The main rationale behind the development of a microwell plate based cell adhesion methodology is due to the effectiveness of the finite bath experiments in studying the degree of cell adsorption to stationary phases, in which Fernandez-Lahore et al. have extensively worked on [11, 24, 25]. Concisely, the methodology involved a cell suspension that was made to contact with an adsorbent (e.g. chromatography support) over a prolonged period under gentle mixing, and it was observed that as the degree of cell adsorption increases, cell concentration in the suspension decreases. This was quantified by calculating the ratio of final cell concentration at the end of the experiment to initial cell concentration.
One major limitation of the finite bath experiments described earlier is that they are time-consuming, laborious and expensive (based on a number of adsorbents involved). The method described here is a scaled-down version of the existing technique making it easier to use, faster and economical by adopting the classical method of a 96-microwell format, where adsorbent surfaces are coupled to the inner walls of the microwell plate.
3.1. Polymer synthesis and 96-microwell plate modification
Dip-coating is a simple method used to modify the surface with polymers, and the procedure is easy to perform, economical, and the surface modification can easily be controlled. To produce a microwell plate with a modified surface, a polymer was initially synthesized. The synthesis of a homopolymer of poly-glycidyl methacrylate was achieved by polymerizing glycidyl methacrylate and 2,3-epoxy-propanol thereby allowing them to react with amino [26] and hydroxyl groups [8] to form a stable covalent bond without any cross-linking agent. These polymers are chemically stable over long storage periods and are relatively resistant to hydrolysis [27]. Additionally, the advantage of end reaction mixture of polymerization reaction contains PGMA-DMSO a solution that is polystyrene-compatible [8]. The polymerization conditions and procedure are described in detail by Eckert et al. [8].
The synthesized polymer was further coupled with DEAE dextran, and the product was analyzed by FTIR. The IR spectra of GMA, PGMA, and DEAE-dextran coupled with PGMA coated on microwell plate are shown in Fig. 1. From the results it can be observed that among the characteristic vibrations, 2900–2800 cm− 1 and 1100–1000 cm− 1 are for the DEAE functionality and the vibration resulting from double bond in the GMA is at 1620 cm− 1, and 730 cm− 1 indicates the carbonyl groups of GMA [8, 28]. The IR spectra noticeably characterize the films of polymerized GMA and introduction of the DEAE functionality to the epoxide group.
3.2. Contact angle measurements
For the measurement of contact angles, diagnostic liquids like water, formamide, and 1-bromonaphtalene were employed on polymer-coated glass slides using the sessile drop technique. The surface free energy components Lifshitz–van der Waals (γLW) and acid–base (γAB) as well as, the electron-donating (γ−) and electron-accepting parameters (γ+) of the liquids used were obtained from the literature [29], while the surface free energy components of biomass were taken from previously published literature [11, 25].
Table 1 shows the contact angle values for S. cerevisiae and CHO cells, which are relevant to process situations and were employed as model biomass system for our studies. Cells were equilibrated in 20 mM phosphate buffer pH 7.4, which offers a chemical environment comparable to that found in ion-exchange sorption processing in industrial practice. When comparing the biomass type, contact angles values for water varied from 15 °C (yeast cells) to 26.1 °C (CHO cells). Similarly, formamide contact angle ranges from 14 °C (yeast cells) to 30.2 °C (CHO cells). This sequence can be interpreted regarding decreased hydrophilic/hydrophobic ratio in agreement with the earlier reports [20, 29]. Polymer contact angles were measured on thin layers of polymers coated on glass slides using spin coating; this enables us to measure the contact angles using the sessile drop method. Dried polymeric surfaces showed a relatively higher affinity for 1-Bromonaphthalene in comparison to hydrated samples. When comparing the polymer type, contact angles values for water varied from 9.2 °C (TWEEN® 20) to 55.5 °C (Poly(methyl vinyl ether) and for formamide, contact angles range from 7.2 °C (Poly(vinyl sulfonic acid)) to 52.6 °C (Pluronic® F-108), while contact angles for 1-Bromonaphthalene range from 7.9 °C (Poly(ethylene glycol) 3550) to 39.3 °C (Poly(methyl vinyl ether)) representing the diversity of the surface energy parameters present in the polymer library tested for antifouling property. Using the contact angle values listed in Table 1, surface free energy components were calculated using modified Young–Dupré equation [30], and all the surfaces analyzed showed hydrophilic in nature. The differences found in the surface energies can be attributed to the functional groups present on the surface.
Table 1
Contact angles of biomass types and polymer surfaces measured using sessile drop method
Material
|
Contact angle (θ)
|
H20
|
FMD
|
ABN
|
Biomass
|
S. cerevisiae cells
|
15 ± 2
|
14 ± 1
|
54 ± 1
|
CHO cells
|
26.1 ± 0.7
|
30.2 ± 0.2
|
42.3 ± 0.5
|
Control
|
DEAE-Dextran
|
18.3 ± 0.6
|
20.6 ± 1.2
|
14.2 ± 0.7
|
Polymeric material
|
Polyacrylic acid 345 (PAA)
|
47.9 ± 2.3
|
20.5 ± 1.7
|
15.6 ± 4.6
|
Poly(methacrylic acid) (PMA)
|
43.5 ± 2.7
|
34.6 ± 2.6
|
36.2 ± 0.4
|
Poly(vinyl sulfate) (PVS)
|
14.2 ± 2.1
|
25.4 ± 6.5
|
14.8 ± 0.7
|
Poly(vinylsulfonic acid) (PSS)
|
11.1 ± 1.3
|
7.2 ± 0.2
|
16.8 ± 0.5
|
Poly(4-styrenesulfonic acid) (PS4)
|
39.6 ± 0.6
|
36.7 ± 1.1
|
24.5 ± 1.8
|
Sulfonated PEEK (sPK)
|
24.9 ± 1.7
|
29.8 ± 2.5
|
13.3 ± 0.8
|
Hyaluronic acid (HLA)
|
32.1 ± 2.4
|
18.6 ± 0.1
|
32.2 ± 0.7
|
Polyvinylpyrrolidone 360 (PVP)
|
50.3 ± 6.7
|
44.5 ± 2.5
|
16.8 ± 1.7
|
Poly(vinyl alcohol) 89–98 (PV1)
|
31.7 ± 1.1
|
31.2 ± 0.2
|
14.1 ± 0.6
|
Poly(propylene glycol) (PPG)
|
41.3 ± 1.4
|
30.9 ± 0.8
|
33.8 ± 1.1
|
Poly(ethylene glycol) 3550 (PG3)
|
16.8 ± 0.3
|
19.1 ± 0.5
|
7.9 ± 1.4
|
Poly(ethylene glycol) 8000 (PG8)
|
14.5 ± 2.1
|
28.7 ± 0.7
|
8.1 ± 2.4
|
Poly(ethylene glycol) 20000 (PG2)
|
30.2 ± 0.9
|
32.4 ± 1.7
|
8.0 ± 0.8
|
Poly(methyl vinyl ether) (PVM)
|
55.5 ± 2.2
|
49.9 ± 0.6
|
39.3 ± 0.7
|
TWEEN® 20 (T20)
|
9.2 ± 0.9
|
15.1 ± 2.0
|
24.3 ± 0.7
|
TWEEN® 80 (T80)
|
17.0 ± 2.0
|
15.4 ± 1.6
|
25.0 ± 1.8
|
Pluronic® F-108 (F18)
|
51.6 ± 0.3
|
52.6 ± 0.2
|
26.0 ± 0.1
|
Brij® 58 (B58)
|
42.2 ± ± 2.6
|
38.9 ± 4.8
|
31.4 ± 1.0
|
Brij® 35 (B35)
|
55.7 ± 2.5
|
29.1 ± 1.1
|
33.1 ± 1.5
|
DData were taken from [11], B data taken from [25], C Own measurements |
Electrostatic interactions are forces applicable over a longer range as compared to Lifshitz–van der Waals (γLW) and acid–base (γAB) interactions and are one of the main factors influencing the colloidal interaction; it can be determined by measuring zeta potential. Zeta potential measurement is usually done using various salt concentration (0, 25, 50, 100 mM NaCl) in the binding buffer and it is then scaled up to any required condition using standard scaling condition [5, 31]. In our work both biomass types showed a similar surface charge of around ~ 20 mV, a negative zeta potential due to the nature of the outer membrane lipid and surface proteins of respective cell type. The control sample of the experiment was a positively charged DEAE-dextran surface having positive surface charge of 5.3 mV, which had the highest tendency for cell attachment. The polymer library zeta potential ranged from − 2.5 mV (PEG 8000) to -37.3 mV for sulfonated PEEK, indicating that polymer library consists of the various degree of magnitude of surface charge.
3.3. Calculation of interfacial free energy between biomass and polymer surface
Surface energies data obtained by contact angle measurement and the surface charge determination by zeta potential measurement can be employed for interaction energies calculation at specific buffer conditions. In our work we used, sphere-to-plate (cell to adsorbent) geometry to mimic the interaction between cells and chromatography adsorbents coated with polymers or polymeric beads of similar sizes and it enabled backward comparison with earlier published data [20]. For calculating intact yeast cells, the diameter was taken as 10 µm, for CHO cells 15 µm and for adsorbent 200 µm was considered.
Anion-exchanger surfaces, such as DEAE dextran are well-known to strongly interact with cells, mostly due to charge-mediated (electrostatic) effects [32]. As shown in Fig. 3, the interaction energy for DEAE surface and yeast cells was − 77 kT and DEAE surface with CHO cells was − 204 kT in 20 mM phosphate buffer, pH 7.4. The interaction between CHO cells and DEAE surface was comparatively much higher than that of with yeast cells. Polymer coating has the capability to alter the interactions between microbial cells and adsorbents in aqueous media [6, 33, 34], by stimulating changes in the free interfacial forces (or free energy) between bodies [4]. However, there has been insufficient research conducted till now. In this work, we successfully performed the xDLVO calculations for twenty-three polymers interacting with yeast and CHO cells. The degree of interaction varied and mainly due to the difference in surface energies, surface charge, and size of the cells. Based on xDLVO calculations for yeast polymer interaction, Poly(methyl vinyl ether) showed lowest interaction with − 25 kT, followed by poly(methacrylic acid) with − 28 kT and PEG 8000 showed highest with − 58 kT. For polymer interaction with CHO cells, poly(methyl vinyl ether) showed lowest interaction with − 76 kT, followed by poly(methacrylic acid) with − 84 kT and PEG 8000 showed highest with − 170kT. Comparing the interaction data, the highest and the lowest interacting polymer for both cell type remained the same. However, we can clearly see the degree of interaction is greater with CHO cells. As mentioned before, surface energy values for polymers are measured in a dry condition, which showed higher affinity to 1-bromonaphthalene, because of this, xDLVO calculation results are more qualitative in nature indicating the trend than absolute interaction. All the polymers showed some degree of cell repellence in comparison to the interaction energies of the naked DEAE dextran surface. A complete list of the interaction energy for polymers and cell types are listed in Table 3.
Table 2
Surface energy parameters and zeta potential values for various biomass types and polymer surfaces
Material
|
Surface energy parameters (mJ m-2)
|
Zeta potential (mV) at 20 mM PB pH7.4
|
γLW
|
γ+
|
γ−
|
γAB
|
γSTotal
|
Biomass
|
S. cerevisiae cells
|
27.9
|
4.4
|
51.5
|
30.1
|
58.0
|
-20.0
|
CHO cells
|
33.6
|
1.2
|
52.1
|
15.8
|
49.4
|
-21.0
|
Control
|
DEAE-Dextran
|
43.1
|
0.4
|
53.8
|
9.5
|
52.6
|
5.3
|
Polymeric material
|
Polyacrylic acid 345 (PAA)
|
42.8
|
1.7
|
22.0
|
12.3
|
55.0
|
-8.6
|
Poly(methacrylic acid) (PMA)
|
36.2
|
1.1
|
34.1
|
12.0
|
48.3
|
-18.1
|
Poly(vinyl sulfate) (PVS)
|
42.9
|
0.2
|
59.6
|
6.4
|
49.4
|
-8.6
|
Poly(vinylsulfonic acid) (PSS)
|
42.5
|
0.9
|
53.2
|
14.0
|
56.5
|
-5.5
|
Poly(4-styrenesulfonic acid) (PS4)
|
40.5
|
0.2
|
41.4
|
5.7
|
46.2
|
-6.6
|
Sulfonated PEEK (sPK)
|
43.2
|
0.1
|
54.0
|
4.7
|
47.9
|
-37.3
|
Hyaluronic acid (HLA)
|
37.8
|
1.9
|
39.1
|
17.1
|
55.0
|
-7.1
|
Polyvinylpyrrolidone 360 (PVP)
|
42.5
|
0.0
|
32.9
|
0.5
|
43.1
|
-4.3
|
Poly(vinyl alcohol) 89–98 (PV1)
|
43.1
|
0.1
|
47.4
|
5.3
|
48.4
|
-5.3
|
Poly(propylene glycol) (PPG)
|
37.2
|
1.2
|
34.7
|
12.9
|
50.1
|
-6.3
|
Poly(ethylene glycol) 3550 (PG3)
|
44.0
|
0.4
|
54.2
|
9.1
|
53.0
|
-3.1
|
Poly(ethylene glycol) 8000 (PG8)
|
44.0
|
0.0
|
62.0
|
2.9
|
46.8
|
-2.5
|
Poly(ethylene glycol) 20000 (PG2)
|
44.0
|
0.0
|
50.2
|
3.1
|
47.1
|
-4.7
|
Poly(methyl vinyl ether) (PVM)
|
32.5
|
0.4
|
29.1
|
6.7
|
39.2
|
-5.9
|
TWEEN® 20 (T20)
|
41.1
|
0.8
|
56.2
|
13.5
|
54.6
|
-8.4
|
TWEEN® 80 (T80)
|
41.2
|
0.8
|
56.2
|
13.5
|
54.7
|
-8.2
|
Pluronic® F-108 (F18)
|
36.1
|
0.0
|
37.5
|
0.0
|
36.1
|
-9.7
|
Brij® 58 (B58)
|
37.4
|
0.4
|
39.4
|
7.8
|
45.2
|
-4.1
|
Brij® 35 (B35)
|
38.2
|
2.4
|
16.2
|
12.4
|
50.6
|
-9.6
|
Table 3
Interaction energies (U) for various polymeric surfaces with yeast and CHO cells at 20 mM phosphate buffer pH 7.4
Material
|
|U| (kT)
|
S. cerevisiae
|
CHO
|
Control
|
DEAE-Dextran
|
-78
|
-204
|
Polymeric material
|
|
|
Polyacrylic acid 345 (PAA)
|
-48
|
-148
|
Poly(methacrylic acid) (PMA)
|
-28
|
-84
|
Poly(vinyl sulfate) (PVS)
|
-47
|
-143
|
Poly(vinylsulfonic acid) (PSS)
|
-50
|
-151
|
Poly(4-styrenesulfonic acid) (PS4)
|
-44
|
-133
|
Sulfonated PEEK (sPK)
|
-37
|
-110
|
Hyaluronic acid (HLA)
|
-37
|
-113
|
Polyvinylpyrrolidone 360 (PVP)
|
-52
|
-158
|
Poly(vinyl alcohol) 89–98 (PV1)
|
-52
|
-155
|
Poly(propylene glycol) (PPG)
|
-36
|
-111
|
Poly(ethylene glycol) 3550 (PG3)
|
-58
|
-169
|
Poly(ethylene glycol) 8000 (PG8)
|
-58
|
-170
|
Poly(ethylene glycol) 20000 (PG2)
|
-55
|
-164
|
Poly(methyl vinyl ether) (PVM)
|
-25
|
-76
|
TWEEN® 20 (T20)
|
-43
|
-131
|
TWEEN® 80 (T80)
|
-44
|
-132
|
Pluronic® F-108 (F18)
|
-31
|
-94
|
Brij® 58 (B58)
|
-39
|
-118
|
Brij® 35 (B35)
|
-36
|
-112
|
3.4. Modified finite bath experiments
Screening of putative antifouling agents based on the effect of different coating on biomass deposition onto custom created dextran DEAE surface can be performed using modified simple partition tests, which were reported earlier and well suited for such applications [4, 13, 24]. DEAE-ligands are known to strongly interact with microbial cells and this property has lead us to generate a DEAE-modified surface to screen antifouling polymer. Partitioning tests are cautiously designed to include a broad range of biomass and polymer interaction pairs. In such experiments, cell deposition to polystyrene surface was less than 10%, based on cell partition index values. Experiments were implemented in 20 mM phosphate buffer with pH 7.4. Contact time was fixed to 30 minutes so as to mimic the process condition to the maximum residence time of the biomass in standard chromatographic runs. If a polymer can show the antifouling property with DEAE, then it poses a higher probability of working on other ligands having lesser cell interaction challenges in expanded bed chromatography. Microbial surfaces are mostly anionic in nature, mainly due to the presence of negatively charged chemical groups like phosphate, carboxylate, and sulfate moieties. Due to the presence of S-layer proteins, amphipathic polymers, and lipids cell envelope can also exert hydrophobic interaction [4]. These properties are unique to each cell type [11, 25] and sometimes the age of the culture also influences such surface properties[35]. Indeed, the naked DEAE surface showed high cell deposition; CPI values were 0.41 for yeast cells and 0.48 for CHO cells. At the control level, CHO cell clearly showed strong interaction with DEAE surface in comparison to yeast cells. Coating of DEAE surface with polymers modified the cell deposition values depending on the polymeric property. Partition test results for yeast indicated that CPI values increased in the presence of all the polymer listed. However, poly(vinyl sulfonic acid), poly(methacrylic acid), poly(4-styrenesulfonic acid) showed the highest CPI increase to 0.54, 0.53, 0.52 respectively and the lowest being TWEEN® 20, Brij® 35, hyaluronic acid with CPI values of 0.43, 0.41and 0.41. Poly(methacrylic acid), polyacrylic acid and poly(vinyl sulfate) showed the highest cell repellence with CPI values of ~ 0.58 and poly(ethylene glycol) 8000, poly(4-styrenesulfonic acid) and poly(ethylene glycol) 20,000 showed the least CPI values of 0.48, which is comparable to naked CPI values. Lowest CPI valued polymers for both CHO and yeast cells mostly failed to avoid cell attachment.
From the previously published literature, it has been well established that xDLVO calculations serve as a valuable tool in understanding biomass adhesion studies. By correlating xDLVO interaction energy with experimentally determined partition values and by using the previously published results as benchmarking to shortlist the potential polymer with cell repellence property enabled us to spot the right candidate using different indicators. In this study, we used polyvinylpyrrolidone 360 as a benchmarking polymer to divide the polymer. The region with green shadow is categorized as safe polymers for further testing. The rationale behind such categorization is that PVP coated anion exchangers could successfully repel yeast cells [4]. However, PVP failed to repel CHO cells in process condition (Unpublished work). The polymer is having interaction energy less than PVP and CPI value higher than PVP in respective biomass types, have a better chance to screen for the best polymer for further studies. Based on this assumption we have observed that PMA, PAA, and PVS are the top three polymers having CPI values higher than PVP and interaction energy less than PVP for CHO cells. PSS, PMA, and PS4 listed as top polymers having better values than PVP for yeast cells. By observing the spread created by CPI and kT values of CHO and yeast cells in Fig. 2, CHO cells showed more interaction with polymers regarding both CPI values and kT values, and they are spread across the plot and have a better correlation with kT and CPI. In the case of yeast cells, the spread is comparably narrow, and CPI and kT correlation are not linear.
The interaction energy as a function of biomass type and adsorbent surface in the feedstock can be observed in Fig. 3. The interaction energy calculation performed for yeast and CHO cell suggest that both cell types interact strongly with DEAE-dextran and top three antifouling polymer surface. This figure helps us to understand the impact of particle size on overall energy vs. distance profile. Lin et al. have recognized the significance of biological particle size, besides the evident electrostatic effects between two opposite charged spheres, during biomass interactions in EBA [36]. Interaction energy vs. distance profile for polymers clearly corroborate the CPI results and present reduction in interaction energy with the cell.
The polymer coating of the support or adsorbents were already performed previously and showed positive results in reducing the impact of fouling. Shortlisted polymers for both yeast and CHO cells are PSA, PMA, PS4, PAA, PMA and PVS. All these polymers have a negative charge on the surface. The thin layered polymer coating on to DEAE surface may be formed due to the electrostatic interaction, this, in turn, modifies the outer surface charge. Some of the polymers we shortlisted have found their application as antifouling agents in other fields including membrane application. PAA has been coated on anion exchanger to reduce fouling in EBA process [6], PAA brushes have resisted the attachment of RBL mast cells [37]. Coating of PAA and PVS reduced fouling in micro and nanofiltration application [14]
Ultimately, shortlisted polymers have to be tested for its cell repellence property by coating on commercial EBA adsorbent. For this purpose, we chose Fastline DEAE adsorbent and yeast cells, and random polymer from the polymer library tested as it houses similar ligand on an agarose gel. Experimental conditions were similar to that of the microplate method. In the end, the effect of polymer coated was visually observed under a microscope, and naked adsorbent showed a strong interaction and aggregation of yeast cells on the adsorbent surface. For Brij® 35 (B35) coated adsorbents, the degree of cell adhesion was lesser than the naked ones but not significant. On PVP coated adsorbents there was very low interaction observed between the yeast cell and adsorbent. PVS, PMA, and PAA showed a significant reduction in cell interaction, in turn, this allows the free ligands to interact with target proteins.