Mar. 26, 2020  9  US 2020/0093894 Al  Example B  Example C  Example D  Example A  Example 1  Example Formulations  [0182]  [0181]  The following example formulations may be pre› pared:  [0185]  [0184]  [0183]  -continued  gently swirled or inverted, ensuring that air bubbles are not  introduced, and observed for about 5 s in front of the white  panel. The procedure is repeated in front of the black panel.  The presence of any particles is recorded.  [0172]  The visual scores are ranked as follows:  [0178]  Whilst the particles in samples with visual scores 4  and 5 are clearly detectable on casual visual assessment  under normal light, samples with visual score 1-3 generally  appear as clear solutions on the same assessment. Samples  with visual scores 1-3 are considered to be "Pass"; samples  with visual score 4-5 are considered to be "Fail"  ( c) Size Exclusion Chromatography  [0179]  Ultra-high performance size exclusion chromatog› raphy of insulin preparations was performed using the  Waters ACQUITY H-class Bio UPLCfi system with a 1.7  µm  Ethylene Bridged Hybrid 125  A  pore packing material in  a 300 mm by 4.6 mm colunm. The column was equilibrated  in 0.65 mg/ml L-arginine, 20% v/v acetonitrile, 15% v/v  glacial acetic acid mobile phase and 10 µI of sample,  acidified with 0.01M HCI, was analysed at 0.4 mL/min, with  276 nm UV detection. All analyses were performed at  ambient temperature. Results are expressed as % high  molecular weight species (HMWS) with respect to the total  protein content.  ( d) Reversed-Phase Chromatography  [0180]  Ultra-high performance reverse phase chromatog› raphy was performed using the Waters ACQUITY H-class  Bio UPLCfi system with a 1.7  µm  Ethyene Bridged Hybrid  particle, 130  A  pore resin trifunctionally immobilised with  a C18 ligand in a 50 mm by 2.1 mm colunm. Insulin samples  were bound in a 82% w/v Na$_{2}$ SO$_{4}$ ,  18% v/v acetonitrile, pH  2.3 mobile phase and eluted in 50% w/v Na$_{2}$ SO$_{4}$ ,  50% v/v  acetonitrile gradient flow. 2µ1 of sample was acidified with  0.01M HCI and analysed at 0.61 mL/min, with 214 nm UV  detection. All analyses were performed at 40°  C.  Insulin compound*  Sodium phosphate  phenol  m-cresol  Ionic zinc (as  ZnC1$_{2}$ )  Nicotinarnide  100 U/ml  2  mM  15.9 mM  15.9 mM  19.7 µg/ml (0.3 mM), equals 0.55% (w/w) based on  the weight of insulin compound in the formulation  80 mM  Insulin compound*  Sodium phosphate  phenol  m-cresol  Ionic zinc (as  ZnC12)  Nicotinamide  NaCl  Dodecyl maltoside  Water for injection  Residual NaCl  pH  1000 U/ml  2  mM  15.9 mM  15.9 mM  19.7 µg/ml (0.3 mM), equals 0.55% (w/w) based on  the weight of insulin compound in the formulation  80 mM  70 mM  0.05 mM  qs  Acidification and subsequent neutralisation during  preparation results in formation of 2-4 mM NaCl  Adjusted to 7.8  Insulin compound*  Sodium phosphate  phenol  m-cresol  Ionic zinc (as  ZnC12)  Nicotinarnide  NaCl  Dodecyl maltoside  Water for injection  Residual NaCl  pH  1000 U/ml  2  mM  15.9 mM  15.9 mM  19.7 µg/ml (0.3 mM), equals 0.55% (w/w) based on  the weight of insulin compound in the formulation  80 mM  70 mM  0.05 mM  qs  Acidification and subsequent neutralisation during  preparation results in formation of 2-4 mM NaCl  Adjusted to 7.4  Insulin compound*  Sodium phosphate  phenol  m-cresol  Ionic zinc (as  ZnC12)  Nicotinarnide  NaCl  Dodecyl maltoside  Water for injection  Residual NaCl  pH  100 U/ml  2  mM  15.9 mM  15.9 mM  19.7 µg/ml (0.3 mM), equals 0.55% (w/w) based on  the weight of insulin compound in the formulation  80 mM  70 mM  0.1 mM  qs  Acidification and subsequent neutralisation during  preparation results in formation of 2-4 mM NaCl  Adjusted to 7.8  NaCl  Dodecyl maltoside  Water for injection  Residual NaCl  pH  70 mM  0.1 mM  qs  Acidification and subsequent neutralisation during  preparation results in formation of 2-4 mM NaCl  Adjusted to 7.4  [0173]  Visual score  1:  Clear solution, virtually free of  particles  [0174]  Visual score 2: - 5 very small particles  [0175]  Visual score 3: - 10-20 very small particles  [0176]  Visual score 4: 20-50 particles, including larger  particles  [0177]  Visual score 5: >50 particles, including larger  particles 