US 2020/0246423 Al  56  Aug. 6, 2020  [0379]  Alternatively, aflibercept is purified from a com› mercially available preparation such as Eyleafi or Zaltrapfi.  Aflibercept is purified away from other formulation com› ponents in Eyleafi or Zaltrapfi using, for example, hydroxy› apatite chromatography, gel electrophoresis, dialysis, affin- B. Purifying Aflibercept from a Commercially-available  Formulation  [0378]  Following any preliminary purification step(s), the  mixture comprising aflibercept and contaminants may be  subjected to low pH hydrophobic interaction chromatogra› phy using an elution buffer at a pH between about 2.5-4.5.  The HIC may be performed at low salt concentrations (e.g.,  from about 0-0.25M salt).  [0377]  The aflibercept composition prepared from the  cells can be purified using, for example, hydroxylapatite  chromatography, gel electrophoresis, dialysis, and affinity  chromatography.  [0370]  Aflibercept may be prepared either de nova or  through purification/isolation from a commercially available  source. The steps below describe production of aflibercept  by culturing cells transformed or transfected with a vector  containing aflibercept nucleic acid. Alternative methods,  which are well known in the art, may also be employed to  prepare aflibercept. For instance, the appropriate amino acid  sequence, or portions thereof, may be produced by direct  peptide synthesis using solid-phase techniques [see, e.g.,  Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman  Co., San Francisco, Calif. (1969); Merrifield, J. Am. Chem.  Soc., 85:2149-2154 (1963)]. In vitro protein synthesis may  be performed using manual techniques or by automation.  Automated synthesis may be accomplished, for instance,  using an Applied Biosystems Peptide Synthesizer (Foster  City, Calif.) using manufacturer’s instructions. Various por› tions of aflibercept may be chemically synthesized sepa› rately and combined using chemical or enzymatic methods  to produce aflibercept.  [0376]  It may be desired to purify aflibercept from recom› binant cell proteins or polypeptides. The following proce› dures are exemplary of suitable purification procedures: by  fractionation on an ion-exchange colunm; ethanol precipi› tation; reverse phase HPLC; chromatography on silica or on  a cation-exchange resin such as DEAE; chromatofocusing;  SD S-PAGE; ammonium sulfate precipitation; gel filtration  using, for example, Sephadex G-75; protein A Sepharose  columns to remove contaminants such as IgG. Various  methods of protein purification may be employed and such  methods are known in the art and described for example in  Deutscher, Methods in Enzymology, 182 (1990); Scopes,  Protein Purification: Principles and Practice, Springer-Ver› lag, New York (1982).  [0375]  Aflibercept is recovered from culture medium or  from host cell lysates. If membrane-bound, it can be released  from the membrane using a suitable detergent solution ( e.g  Triton-X 100) or by enzymatic cleavage. Cells employed in  expression of aflibercept can be disrupted by various physi› cal or chemical means, such as freeze-thaw cycling, soni› cation, mechanical disruption, or cell lysing agents.  Example I-Preparation of Aflibercept  [0374]  Aflibercept may be expressed in a mammalian cell  line, such as a CHO cell and may be modified post› translationally.  [0369]  The samples may also be assessed by starting the  experiment at one temperature, e.g., 40°  C., and then step› ping or ramping to a different temperature, e.g., 25° C.,  and/or 2-8°  C., for a selected period of time. For example,  the samples may be stored at -20°  C. or 2 months; and/or at  40°  C. for 1 and 2 weeks; and/or at 25°  C. for 2 and 4 weeks,  and/or at 2-8°  C. for 2 months. A drop test can be carried out  at 25°  C.  [0368]  Unless otherwise indicated, formulations may be  maintained at an indicated temperature, e.g., 40°  C., 25°  C.,  2-8°  C., or a freezing temperature (e.g. 0°  C. and below) for  a period of time, e.g., days, weeks, or months, and assessed  for stability and/or other parameters. For example, the pH,  conductivity and/or viscosity of the samples may be mea› sured at time zero and periodically throughout the experi› ment. The material may be analyzed for stability and other  properties using, e.g., chromatography or other methods;  non-limiting examples include SEC, RP, HIC, CEX, AF4  and/or CE-SDS. These analyses are typically carried out at  time zero and then periodically during the experiment.  Stability Testing Conditions  [0373]  Methods of eukaryotic cell transfection and pro› karyotic cell transformation, which means introduction of  DNA into the host so that the DNA is replicable, either as an  extrachromosomal or by chromosomal integrant, are known  to the ordinarily skilled artisan, for example, CaC1$_{2}$ ,  CaPO$_{4}$ ,  liposome-mediated, polyethylene-gycol/DMSO and elec› troporation.  [0372]  Host cells transfected or transformed with expres› sion or cloning vectors described herein for aflibercept  production is cultured in nutrient media modified as appro› priate for inducing promoters, selecting transformants, and/  or amplifying the genes encoding the desired sequences. The  culture conditions, such as media, temperature, pH and the  like, are selected by the skilled artisan using well-known  approaches without undue experimentation. For example,  principles, protocols, and practical techniques for maximiz› ing the productivity of cell cultures can be found in Mam› malian Cell Biotechnology: a Practical Approach, M. Butler,  ed. (IRL Press, 1991) and Sambrook et al., supra.  [0367]  A full cross validation was performed on all cali› bration models using standard techniques. Briefly, one  sample is removed at a time, the data set is recalibrated, and  a new model is constructed. This process is repeated until all  of the calibration samples are removed once and quantified  as a validation model. Therefore, the first set, containing all  samples is referred to as the calibration set and the one after  cross-validation as the validation set. The jack-knife algo› rithm was used to determine statistical significance for any  factor used in constructing the PLS models described above.  ber of PCs, and constructs a suitable model. The model that  includes all of the samples is termed a calibration model.  The overall coefficient of determination (r 2 )  indicates the  quality of the model. All PLS calculations were conducted  using Unscramblerfi software (CAMO, Corvallis, Oreg.). A  PLS analysis done with a single variable in the Y-matrix is  termed PLSl analysis. Building a model that fits multiple  variables in the Y-matrix is called PLS2 analysis.  [0371]  DNA encoding aflibercept is inserted into an  expression vector appropriate for the host cell to express  aflibercept.  A. Recombinant Production 