1. In biotechnology industry, a visual method is mostly utilized for color characterization of liquid drug product solutions. In this method an analyst visually determines the color of the sample by choosing the closest match to standard color series. This method can be subjective due to the requirement of an analyst to make a judgment of the best match of color of the sample to the standard color series. Furthermore, variability in preparation of the color series solutions exists. In addition, the current method does not capture data on hue and chroma that would allow for improved product characterization and the ability to detect subtle differences between samples. To overcome these challenges, we describe a quantitative method for color determination that greatly reduces the variability in measuring color and allows for a more precise understanding of color differences. Following CIE color industry standards, this method converts a protein solution’s visible absorption spectra to L*a*b* color space. Color matching is achieved within the L*a*b* color space. We describe here the algorithm used such that the quantitative method correlates with the current visual method. In addition we provide the L*a*b* values for the EP color standards required for the Visual color assessment method. We have determined the L*a*b* values of the EP color standards by gravimetrically preparing and measuring multiple lots of the color standards. We demonstrate the correlation between the visual and quantitative method using both low and high concentration antibody solutions. Spectral Method for Quantitation of Color in Protein Drug Solutions Jian Yin, Tom Patapoff, Travis Horst, Kimia Rahimi, Inna Notkin, Jian Zhang, Joseph Marhoul, Sue Skieresz, Bruce Kabakoff, Sarah Du and Trevor Swartz (Genentech Inc.) Gordon Leggett and Paul Barnes (HunterLab) Jian Yin, Tom Patapoff, Travis Horst, Kimia Rahimi, Inna Notkin, Jian Zhang, Joseph Marhoul, Sue Skieresz, Bruce Kabakoff, Sarah Du and Trevor Swartz (Genentech Inc.) Gordon Leggett and Paul Barnes (HunterLab) Visual assessment and quantitative method correlate for both high and low concentration protein solutions (low and high turbidity) There is a slight bias of quantitative assessment method towards Brown color series vs. Brown Yellow for visual assessment   This is partially attributable to light scattering that occurs from light entering the sides of the sample tube.   This is demonstrated by altering visually observed data when tube is wrapped in foil (data not shown). Quantitative method has much higher precision as compared to visual assessment (see associated poster; ”Accuracy and Precision Assessment of a Spectrophotometric Method for Quantitative Measurement of Color in Protein Drug Solutions”) Vitamin B2 Transmission X Solar Spectrum Tristimulis value Tristimulis value Blue Wavelength (nm) Green Red Wavelength (nm) Relative intensity Riboflavin observed through daylight illuminant Absorption (OD) Wavelength (nm) R1 R7 Absorption of red color series a* b* L Water R7 R1 Green Blue Red Yellow Yellow quadrant 100 0 -110 110 - 110 a* Color series is all within the ‘yellow quadrant’ R7, B7, BY7,Y7, G7 are all tightly clustered correlates with difficulty in differentiating when compared to actual color standards Larger spacing are found between more intensely colored samples Color space is uniform in perception b* Red BrownYellow Green Yellow Brown Yellow Take the absorption spectrum and transform to L*a*b* values a b Red Brown Brown Yellow Green Yellow Yellow Protein solution Shortest distance correlates to best match of hue and chroma (intensity) in visual assessment method Protein solution assigned to yellow series, less intense than Y3 a b Yellow 3 Yellow 4 Green Yellow 4 Green Yellow 3 Protein solution B1 B9 BY1 BY7 Brown Brown Yellow Protein solution test sample Color characterized by a visual determination of color using EP color standards Basis for quantitative color method: Information about color is captured in the absorption spectrum of color standards Introduction Quantitative method describes relative perception of color standards BLUE, GREEN and RED values transformed to visually uniform L*a*b* color space Transmission is transformed to BLUE, GREEN and RED values using illuminant and human observer tristimulis values Implementation of a quantitative color determination method for a protein solution Quantitative method correlates to visual assessment method with improved precision Protein solution test sample HunterLab Software has been customized such that the readout correlates to the current visual assessment method Developed daily system suitability protocol Developed validation protocol Goal is to be operational in GMP environment in 2015 Quantitative method report is consistent with visual assessment method with improved precision Sample L*a*b* values will be recorded to allow for tracking /trending of color (Lot to Lot variability and on stability) Implementation of a quantitative color test method for GMP testing of products (COA and stability) Challenges: Current visual color assay has inherent variability Current color measurement: Color (hue) readout: An analyst picks the best match of sample to a color series Intensity readout: An analyst picks the next darkest color standard as test result Variability in making up standard color solutions Subjectivity in picking best match color Wavelength (nm) Absorption (OD) Vitamin B2 R7 Absorption of a sample from each color series X = Absorption spectrum is mathematically converted to Transmission Note: OD of 1 is equal to 10% Transmission Color vision initiated with blue, green and red photoreceptors Wavelength (nm) % Transmission Absorption can be converted to transmission (aligns with our perception) Absorption (OD) Absorption spectrum Vitamin B2Transmission spectrum Vitamin B2 Wavelength (nm) Correlation of instrument readout with visual assessment for samples with little color b* a* water B6 R7 Allowed Hue and Chroma will match visual assessment method Less colored than B6 but very Red or very Green hue is allowed Less colored than R7 reports to the Brown color series Y 3.8 Y 3.6 Y 3.4 Y 3.2 Sample IDConc.TurbidityEP visual assessment report color (≤) HunterLab report color (≤) mg/mL Analyst ID 123451 mAb 120Ref IIB7BY6B7 B6 mAb 222Ref IB9 B8B9 mAb 325Ref IIB7BY6B7B8B6 mAb 450Ref IBY4Y4BY4Y4 B5 mAb 555Ref IIIB6Y5B6 B5 mAb 650Ref IIBY5Y5BY5Y4BY5B5 mAb 760Ref IB6 B5 R6 mAb 8125Ref IVB6Y5B6 B5 mAb 9125Ref IBY5 B5 BY4B5 mAb 10150Ref IIB5 mAb 11150Ref IIBY5 B6Y5B5 mAb 12150Ref IIIY1BY2BY1 Y1BY1 mAb 13160Ref IIIBY5Y5B5 BY4B5 mAb 14200Ref IIBY3BY4BY3B4BY4B4 mAb 15206Ref IIB5BY4B4BY4 B4 Locking L*a*b* values for EP color standards L*a*b* values for EP color standards have been locked and will be published: Gravimetrically prepared and measured L*a*b* values of 5 batches of each color standard Variability of 5 batches of color standards measured by multiple analysts is less than visually discernable (delta E<0.5) Statistically robust method used to lock L*a*b* values