US 2020/0246423 Al  54  Aug. 6, 2020  [0350]  For the example below, the levels of subvisible  particles (SVPs) were monitored for each of the formula› tions in Blocks A through G at all of the time points using  micro-flow imaging (MFI). Total particle levels are reported,  along with a corrected particle concentration once the cir› cular particles likely due to air bubbles and oil droplets are  [0332]  The SEC analysis parameters are described below  [0331]  Size exclusion chromatography (SEC) analyses  were conducted on formulations to measure the changes in  colloidal stability. SEC can quantify oligomers and soluble  aggregates, as well as detect fragmentation of proteins.  [0330]  N. Size Exclusion Chromatography Method  [0349]  Prior to analysis, approximately 10 mL of MilliQ  water was flushed through the MFI system using a 100 uM  FlowCell (FlowCell, 100 uM, 1.6 mm, Silane Coating). For  each sample, 350 µL  was pipetted and sample-filled pipette  was placed in the MFI inlet port. 0.03-0.05 mL manual purge  (by volume) was performed followed by selecting "Opti› mize illumination" to calibrate and set the background.  Analysis was started by selecting "Start analysis". A total of  180 ul of sample was analyzed for each measurement. After  each measurement, 1-4 ml of MilliQ water was flushed  through the flow-cell.  [0348]  MFI Analysis.  [0329]  The samples were sterile filtered in a clean hood  that was wiped down with 70% ethanol. Each formulation  was loaded into a sterile syringe with sterile filter attached.  The sample was then slowly pushed through filter into a  sterile container. After the samples had been sterile filtered,  they were loaded into autoclaved vials and caps  [0328]  M. Sterile Filtering and Sample Filling  [0347]  Samples were placed in the BSC and allowed to  equilibrate to room temperature for about 30 minutes. Using  Neptune Barrier Tips (1000 uL Barrier Tip, Cat #1000.96N),  samples were diluted 1: 1 with MilliQ Water, specifically 190  uL MilliQ water+190 uL Samples. All samples were  degassed for 20 min  @ 70 cmHg.  [0346]  Sample Preparation.  [0345]  In order to ensure the MFI was counting and  assessing particle size accurately, 5 uM size/concentration  standards (5 um polystyrene beads for MFI system suitabil› ity Count-cal Cat# CC05) and 10 uM size standards (10 um  polystyrene beads for MFI system suitability Duke Std Cat  #4210A) were used. Triplicate analyses for each standard  were performed and the average of each standard were  within 10% of true/given manufacture value.  [0344]  MFI System Suitability.  [0327]  The bulk processed aflibercept material was for› mulated following the procedures below. The aflibercept  material was buffered exchanged following the dialysis  procedure. After dialysis the osmotic pressure and the pH  was checked, if the pH of the sample was not within –0.1 pH  units, the formulation buffer was made more acidic or basic,  by repeat dialysis or by addition of HCl or NaOH, until the  pH target was reached. Following pH adjustment if needed,  the sample was concentrated above the target formulation  concentration. The pH, osmotic pressure and the protein  concentration was then measured once more. The sample  was then diluted with the formulation buffer to reached the  target formulation protein concentration within 10%. The  protein concentration was then measured once more to  insure the diluted sample was within 10% of the target  concentration. The last step was the addition of the 20% PS  20 (diluted in water) to the sample based on weight, if  required.  [0326]  L. Formulating  [0343]  To evaluate sub-visible particles in formulations  Micro Flow Imaging analysis was conducted. The MFI  analysis was performed on a MFI 5200 Protein Simple  system following the procedure described below.  [0325]  The osmotic analysis was performed using an  Advanced Instruments, Osmo 1. At the start of analysis, a  reference standard at 290 mOsm is analyzed to insure the  instrument is working properly. After the reference standard  has passed the samples are then analyzed. 20 uL of material  is removed and analyzed by the Osmo 1, after analysis the  chamber is cleared by a using a chamber cleaner. This  procedure is repeated for each sample.  [0324]  K. Osmotic Analysis  [0342]  In the examples below, screening studies were  conducted to identify formulations with particle counts  indicating that the formulation is sufficiently stable and  therefore suitable as commercial aflibercept product. For› mulations with exceptionally high particle counts ( e.g.  > 1,000,000) may not be sufficiently stable. The particle  counts reported in the examples below for a formulation  may not be the particle counts for a commercialized afliber› cept product. For example, a commercialized product is  manufactured under strict GMP conditions which is  expected to reduce the number of particles found in the  formulation. Moreover, USP guidelines (e.g. USP <788>,  <789>, which are hereby incorporated by reference in their  entirety) limit the number of particles that can be present in  certain products. When manufactured and handled under  GMP conditions, it is expected that the formulations herein  that are identified to be sufficiently stable based on the  screening test of sub-visible particles will be suitable for a  commercial aflibercept product.  [0341]  0. Sub-Visible Particle Evaluation by MFI  [0323]  After sample preparation the pH will be checked  for each formulation and be within –0.1 of the target pH.  Before the start of analysis, the pH probe was calibrated with  three pH standards ordered from fisher. The pH of the  formulation will be measured by inserting the pH probe in  to the sample and waiting until the measured value has  stabilized, which can take up to 1 to 2 minutes. After the  analysis the pH probe is washed with 18Q water for one  minute and stored in the pH storage solution.  [0322]  J. pH Analysis  lation was measured using the Solo VP. The concentration of  the sample was measured by added 50 to 100 µL  of material  into a Solo VP small UV disposable vessel. A new fibrette  was installed and the sample absorbance was measured by  the instrument, using an extinction coefficient of 1.55 mL.  mg- 1  cm- 1 , and correcting background scattering. After  analysis the sample is removed with a pipette the disposable  vessel and fibrette are both disposed. This procedure is  repeated for each sample.  [0340]  Data Collection Time: 15 minutes  [0339]  UV Setting: 280 nm  [0338]  Injection Vol: 5 µL  [0337]  Flow Rate: 0.25 mL/minute  [0336]  Column Temp.: 30°  C.–2°  C.  [0335]  Autosampler Temp: 5°  C.–3°  C.  [0333]  Column: ACQUITY UPLC BEH SEC (CAT.  186005225), 220  A  1.7 µm, 4.6 mmx150 mm  [0334]  Mobile Phase: 20 mM Sodium Phosphate, 250  mM Sodium Chloride, pH 7.55* 