, the concentration of glucose is considerably higher than in S1.. Reference: L. Yee and H. W. Blanch. Recombinant Trypsin Production in High Cell Density Fed-Batch Cultures in Escherichia coli. Biotechnology and Bioengineering. 1993; 41: 781-790

Table 4: Composition and conditions for A2

	S5: Feed 2	S6: Feed 2
Total mass [kg]	18.89	18.89
Temperature [°C]	21	121
Pressure [bar]	1	2
WT% H2O	30.54	30.54
WT% Glucose	65.00	65.00
WT% NH4Cl	0.50	0.50
WT% KH2PO4	0.17	0.17
WT% MgS·7 H2O	0.75	0.75
WT% L-arginine·HCI	3.00	3.00
WT% Ampicilin	0.04	0.04

The substrate of the fed-batch has to be cooled as in the case of E1. The same conditions are chosen, see table 5, and the exchanger was simulated in Aspen Plus. The simulation showed that 348 [L/batch] cooling water is needed, and that the heat duty is 4,016,502 [kJ/batch].

Table 5: Composition and conditions for E1

	S6: Feed 2	S7: Feed 2
Total mass [kg]	18.89	18.89
Temperature [°C]	121	37
Pressure [bar]	2	1
WT% H2O	30.54	30.54
WT% Glucose	65.00	65.00
WT% NH4Cl	0.50	0.50
WT% KH2PO4	0.17	0.17
WT% MgS·7 H2O	0.75	0.75
WT% L-arginine·HCI	3.00	3.00
WT% Ampicilin	0.04	0.04

As mentioned above, the fed-batch facilitate high yield of dry cells. The experimental data from the research paper showed that a concentration of 75 [g dry cells/L] was reached after 27 [h]. This was deemed an appropriate level to start induction. The results also showed that substrate was fed in a rate, where glucose did not accumulate in the broth, and furthermore that the levels of acetate after an initial rise was non- existent in the end of the fed-batch phase, see figure 5: Batch fermentation. The composition of the fermentation broth before induction can be viewed in table 6. Reference: L. Yee and H. W. Blanch. Recombinant Trypsin Production in High Cell Density Fed-Batch Cultures in Escherichia coli. Biotechnology and Bioengineering. 1993; 41: 781-790

Michael L. Shuler and Fikret Kargi. Bioprocess Engineering - Basic Concepts; second edition; Pearson Education International; 2002

Table 6: Composition and conditions for R1.2

	S7: Feed 2	R1: Second phase
Total mass [kg]	18.89	44.65
Temperature [°C]	37	37
Pressure [bar]	1	1
WT% Dry cells		7.50
WT% H2O	30.54	84.59
WT% Glucose	65.00
WT% NH4Cl	0.50	0.29
WT% KH2PO4	0.17	0.94
WT% MgS·7 H2O	0.75	0.32
WT% CaCl2·2H2O		0.004
WT% FeSO4		0.01
WT% L-arginine·HCI	3.00	1.73
WT% TraceMetals		4.61
WT% Ampicilin	0.04	0.02

Now the production of peptide aptamers can begin. The production is induced by Isopropyl-β-D- thiogalactoside (IPTG), which is added to a concentration of 2 [mM]. The target concentration of product will be discussed during the elution phase of the product recovery. The composition at start of induction and of the fermentation broth can be viewed in table 7. It was shown that inducing in the late log phase let to high yields of production, reducing the production phase to 4 [h]. Another benefit of the shortened residence time of the product, which reduces the risk of degradation by for example proteases. In the research paper the yield of product was reported to be 56 [mg/L] and the dry cell concentration was 92 [g/L]. Reference: L. Yee and H. W. Blanch. Recombinant Trypsin Production in High Cell Density Fed-Batch Cultures in Escherichia coli. Biotechnology and Bioengineering. 1993; 41: 781-790

Michael L. Shuler and Fikret Kargi. Bioprocess Engineering - Basic Concepts; second edition; Pearson Education International; 2002

Table 7: Composition and conditions for R1.3

	R1: induction start	S8: Fermentation broth
Total mass [kg]	44.64	44.64
Temperature [°C]	37	37
Pressure [bar]	1	1
WT% Peptide aptamer		0.01
WT% Dry cells	7.50	9.20
WT% H2O	84.54	82.89
WT% NH4Cl	0.29	0.29
WT% KH2PO4	0.94	0.94
WT% MgS·7 H2O	0.32	0.32
WT% CaCl2·2H2O	0.004	0.004
WT% FeSO4	0.01	0.01
WT% L-arginine·HCI	1.73	1.73
WT% TraceMetals	4.61	4.60
WT% Ampicilin	0.02	0.02
WT% IPTG	0.05

This is the first step of the product recovery. Before applying the fermentation broth to the column it needs to be fluidized. The fluidization process is described in greater detail during the next step. An important parameter for the creation of a stable bed is viscosity, should viscosities of the different buffers used vary in a degree that destabilizes the bed an inert viscous liquid will be added.. Reference: Pharmacia. Introduction to Expanded Bed Adsorption.
(Link) Accessed July 18^th 2015]

The expansion is achieved be applying 10 bed volumes (BV) of column buffer (20 mM (4-(2- Hydroxyethyl)piperazine-1-ethanesulfonic acid sodium salt) (Na-HEPES) and 500 mM Sodium chloride (NaCl)) at a flowrate of 300 [cm/h], which is recommended, though flows between 200 and 400 [cm/h] has been shown sufficient. The composition can be viewed in table 8. Determination of the BV relies on the production (see equation 1), it has been decided to produce 2 [g/batch] of the peptide aptamer. This decision will be elaborated in the decription of the elution step (C1.4). Reference: NEB-impact

Pharmacia. Introduction to Expanded Bed Adsorption.
(Link) Accessed July 18^th 2015]

BV [L]=(binding capacity of the beads [g/L])/(mass of product [g] ) = 1.19 L (1)

To estimate the time needed for equilibration, we need to estimate the volume of the column. Expansion is reported to be 2-3 BV, therefore an expansion of 2.5 BV was chosen for calculations. Thus the column should be able to contain approximately 3 L. A column with a diameter of 50 [mm] is deemed to be appropriate for this process. To transfer this flowrate to a volumetric, it is calculated how large the volume is per cm, which is approximately 0.02[L]. This corresponds to a volumetric flowrate of 6 [L/h]. Now the time of application can be calculated to be 2 [h] and the height of the column to be 1.5 [m]. Reference: NEB-impact

Pharmacia. Introduction to Expanded Bed Adsorption.
(Link) Accessed July 18^th 2015]

Table 8: Composition and conditions for C1.1

	S9: equilibration	S10: Waste
Total mass [kg]	11.88	11.88
Temperature [°C]	21	21
Pressure [bar]	1	1
WT%	96.60	96.60
WT% NaHEPES	0.48	0.48
WT% NaCl	2.92	2.92

The benefit of the EBA is, that the fluidization leaves space between the beads for particulate matter to pass through. Rendering the solid separation steps of traditional product recovery unnecessary. This type of chromatography has been reported to be a one-step purification. Another benefit is the time reduction before the protein is bound to the beads, further reducing the residence time.. Reference: Pharmacia. Introduction to Expanded Bed Adsorption.
(Link) Accessed July 18^th 2015]

The bed is fluidized by controlling the density of the beads, and thereby the downward force of sedimentation, and the upward force created by the flow. The sedimentation velocity is controlled by the design of the beads. In a stable bed the beads follow a Gaussian distribution. This is obtained by varying the density of the beads. Therefore the ordinary agarose beads are fitted with inert cores of varying diameters. An unstable bed leads to risk of turbulence or channelling, which will say that the feed does not have sufficient contact with the beads to bind the peptide aptamers efficiently. Reference: Pharmacia. Introduction to Expanded Bed Adsorption.
(Link) Accessed July 18^th 2015]

In this step the fermentation broth is applied first, after which all impurities are washed out of the column by applying 20 BV of column buffer. The streams can be viewed in table 9. Reference: NEB-impact

Table 9: Composition and conditions for C1.2

	S8: Fermentation broth	S11: Wash	S12: Waste
Total mass [kg]	44.64	23.75	68.39
Temperature [°C]	37	21	21
Pressure [bar]	1	1	1
WT% Peptide aptamer	0.01
WT% Dry cells	9.20		6.01
WT% H2O	82.89	96.60	88.83
WT% NH4Cl	0.29		0.19
WT% KH2PO4	0.94		0.61
WT% MgS·7 H2O	0.32		0.21
WT% CaCl2·2H2O	0.004		0.0026
WT% FeSO4	0.01		0.01
WT% L-arginine·HCI	1.73		1.13
WT% TraceMetals	4.60		3.00
WT% Ampicilin	0.02		0.01
WT% NaHEPES		0.48	0.17
WT% NaCl		2.92	1.01

It is assumed that all cells will be contained in S10 and S12 and should be sterilized.. The conditions during sterilization is the same as that of the A1 and A2. The simulation in Aspen Plus yields the following results: a volume of 80.2 [L] and a heat duty of 167,301,000 [kJ/batch].

Table 10: Composition and conditions for A3

	S21: Waste	S22: Waste
Total mass [kg]	80.27	80.27
Temperature [°C]	21	121
Pressure [bar]	1	2
WT% Dry cells	5.12	5.12
WT% H2O	89.98	89.98
WT% NH4Cl	0.16	0.16
WT% KH2PO4	0.52	0.52
WT% MgS·7 H2O	0.18	0.18
WT% CaCl2·2H2O	0.002	0.002
WT% FeSO4	0.005	0.005
WT% L-arginine·HCI	0.96	0.96
WT% TraceMetals	2.56	2.56
WT% Ampicilin	0.01	0.01
WT% NaHEPES	0.21	0.21
WT% NaCl	1.30	1.30

Before releasing the waste stream to the ordinary waste water system, the pressure has to be lowered. Therefore the stream is cooled to ensure that the waste leaves as a liquid. This unit was simulated in Aspen Plus, the results are as follows: 14,854 [L/batch] cooling water is needed, and the heat duty is 172,581,480 [kJ/batch]. The composition does not change and can be viewed in table 11.

Table 11: Composition and conditions for E3

	S22: Waste	S23: Waste
Total mass [kg]	80.27	80.27
Temperature [°C]	121	99
Pressure [bar]	2	2
WT% Dry cells	5.12	5.12
WT% H2O	89.98	89.98
WT% NH4Cl	0.16	0.16
WT% KH2PO4	0.52	0.52
WT% MgS·7 H2O	0.18	0.18
WT% CaCl2·2H2O	0.002	0.002
WT% FeSO4	0.005	0.005
WT% L-arginine·HCI	0.96	0.96
WT% TraceMetals	2.56	2.56
WT% Ampicilin	0.01	0.01
WT% NaHEPES	0.21	0.21
WT% NaCl	1.30	1.30

In this step Intein selfcleaves releasing the peptide aptamer. This is achieved by flushing the column with 3 BV of cleavage buffer (20 mM Na-HEPES, 500 mM NaCl, 50 mM threo-1,4-Dimercapto-2,3- butanediol (DTT)), where after the resin is left at room temperature for 16 h, therefore it is assumed that all buffer leaves the column as waste in this step. It has been reported, that product recovery is above 95%, therefore it is assumed that 95% will be cleav ed.. Reference: NEB-impact

Table 12: Composition and conditions for C1.3

	S13: Cleavage	S14: Waste
Total mass [kg]	3.76	3.76
Temperature [°C]	21	21
Pressure [bar]	1	1
WT% H2O	95.83	95.83
WT% NaHEPES	0.48	0.48
WT% NaCl	2.92	2.92
WT% DTT	0.77	0.77

Now the product is ready to be eluted with column buffer. Since the peptide aptamers are an alternative to monoclonal antibodies, the concentration of the end product has been chosen by comparison of 50 products from Sigma-Aldrich. Concentrations of the antibodies range from 0.5-2 g/L, the most common concentration (39 out of 50) was found to be 2 g/L, thus making this the target concentration of PAST’s products. See pdf (Sigma-Aldrich antibodies) for details. Reference: NEB-impact Pharmacia. Introduction to Expanded Bed Adsorption.
(Link) Accessed July 18^th 2015

Sigma-Aldrich. (Link) Accessed August 1^st 2015.

Due to the changing productions and the decision to release the broth at a predefined viscosity, concentrations of the product in each batch will fluctuate. Therefore the aim is to produce 2.5 g of the peptide aptamer per batch. The wanted concentration is reached by measuring the concentration at the release of the broth and adjust the elution accordingly. This determines the volume of the elution, the result can be viewed in table 13. Elution is performed with column buffer. If this buffer is not suited for storage, it can be changed by dialysis, though for now it is assumed appropriate. Reference: NEB-impact

Pharmacia. Introduction to Expanded Bed Adsorption.
(Link) Accessed July 18^th 2015]

Table 13: Composition and conditions for C1.4

	S15: Elution	S16: product
Total mass [kg]	1.19	1.19
Temperature [°C]	21	21
Pressure [bar]	1	1
WT% Peptide aptamer		0.20
WT% H2O	96.60	96.40
WT% NaHEPES	0.48	0.48
WT% NaCl	2.92	2.92

The last step of the product recovery is regeneration of the column, which prepares it for the next batch. It has been reported that the resin can be reused 4-5 times, it is assumed that 4 times is appropriate. Regeneration is performed by washing the bed with 3 BV of stripping solution (0.3 M Sodium hydroxide (NaOH)) and then leaving the bed to soak for 30 minutes. After which the column is washed with additional 7 BV of the stripping solution, followed with 20 BV of water. Lastly 5 BV of column buffer is applied. Reference: NEB-impact

Table 14: Composition and conditions for C1.5

	S17: Stripping	S18: Water	S19: Column buffer	S20: Waste
Total mass [kg]	11.88	8.31	5.94	26.13
Temperature [°C]	21	21	21	21
Pressure [bar]	1	1	1	1
WT% H2O	99.64	100	96.60	99.06
WT% NaHEPES			0.48	0.11
WT% NaCl			2.92	0.66
WT% NaOH	0.36			0.16