Journal of Annals of Bioengineering

Research Article

Ultra Scale-Down Characterization And Stability Of Monoclonal Fusion Proteins Used To Treat Cancer

Peter Blas*

The Kibworth School, Kibworth, Leicester, England, United Kingdom

Received: 21 July 2019

Accepted: 12 September 2019

Version of Record Online: 07 October 2019

Citation

Blas P (2019)Ultra Scale-Down Characterisation and Stability of Monoclonal Fusion Proteins Used to Treat Cancer. J Ann Bioeng2019(1): 69-80.

Correspondence should be addressed to
Peter Blas, United Kingdom

E-mail: peter_blas@hotmail.com
DOI: 
10.33513/BIOE/1901-07

Copyright

Copyright © 2019 Peter Blas. This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and work is properly cited.

Abstract

Monoclonal antibodies and fusion proteins are increasingly being engineered to treat an extensive array of diseases including Alzheimer’s, Cancerand Parkinson’s. Characterisation at small scale is vital to ensure the product is not lost during large scale manufacture. Very high valued process material can be characterised in an Ultra Scale-Down (USD) shear device to identify ways to improve its large-scale bioprocessing. The device can be used to uncover parameters that degrade or protect the protein during the manufacturing of this biopharmaceutical. This article presents that the presence of shear and varying bioprocess conditions degrades a monoclonal fusion protein this degradation can be reduced by chemical protection. This work shows that USD studies can be used to uncover protective agents of a therapeutic used in cancer therapy, which may inform how to improve the large-scale productivity of monoclonal antibody fusion proteins.

Keywords

Biopharmaceutical; Fusion Proteins; Monoclonal Antibodies Ultra Scale-Down; Shear

Introduction

The ultra scale-down characterisation of the protein solutions is an important part of biopharmaceutical development, as it could uncover how proteins are degraded in shear associated fields that are prevalent in bioprocessing environments [1-4]. As monoclonal antibody fragments are being used more commonly all the time [5], categorisation of complicated fusion proteins fragments and how they interact with robust bioprocess conditions needs to be achieved [6]. Detailed characterisation of complex protein structures during their interactions with harsh bioprocessing environments could ensure high yields are produced in the final protein product [4].

Various research groups have shown that fragile fusion proteins and monoclonal antibodies are degraded by shear forces and harsh hydrodynamic conditions [7-10]. The first scientists who published that enzymes lost their activity in high shear conditions wereCharm and Wong [11], they concluded that carboxypeptidases and two other proteins were denatured when shear with no air/liquid interface was present in the system. These findings were confirmed by Tirrell and Middleman [12]. Conversely other scientists Thomas and Dunnill [13], and Maa and Hsu [14] have shown that shear alone has little effect on protein stability, they have proposed that shear (without air liquid conditions) has little effect on the proteins they investigated for example alcohol Dehydrogenase (ADH) and recombinant human Growth Hormone (rhGH) did not denature or break down, but shear forces with bubble entrapment lead to the deactivation of these proteins, it was unsure if denaturing/deactivation or aggregation was occurring [2,12]. The variation in research results could be because different shear devices have varying levels of solid/liquid or air/liquid interfaces found within the system [15].

Work by Boychyn [16], described how different air/liquid interfaces within an industrial disc-stack centrifuge can produce immense shear stresses. Variations in protein stability could be attributed to different protein structures interacting with solid/liquid and air/liquid interfaces with shear stress. It has been hypothesised that the hydrophilic and hydrophobic nature of proteins could allow them to unfold at the interfaces [17]. Therefore, additional shear effects like these could be important in the way that proteins are deactivate [18,19].

The biopharmaceutical, MFECP1, investigated in this study was a therapeutic used in Antibody-Directed Enzyme-Prodrug Therapy(ADEPT),this worked in a two-step drug delivery system, in which an enzyme was directed to a tumour because of its attachment to an antibody, where it converted a prodrug into a toxic chemical that destroys cancer cells [20]. This process was proposed byBagshawe [21], and functions as another way to target antibody to tumour cells, like guided radiation (RIT) or other radio immuno-conjugates. The ADEPT therapy worked by injecting the patient with a MFECP1 fusion protein, which comprises of a ScFv antibody fragment (called MFE-23) fused to an enzyme, in this case Carboxypeptidase (CPG2) [5]. Next the ScFv antibody fragment recognises and attaches to a cancer glycoprotein Carcino-Embryonic Antigen (CEA) [22], which was highly expressed on the surface of cancer cells in patients with colorectal carcinoma [23].

The specific affinity of the antibody fragment to the cancer antigen marker CEA allows the build-up of the MFECP1 fusion protein precisely at the site of action. After adequate time for clearance of unbound antibody fragments from the rest of the body, a prodrug is injected. This prodrug is cleaved into a cytotoxic drug by the CPG2 enzyme, at the tumour location resulting in the destruction of cancer cells. The system can only work if the fusion protein is intact, the enzyme cannot be directed to the cancer site if the MFECP1 has under gone breakdown due to shear hence the reason the present line of work.

This article addresses how the monoclonal MFECP1fusion protein therapeutic used in ADEPT in small scale experiments responded to a range of shear-associated affects in the USD device [5,21,22]. It examines how the purified fusion protein may be characterised and explains why the ELISA was the best procedure to detect protein in degraded sheared solutions. It also describes how the same fusion protein was used to establish the optimum parameters to observe changes due to shear associated degradation in a shear device. A first order kinetic relationship first published [24] was used to classify the rate of fusion protein deactivation and this was then used to quantify the rate of protein loss during different shear environment. The affect of air/liquid interfaces, protein concentration, change in protein structure concentration and the addition of protective agents was investigated and the rate of degradation was assessed. This work identifies factors that might help with the large-scale manufacture of monoclonal fusion proteins.

Materials and Methods

Preparation of MFECP1 fusion protein stocks

All MFECP1 fusion protein stock solutions were made up in a 0.01M Phosphate Buffer Saline solvent which had a p Hof 7 (PBS); 0.003M KCl; 0.1M NaCl and made up with de-ionized water v/v (18.2 Ω, Milli-Q system).MFECP1 fusion protein stock solutions were made up to around, 500 ng/mL and were stored on ice when not in use during shear experiments. GMP fermentations were conducted in the Copy May Production Unit at the Royal Free Hospital, London, England.

Shear device experiments

The therapeutic fusion protein used in ADEPT identified [25,26], was exposed to measured shear stress with a spinning disc shear device, the dimensions of which can be found in figure 1. This shearing equipment was produced in stainless steel and contained a revolving disc with an empty concentric cylinder. The diameter of the disc was 40mm with a thickness of 1.5 mm. The rotation of the disc was achieved by attachment to a 7-volt motor with a stainless-steel shaft going through a water proof plastic stopper. The shear stress device could hold a maximum volume of around 20 mL. Throughout the shear stress experiments the internal temperature of the chamber was maintained with an ice cooled water bath and monitored with a thermocouple at 4°C, a revolution counter measured the disc speed in rpm made in the Department of Biochemical Engineering, UCL, London.

Ultra Scale-Down Characterization And Stability Of Monoclonal Fusion Proteins Used To Treat Cancer

Figure 1: Shows the internal dimensions of the ultra scale-down shear device with a tapered top inside the device. Characterization of the monoclonal fusion protein solution with and without an air/Liquid interface was performed in the shear device. All shear experiments were conducted at a constant temperature of 4°C maintained by an ice-cooled water bath. All dimensions shown are in millimeters, the device held a total volume of approximately 20 mL of protein solution.

GMP manufactured therapeutic pure standard solutions of the protein, (20 mL, at around 0.50 µg/mL) were subjected to shear stress, at a constant internal temperature, for a 1-hour period, at varying rotational speeds. Protein samples were taken over a 1-hour period and assayed by ELISA within 2h of the start of the experiment. Each time a shear stress protein sample was taken it was replaced with equivalent volumes of un-sheared standard protein solution to maintain a consistent air/liquid interface. Samples of sheared fusion protein material was frozen for analysis later by Enzyme Assay (EA) and SDS-PAGE gel analysis. The samples were stored at -80°C because the range of analytical techniques used for analysis took several hours to perform and could not all be completed within a day.

Air/liquid interface

As well as shearing with no air, the USD device was able to shear protein samples with different percentage air/liquid interfaces. By half filling the shear device with 10 mL of GMP manufactured therapeutic pure standard protein solution it was possible to generate a 50% air/liquid interface and with 15 mL of protein solution it was possible to generate a 25% air/liquid interface. A plastic bottom plate was fabricated from transparent Perspex for easy visualisation of the different interfaces produced in the shear stress device.

ELISA

The Enzyme Linked Immunosorbent Assay (ELISA) used to analyse the MFECP1 fusion protein under investigation is described [20]. Plates (NUNC 96 well ImmunoplatesMaxisorp, SLS, Denmark) were coated with 100 µL of N-A1 (1 µg/mL) and incubated for 1h at room temperature. N-A1 is the known functional domain on the carcinoma-Embryonic Antigen (CEA) that interacts with MFE-23 antibody fragment [21]. This domain was produced by fermentation at a final concentration of 0.5 mg/mL, supplied by the Royal Free Hospital, (Oncology, UK). Control wells were coated with PBS only under the same conditions and emptied before blocking with milk proteins. All wells were blocked with 5% milk proteins (Marvel Milk powder, UK)/PBS (150 µL/well) for 12h to eliminate non-specific binding of proteins. Sheared MFECP1 fusion protein samples (100 µL samples) were applied and incubated for 1h, after which samples were removed and wells were washed with PBS solution. Detection of the intact MFECP1 fusion protein was carried out by incubating for 1h with polyclonal anti-CPGprimary antibody raised in rabbit, diluted 1/25,000 in 1% milk proteins/PBS (100 µL/well), followed by incubation with anti-Horse Radish Peroxidase (anti-HRP) diluted 1/1,000 in 1% milk proteins/PBS (100 µL/well). Washing steps consist of four washes with 0.1% Tween 20/PBS (v/v), followed by three PBS washes.Plates were developed with o-phenylenediamine (C6H4(NH2)2·2HCl) in phosphate citrate buffer (Na2HPO4.7H2O) (C6H8O7.H2O) with sodium perborate (NaBO3·H2O), (100µL/well) and the reaction was stopped after 3 minutes with 4 M HCl, (100 µL/well). Optical density was measured at 490 nm on an Opsys MR ELISA plate reader (Dynex Technologies Limited, UK). The sandwich ELISA worked by producing the response when intact MFECP1 fusion protein was present. To calculate the approximate concentration of intact MFECP1 fusion protein in the sheared samples, a calibration curve was set up.  Absorbance’s were measured at 490 nm of serial stock solutions from 700-31 ng/mL producing a calibration line giving a predictable relative error of +/-10%. Control experiments showed that the reagents F68 (0.01% v/v) and BSA gave a zero response at 490 nm.

CPG2 Enzyme assay

The Enzyme Assay (EA) used to measure the Units (U) of CPG2 activity present in unknown sheared samples has been described previously [20]; 1 U is equal to the amount of CPG2 enzyme required to hydrolyze 1 mmol of methotrexate per minute at 37°C. The CPG2 enzyme breaks down the prodrug into a cytotoxic entity and also shows its chemical homology with methotrexate[5,20]. Note, the rate of enzyme activity was dependent on temperature therefore it was critical that all solutions were maintained at 37°C. This was achieved with the use of a thermocouple sensor (PicoLog, USA), which was inserted into a quartz cuvette containing 990 µL of CPG2 assay buffer within the UV/Visible, spectrophotometer (F6500, Hitachi, Japan). The assaying could begin when buffer in the cuvettes reached 37°C. CPG2 assay buffer composition (Trizma base 12.11g.L-1 Zinc Chloride 0.025g.L-1). The assay buffer was prepared by dissolving the Trizma base and Zinc Chloride in 1 L of deionised water; pH was adjusted to 7.3 with HCL.Methotrexate solution was prepared by adding 50 µL of methotrexate to 50 mL of CPGbuffer. All dilutions were made with PBS. The assay procedure was carried out as follows: 990 µL of the pre-warmed (37°C) diluted methotrexate solution was added to a quartz cuvette and left to reach 37°Cin the temperature regulated spectrometer. Once the temperature reached 36.9°C, 10 µL of the sheared sample was added to the cuvette and mixed with a glass pipette. After mixing the spectrometer shutter was closed and the CPG2 assay software calculated the rate of methotrexate disintegration by a colour change at wavelength 320 nm. The rate of this conversion was directly related to the units of CPG2 enzyme activity present in the unknown sample. All enzyme assays were conducted in triplicate (n=3) standard deviations are shown with +/- error bars in figures [20].

Results

Figure 1 shows the dimensions of the USDshear device used to inflict appropriate shear forces on the MFECP1 fusion protein. The shear device in figure 1 was used to test the robustness of a fusion protein solution with an air/liquid interface and a starting concentration ranging from 450-500 ng/mL. The results show that a 50% air/liquid interfaces degraded the fusion protein in a USD shear device, shear stress 10,000 rpm (Figure 2).Around~80 % of the starting fusion protein was degraded after 20 minutes of shearing, generating a rate constant, k, of around 5.36 (+/- 0.22) h-1 and an ending steady value of 117 ng/mL. In contrast, 60 minutes of shear (10,000 rpm) with no interface resulted in a finishing concentration of 392 ng/mL, i.e., 20% of the protein was lost, generating a rate constant, k, of 0.39 (+/-0.72) h-1 table 1. However, there is evidence of an initial period (0 and 0.4 h) with no protein breakdown.

Ultra Scale-Down Characterization And Stability Of Monoclonal Fusion Proteins Used To Treat Cancer

Figure 2: The effect of air/liquid interface and shear on fusion protein degradation. Experimental conditions were: (____)Co = 522 ng/mL, no air liquid interface, 10,000 rpm; and () Co = 534 ng/mL, 50 % air liquid interface, 10,000 rpm. Co = initial concentration of intact MFECP1 fusion protein as measured by ELISA. All shear experiments were conducted at a constant temperature of 4oC maintained by a cooled water bath. Curves are lines of best fit for a first order kinetic relationship to an equilibrium (non-zero value). Degradation constants and final equilibrium values are reported in table 1.

Shear condition

N

(rpm)

Initial concentration

Co

(ng/ml)

Final Equilibrium Concentration

C

(ng/ml)

Final Fraction

Value

(C /co)

 

Air Volume

(%)

Calculated Constants

(k)

(h-1)

Standard Error

(+/-)

(h-1)

Figure 2

10,000

522

392

0.75

0

0.39

0.72

10,000

534

117

0.22

50

5.36

0.22

Figure 3

5000

459

431

0.94

0

0.05

0.25

5000

436

79

0.18

50

5.36

0.30

5000

473

90

0.19

25

2.81

0.45

Figure 4

5000

440

348

0.79

0

2.80

1.56

5000

458

54

0.12

50

33.4

0.80

Figure 5

5000

100,000

84

0.84

0

1.37

1.29

5000

100,000

45

0.48

50

2.88

0.92

Figure 6

5000

100,000

8.26

6.2

0

0.82

0.19

5000

100,000

8.68

4.7

50

0.92

0.29

Figure 7

5000

450

180

0.40

50

3.80

0.49

5000

473

345

0.73

50

2.30

0.55

Figure 8

5000

450

180

0.4

50

3.80

0.49

5000

474

480

1

50

0

0

Table 1: Shows shear conditions, initial concentrations and final equilibrium values with calculated rate constants.

Next, the effect of air was investigated, it was hypothesised that the rate of protein disintegration might be directly proportional to the percentage of air in the system. If this was true it could mean that bioprocessing of a fusion protein at large scale would result in significant losses if this aspect was not taken into consideration. Different percentage air/liquid interfaces could be created in the shear device by removing the corresponding amounts of liquid. Shear was inflected on protein stock solutions around 500 ng/mL to observe how robust the protein was at a shear stress of 5,000 rpm. Duplicate samples were taken and tested from the USD device over a one-hour period. Results show that a 25% v/v air to liquid interface was as devastating to the viability of the fusion protein as a 50% interface (Figure 3). The results here show that a control MFECP1 fusion protein solution containing no air/liquid interface was degraded by 6% from the initial protein concentration of 459 ng/mL producing a breakdown rate constant, k, of 0.05 (+/-0.25) h-1 and a final equilibrium concentration of 431 ng/mL. However, air/liquid interfaces of 50% and 25 % gave an~80 % loss from the initial concentration of the protein, showing that both were equally detrimental in protein loss. When the rate constants for 50% and 25% air/liquid interfaces are compared (Table 1) no direct conclusions can be made that the rate of protein breakdown is related to the amount of air. If the curves of each experiment are compared one can assume the effect of air in the system has an affect on the stability of the protein, however, the amount of air present does not affect the rate at which fusion protein degrades.

Ultra Scale-Down Characterization And Stability Of Monoclonal Fusion Proteins Used To Treat Cancer

Figure 3: The effect of percentage air/liquid interface on the rate of fusion protein degradation. Experimental conditions were: (____)Co = 459 ng/mL, no air liquid interface, 5,000 rpm; (____) Co = 436 ng/mL, 50% air liquid interface, 5,000 rpm; and (__u__) Co = 473 ng/mL, 25 % air liquid interface, 5,000 rpm. Co = initial concentration of intact MFECP1 fusion protein as measured by ELISA. All shear experiments were conducted at a constant temperature of 4oC maintained by a cooled water bath. Curves are lines of best fit for a first order kinetic relationship to an equilibrium (non-zero value). Degradation constants and final equilibrium values are reported in table 1.

It was hypothesized that the protein degradation reported in figure 2 and 4 when the protein was exposed to air/liquid interfaces and a constant shear field protein deactivation was due to potential fragmentation between the MFE and CPG2 domains. These two fragments are fused by a single peptide bond and are not normally found together in nature. To test if the design of the linker region between the two domains was influential in the rate of protein breakdown, a fusion protein formed with a 10 amino acid linker chain was tested with the same shear conditions that degraded the MFECP1 fusion protein. The linker region was comprised of a (Gly4 Ser)n amino acid sequence where n=2. This peptide chain linked fusion protein was thought to be more shear sensitive and fragile than the peptide bond linker fusion protein (MFECP1), which contains just a peptide bond linking the two domains together. The results in figure 4 show that without an air and liquid interface the peptide chain linked protein degraded at a rate of 2.8 (+/- 1.56) h-1 producing a 21% breakdown from the original stock solution (Table 1).

Ultra Scale-Down Characterization And Stability Of Monoclonal Fusion Proteins Used To Treat Cancer

Figure 4: The effect of air/liquid interface on the rate of peptide chain linked fusion protein degradation. Experimental conditions were: (____)Co= 440 ng/mL, no air liquid interface, 5,000 rpm; and ()Co = 458 ng/mL, 50 % air liquid interface, 5,000 rpm. Co = initial concentration of intact MFECP1 fusion protein as measured by ELISA. All shear experiments were conducted at a constant temperature of 4oC maintained by a cooled water bath. Curves are lines of best fit for a first order kinetic relationship to an equilibrium (non-zero value). Degradation constants and final equilibrium values are reported in table 1.

The generation of a 50/50 air to liquid interface tarnished the protein at a rate of 33.4 (+/-0.80) h-1 and generated an 88% breakdown. If we compare the data from figure 3and 4we observe that the peptide chain linked fusion protein was more fragile than the peptide bond linked fusion protein(MFECP1) at all the shear conditions tested.It was important to establish that breakdown occurred at large scale, so the results could be varied. The results in the figure 2-4were for a dilute protein solution not normally found in the full-scale production process. These low concentrations were used because the fusion protein was a precious and expensive product to produce. Protein solutions of ~100 µg/mL (which are similar to the concentrations found at production scale), were subjected to a shear rotational speed of 5,000 rpm, together with and without a 50% air/liquid interface. The stability of the protein can be seen in figure 5. Results show the addition of a 50/50air to liquid interface amplified the rate of protein deactivation over a 1-hour period as seen previously in the small-scale experiments conducted at lower concentrations (Figures 3,4). A first order rate constant of 2.88 h-1 (+/-0.92) was generated when a 100 µg/mL stock solution was sheared with a 50% air/liquid interface; this resulted in 55% degradation in protein integrity. Protein was degraded at a rate of 1.37 h-1 (+/-1.29) without an air/liquid interface, resulting in a 20% loss in protein integrity. The rate of protein deactivation increased from 1.37 h-1 (+/-1.29) when no air to liquid interface was present down to 2.88 h-1 (+/-0.92) when a 50% air/liquid interface was applied. Final equilibrium concentrations for no air were 84 µg/mL and 45 µg/mL with air. It can be seen that the rate of breakdown with the addition of the 50% air/liquid interface at 100 µg/mL was not as fast as in the lower concentration studies (Figure 2 and 4)but did occur.

Ultra Scale-Down Characterization And Stability Of Monoclonal Fusion Proteins Used To Treat Cancer

Figure 5: Shows the effect of air/liquid interfaces on fusion protein degradation. Initial concentrations and shear conditions were: (____) Co = 100 μg/mL, no air/liquid interface, 5000 rpm; and (____) 50% air/liquid interface, 5000 rpm. Co= initial concentration of intact MFECP1 fusion protein as measured by ELISA. All shear experiments were conducted at a constant temperature of 4°C maintained by a cooled water bath. Curves are lines of best fit for a first order kinetic relationship to equilibrium (non-zero value). Degradation constants and final equilibrium values are reported in table 1.

Enzyme activity was also monitored during the shearing of the 100 µg/mL MFECP1 fusion protein stock solution with a 50/50 air to liquid interface. Results in figure 6 show that an addition of an interface increased the rate of enzyme deactivation from 0.82 h-1 (+/- 0.19) without air, to 0.92 h-1 (+/- 0.29) with air. The addition of the air also resulted in a lower final equilibrium activity of CPG2, the presence of the interface increased the percentage of protein degradation from 25% without air to 45% with air. The results show that the addition of the air increased the apparent deactivation in enzyme activity; it was thought that this may be due to aggregation as CPGis quite a robust enzyme and less likely to be fragmented by shear.

Ultra Scale-Down Characterization And Stability Of Monoclonal Fusion Proteins Used To Treat Cancer

Figure 6: Shows the effect of an air/liquid interface on CPG2 enzyme activity at 100 μg/mL. Initial activity and shear conditions were: (----) Eo = 8.26 U/mL, no air/liquid interface, 5000 rpm; and (_O_) Eo = 8.68 U/mL, 50% air/liquid interface, 5,000 rpm. Data points show the mean values, error bars show +/-S.D, curves are lines of best fit for a first order kinetic relationship to an equilibrium (non-zero value). All shear experiments were conducted at a constant temperature of 4°C. Degradation constants and final equilibrium values are reported in table 1.

An air/liquid interface was created in a shear device while exposing the MFECP1 fusion protein to shear with 0.01% BSA w/v. The results in figure 7show that the addition of BSA to the fusion protein solution was particularly beneficial to the integrity of the protein over 60 minutes of shear at 5000 rpm, resulting in a lower rate constant, k, of 2.3 (+/- 0.55) h-1 and a final protein concentration of 345 ng/mL (Table 1). The absence of BSA at same shear conditions led to a higher rate constant, k, of 3.8 (+/- 0.49) h-1 and a lower finishing concentration of 180 ng/mL So the addition of this protein agent reduced the amount of fusion protein breakdown and protected it by approximately a half, from 60% without BSA to ~30% with BSA.

Ultra Scale-Down Characterization And Stability Of Monoclonal Fusion Proteins Used To Treat Cancer

Figure 7: The effect of BSA on the rate of fusion protein degradation.Experimental conditions were: (____) Co = 473 ng/mL, 50% air liquid interface, 5,000 rpm, 0.01% BSA and; () Co = 450 ng/mL, 50 % air liquid interface, 5,000 rpm. Co = initial concentration of intact MFECP1 fusion protein as measured by ELISA. All shear experiments were conducted at a constant temperature of 4°C maintained by a cooled water bath. Curves are lines of best fit for a first order kinetic relationship to equilibrium (non-zero value). Degradation constants and final equilibrium values are reported in table 1.

Serum albumins are regularly used in mammalian cell cultures to improve cell cultivation [27]. However, it would be very difficult to get FDA approval for the addition of an animal derived substance into a process train generating an inject able human therapeutic due to any possible cross contamination of diseases, for example BovineSpongiform Encephalopathy (BSE) [28]. Even if human sera were considered to be safe and could be added at large scale to improve yields, (by reducing disintegration of the protein), one would have to consider the overall economic benefits because of the very high cost of this particular additive [27]. Therefore, because of these cost considerations and possible regulatory concerns, a commonly used and cheap surfactant known to act at the air/liquid surface was investigated as a stabilising agent that could reduce MFECP1 fusion protein breakdown. The surfactant F68 is used throughout shear sensitive mammalian cell culture [29], and as a result was tested next as a possible protective agent to reduce protein degradation. A 50% air/liquid interface was created in a shear device while exposing the MFECP1 fusion protein to shear at, around 500 ng/mL plus the addition of 0.01% F68 v/v (Figure 8). MFECP1 fusion protein sheared with no agent present produced a lower rate constant with a lower overall equilibrium concentration when compared to the control. However, the addition of the surface-active agent, F68, generated a first order rate constant of zero (Table 1). This shows that the addition of F68 was beneficial in preserving the integrity of the protein over the 1h shearing time. The final equilibrium concentration was improved with the addition of the agent showing it reduced breakdown. Shear experiments conducted with protein concentrations 500ng/ml showed that the addition of the detergent should stabilise the fusion protein with the presence of shear and an interface. The results showed how ELISA and EA protein fractions over a 1h period were improved with the addition of BSA and F68, thus showing similar results to [30]. The results in figure7 and 8 show that the addition of this agent to the large-scale process could be beneficial for the stability of the protein during manufacture.

Ultra Scale-Down Characterization And Stability Of Monoclonal Fusion Proteins Used To Treat Cancer

Figure 8: The effect of surfactant F68 on the rate of fusion protein degradation. Experimental conditions were: (____) Co = 474 ng/mL, 50% air liquid interface, 5,000 rpm, 0.01% F68 and; () Co = 450 ng/mL, 50 % air liquid interface, 5,000 rpm. Co= initial concentration of intact MFECP1 fusion protein as measured by ELISA. All shear experiments were conducted at a constant temperature of 4°C maintained by a cooled water bath. Curves are lines of best fit for a first order kinetic relationship to equilibrium (non-zero value). Degradation constants and final equilibrium values are reported in table 1.

Discussion

The fusion protein under investigation has been quantified using a very specific antibody-based assay and characterised in a USD shear device. An ELISA quantification method was used to accurately and specifically measure the amount of unbroken fusion protein present in samples that have been sheared. Shear experiments in figure 2 monitoring the stability of a fusion protein over a 60 minutes time period and found changes in protein integrity over time during different shear conditions. As described in the model by Oliva et al., [24] a first order relationship was used the measure the protein kinetic degradation over the shearing time[4]. This model generated constants that could be compared between different experiments when the MFECP1 fusion protein was introduced to different process environments. The results showed the addition of a 50/50 air/liquid interface to the device increased the rate of protein breakdown over a 60-minute period. When rate constants for 25% and 50% were compared and repeated, no differences were observed to prove that the rate of protein loss was linearly (synergistically) related to the percentage of air in the system (Figures 2,3). However, the mere presence of air in the system was shown to be important in protein stability. The stability of a 10 amino acid peptide chain linked fusion protein was assessed and results show that the addition of an air/liquid interface degraded this protein at a faster rate than standard fusion protein (MFECP1) showing that it was more shear sensitive and fragile. The additions of different percentage air/liquid interfaces showed the same results as seen with normal fusion protein (MFECP1) figure 3, for example the rate of peptide chain linked fusion protein loss was not directly related to the percentage of air in the system. The shear experiments outlined were conducted at 500ng/ml due to the precious nature of the therapeutic protein solution but we needed ensure the same effect was occurring at concentrations found at large scale. The shear device was used to assess the integrity of protein stock concentrations that were normally found during the large-scale production. The results showed that the addition of an 50/50 air/liquid interface degraded the fusion protein over a 60 minutes time period at 100 µg/mL concentrations (Figure 6). However, the rate of degradation was slower than experiments performed at lower concentrations at around 500 ng/mL, suggesting that excess protein may be protecting the effect of protein breakdown/or loss. Detailed enzymatic analysis of CPG2 enzyme illustrated that activity was lost with the addition of an 50% air/liquid interface during the shearing time (Figure 6). The rate of CPG2 deactivation was much lower than pervious experiments (but did occur) and this supports that deactivation of CPG2 is unlikely as it is a robust enzyme, therefore it was supposed that aggregation could be occurring.

The results in figure 6 and 8have shown that MFECP1 fusion protein stability was improved by a variety of surface-active agents under shear associated conditions. It was identified that BSA, and F68 helped stabilise a MFECP1 fusion protein stock solution by varying levels. The level of stability was quantified by comparing the rate of the first order breakdown constants generated over a 1-hour time period. It was found that F68 seemed to be the best reagent at reducing protein breakdown. It was hypothesised that this agent could reduce high shear associated effects and generate low breakdown constants giving good protein stability. The results indicate that adding of an agent like Pluronic (F68) to large scale bioprocessing could improve yields by decreasing the amount of fusion protein lost from shear related degradation.

Conclusion

In conclusion USD experiments have shown that the addition of various interfaces can be detrimental to the stability of the fusion protein. This effect is also seen with a modified chain linked fusion protein and with fusion protein at 100 µg/ml higher concentration stock solutions normally found at production scale. The addition of F68 and BSA had a protective effect on the integrity of the protein. These reagents could be added to the large-scale process of complicated fusion proteins to improve process yields.

Acknowledgements

The present worldwide publication is wholly dedicated in honour of my late mother and father Baljinder Kaur Blas and Ram Blas who both passed away during the course of my PhD. It is also dedicated to my beautiful children, Leah Sophie and Theodore Colin Blas who inspire me to achieve the very best in life. I would also like to thank my fiancée Miss Tiffany Amelia Greenwood, your unconditional support means the world to me.

Furthermore I would like to thank the following scientists for their trust, guidance and the opportunity to complete this study:

Professor Gary Lye (Head of Department, Biochemical Engineering at UCL)

Professor Nigel Titchener-Hooker (Dean of Faculty of Engineering Sciences at UCL)

Professor Kerry Chester (Research Department of Oncology, Cancer Institute, UCL)

Professor John Ward (Synthetic Biology for Bioprocessing, UCL)

Professor John Mitchell (Communications Systems Engineering, Vice Dean Education, UCL)

Professor Nik Willoughby of Bioprocessing, Heriot-Watt University

Professor Mike Hoare, UCL.

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