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. 

[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. 

Stability Testing Conditions 

[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. 

[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. 

[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. 

## Example I-Preparation of Aflibercept 

A. Recombinant Production 

[0371] DNA encoding aflibercept is inserted into an expression vector appropriate for the host cell to express aflibercept. 

[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. 

[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. 

[0374] Aflibercept may be expressed in a mammalian cell line, such as a CHO cell and may be modified post› translationally. 

[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. 

[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). 

[0377] The aflibercept composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography. 

[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). 

B. Purifying Aflibercept from a Commercially-available Formulation 

[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- 

