Table 1. Advantages and disadvantages of identified separating techniques used for ovalbumin

Description	Advantages	Disadvantages	References
Electrophoretic separations
1. Native PAGE			Desert et al. (2001), Geng et al. (2012), Miguel et al. (2005)
Stacking gel - 4% acrylamide, separating gel - 7.5% acrylamide, pH 8.8	- Proteins did not undergo denaturation - Reflected the coexistence of A₁, A₂, and A₃ isoforms of ovalbumin	- Did not possess the ability to calculate the pI values of the molecules
2. 2D electrophoresis
pH gradient: 4-7Subjected to SDS-PAGE (12%)	- Enabled the visualization of ovalbumin not corresponding to any known egg white molecule - Considering both the pI and molecular weight of the protein	- Limited resolution - Denaturation of polypeptides occurred when subjected to SDS-PAGE
3. SDS-PAGE
Stacking gel - 4%, separating gel - 12% acrylamide	- Abled to localize ovalbumin considering molecular weight	- Denaturation of protein
4. Iso-electric focusing
7.5% acrylamide gel pH gradient: 3-7Running conditions - 1 h at 100 V, 1 h at 250 V, and 30 min at 500 V	- Visualization of ovalbumin did not correspond to any known egg white molecule - Identification of the availability of isoforms of ovalbumin - Can calculate pI values of isoforms	- Not suitable for egg white protein separation because iso-electric points (pI values) of most proteins are closer values
Chromatographic techniques
1. RP-HPLC
C4 supelcosil LC-304 column, at 214 nm	- Better separation when compared to gel permeation chromatography	- Approximately 30% retention of ovalbumin on column	Awade and Efstathiou (1999)
2. Gel permeation
With superose 12 HR 10/30 column, at 280 nm with FPLC system		- Not recommended (Ovalbumin was eluted together with ovotransferrin, ovomucoid, avidin, and ovoglobulin)	Awade and Efstathiou (1999)
3. Anion exchange chromatography
i) Dimethylaminoethyl (DEAE) - cellulose (Whatman DE92 anion exchange cellulose)	- Better to use than Whatman DE52 because resin allows the application of a higher flow rate	- Adsorption of ovalbumin was observed - Lower capacity and protein recovery when compared with Whatman DE52	Awade (1996)
ii) Mono Q HR 5/5 using HPLC system (0.02 M Tris-HCl, pH 9, increasing NaCl concentration, detecting at 280 nm)	- Higher resolution compared with gel permeation and RP-HPLC and higher recovery than RP-HPLC (30% retention in RP-HPLC) - Low time consumption	- Sample preparation was essential for removing ovomucin - The complex separation procedure	Abeyrathne et al. (2013b), Awade and Efstathiou (1999)
iii) Q-Sepharose fast flow Mucin-free egg white is loaded to a cation exchanger (S Ceramic Hyper DF) to separate, and the resulting elute used in anion exchange chromatography (0.14 M NaCl isocratic elution, detected at 280 nm)	- Higher purity (91%) and yield (80%)	- Sample preparation was essential because mucin-free egg white was loaded, use of water in precipitation of ovomucin (Cause huge loss of proteins such as ovalbumin and lysozyme in the process)	Guerin-Dubiard et al. (2005), Omana et al. (2010)
iv) Q-Sepharose fast flow (Based on frontal chromatography)	- Simple - Low time consumption - High purity (94% in 0.14 NaCl and 83% in 0.5 M NaCl isocratic elusion) - Non-altered by-product	- Laboratory scale method - Sample preparation was required - Not following the two-step precipitation method for ovomucin. So, contamination of ovalbumin and lysozyme can occur during precipitation of ovomucin	Croguennec et al. (2000), Omana et al. (2010)
v) Q-Sepharose fast flow FPLC system (0.05 M Tris-HCl, pH 9, 0.3 NaCl)	- Non-denaturing method - Low time consumption	- Low protein recovery (54%) and purity (70%) - Laboratory scale - Sample preparation was essential	Belchior and Freire (2021), Tankrathok et al. (2009)
vi) Q Sepharose fast flow with FPLC (Co-extraction of egg white proteins) Two-step precipitation method for ovomucin precipitation and cation exchange chromatography (SP Sepharose FF) for separation of lysozyme, ovotransferrin, ovoinhibitor mainly	- High purity (estimated purity from 100 mM supernatant was 91.2%, estimated purities for three ovalbumin fractions resulted from 500 mM supernatants were 82%, 100%, and 100%) - High yield (yield of ovalbumin from 100 mM supernatant-40.70% from total egg white protein)	- High salt concentration in 500 mM supernatant demands dialysis prior to ion-exchange chromatography - Recovery of ovalbumin from 500 mM supernatant was practiced after cation exchange chromatography and resulted in low yields (0.40%, 0.38%, 1.62% from total egg white protein)	Omana et al. (2010)
4. Cation exchange chromatography
CM-Sepharose FF chromatography	- Single protocol, purity was higher than commercial ovalbumin, low cost	- Egg white pretreatment was needed (removal of ovomucin and globulins)	Ma et al. (2020)
Salt Precipitation
1. (NH₄)₂SO₄ or Na₂SO₄	- Separated ovalbumin successfully for the first time	- Product with high salt concentration - Irreversible unfolding of proteins can occur due to high salt concentration - Difficulties in the separation of lysozyme and ovalbumin	Datta et al. (2009), Hopkins (1900), Pereira et al. (2016)
2. Sequential separation of lysozyme, ovotransferrin, ovomucin, and ovalbumin [Amberlite FPC 3500 ion exchange resin iso-electric precipitation of ovomucin → 2.5% (w/v) citric acid and 5% (w/v) ammonium sulfate combination → 1.5% (w/v) citric acid and 2% (w/v) ammonium sulfate combination → ultrafiltration]	- Simple, non-toxic method, Better than ammonium sulfate and acetic acid combinations, which were practiced earlier (A lower percentage of ammonium sulfate has been utilized) - Effective separation of ovalbumin (purity was greater than 87%, yield greater than 97.7%) - The possibility to scale up can be used in the laboratory as well as on an industrial scale, provides cost benefits due to sequential separation of highly purified multiple proteins, within four days separation of four proteins can be accomplished	- Several stages of equilibrium/purification of proteins correspond to a multiple-step process	Abeyrathne et al. (2013b), Abeyrathne et al. (2014a), Belchior and Freire, (2021), Pereira et al. (2016)
3. Successive extraction of egg white proteins (Modified from Abeyrathne et al., 2014)	- Higher purity (~95%) - Low cost - Simple	- Changes in the secondary structure of ovalbumin were identified	Ji et al. (2020)
Ultrafiltration
1. Amicon stirred cell fitted with polyether sulfone (PES) membrane (Two-stage ultrafiltration scheme; 30 and 50 kD flat disks)	- High purity (98.7%)	- Preparation of the sample was required by steps such as centrifuging, dilution, and pH adjustments, can be highly affected by the operating and physicochemical conditions, difficult to use in the scaling-up process	Abeyrathne et al. (2013b), Datta et al. (2009)
Aqueous Biphasic Systems (ABS)
1. High-speed counter-current chromatography; aqueous polymer two-phase system (polyethylene glycol and potassium phosphate)	- Proteins were purified from the crude solution of fresh egg white in one step, short elution time. denaturation and adsorption loss are lower compared to column chromatography, 95% purification recovery [16% (w/w) polyethylene glycol and 17% (w/w) potassium phosphate, pH 9.2]	- Require specialized equipment, environmental and economic problems have been identified in polyethylene glycol-salt (PEG-salt) systems. This is due to the large consumption of phase-forming chemicals and possesses difficulties in regeneration, waste disposal problems, scaling up is not practical	Abeyrathne et al. (2014a), Saravanan et al. (2008), Shibusawa et al. (1998), Shibusawa et al. (2001), Pereira et al. (2016)
2. The aqueous biphasic system coupled with high-speed counter-current chromatography [16% (wt) Polyethylene glycol/ PEG-1000 and 17% (wt) potassium phosphate, pH 9.2]	- Purified to 95% - Absence of solid support when compared to other chromatographic techniques	- Higher toxicity because it contains a phosphate-based system - High molecular weight polymer usage	Pereira et al. (2016), Zhi et al. (2005)
3. Single step purification [Polyethylene glycol/PEG-400 (25% (wt)), potassium citrate/citric acid aqueous biphasic system (25% (wt), 50% (wt) egg white aqueous solution (1:10v/v)), pH 7]	- A simple approach with a single step - Low time consumption and low cost - Sustainable method (recycling PEG phase during purification of ovalbumin by inducing precipitation at low temperature and centrifugation) - Better compared to other polymer salts ABS - Minimum loss of protein due to denaturation, minimized adsorptive loss, (absence of solid support when compared to other chromatographic techniques) - Do not require specialized equipment, can perform on an industrial scale - Biodegradable and less toxic (because ABS is citrate-based, not phosphate as in Zhi et al., 2005)	- The recovery yield is 65%	Pereira et al. (2016)
4. Aqueous two-phase flotation (ATPF) (Polyethylene glycol 1000, ammonium sulfate)	- Low cost (Developed for separation of ovalbumin from byproduct formed in salted egg yolk production), simple, purity is approximately 92%, no difference in the ovalbumin structure or functional properties (Tested properties; oil binding capacity, foam capacity, emulsion ability, viscosity)	- Developed for the separation of ovalbumin from salted egg white	Jiang et al. (2019)
5. Simultaneous separation of egg white proteins by using ABS/TPP [30% (wt) PEG 2000, 13% (wt) K₂H₂PO₄/KH₂PO₄, pH 7.0]	- Recovery yield 82% - Can apply for crude suspensions directly - A single step to recover more than one protein	- Loss of ovalbumin due to use of high molecular weight PEG	Belchior and Freire (2021)
Polyethylene glycol precipitation combined with chromatography
1. Co-purification using polyethylene glycol precipitation (PEG-8000) and anion exchange chromatography; Q Sepharose Fast Flow (20 mM Tris-HCl buffer, pH 8, 0.18 M NaCl)	- Simple - Desalination was not required prior to anion exchange chromatography - Has the potential to use at industrial level - Higher purity (three peaks with purity percentages 90.77%, 96.45%, 94.84%) and low cost since only one column is used	- Recovery percentage of ovalbumin is 88.64%	Geng et al. (2012)