1. A quantitative spectral method has been developed to precisely measure the color of protein solutions. In this method, a HunterLab UltraScan Vis spectrophotometer is utilized for capturing the visible absorption spectrum of a protein solution, which can then be converted to color values (L*a*b*). These quantitative values (L*a*b*) allow for calculating the best match of a sample’s color to an EP reference color solution. In order to qualify this instrument and assay for IMP QC use, an assessment was conducted to evaluate the system suitability and assay accuracy and precision. Standards for system suitability testing were developed in preparation for moving this assay into a GMP testing environment. Different instruments, cuvettes, protein solutions and analysts were compared in this study and the instrument accuracy, repeatability and assay precision were determined. The instrument and assay are found suitable for use in measuring color of drug substances and drug products. The quantitative spectral method is comparable to the current EP visual method, with significantly improved reproducibility. These studies demonstrate that this assay is suitable for use in a GMP laboratory for lot release and stability testing. Accuracy and Precision Assessment of a Spectrophotometric Method for Quantitative Measurement of Color in Protein Drug Solutions Jian Yin, Bruce Kabakoff, Tom Patapoff, Bartolo Chen, Joseph Marhoul, Norman Shih, Jian Zhang, Trevor Swartz and Kimia Rahimi Genentech Inc. Jian Yin, Bruce Kabakoff, Tom Patapoff, Bartolo Chen, Joseph Marhoul, Norman Shih, Jian Zhang, Trevor Swartz and Kimia Rahimi Genentech Inc. ASSAY ACCURACY ASSAY PRECISION : INTERMIDIATE PRECISION Wavelength accuracy check: Use a didymium solid color standard to verify the wavelength accuracy at two specific wavelength: 430 nm and 570 nm. Photometric response check: Use a neutral density filter to verify the midrange photometric response over the whole visible range (360 nm-780 nm) Target values EP Visual assessment HunterLab Spectral method BY1 BY1.0 B1 B1.0 B5 B5.0 R5 R5.0 GY4 GY4.0 BY4 BY3.9 The spectral method correlates well with the EP visual assessment method. Both the spectral method and the visual assessment method matched the target color values when using EP color reference solutions as testing samples The acceptance criteria is set at Max ΔE*2000≤0.5 The Max ΔE*2000 between each measurement to the mean of the replicates was used as a single matrix to evaluate the assay variability The smaller the Max ΔE*2000, the smaller the assay variability Human eyes can only note the color differences when the ΔE*2000≥1 The assay precision was determined using three representative protein solutions with different concentrations and turbidities. (Mab 3, Mab 5 and Mab 14 in above table) Sample IDMab 3Mab 5Mab 14 Reading #L*a*b* color (≤)L*a*b* color (≤)L*a*b* color (≤) 199.14-0.141.87 B697.87-0.215.16B597.42-0.598.30 B4 299.24-0.141.86B697.92-0.205.15B596.95-0.558.34B4 399.30-0.141.89B697.95-0.225.17B597.41-0.578.32B4 499.16-0.151.88B698.03-0.225.17B597.39-0.578.33B4 599.04-0.131.86B697.93-0.235.14B597.32-0.578.32B4 699.35-0.141.88B698.05-0.235.16B597.52-0.588.31B4 Max ΔE*2000 0.030.040.05 Max ΔE*2000 ≤0.16 Instrument to instrument variability-Max ΔE*2000 ≤0.17 Sample ID Mab 3Mab 5Mab 14 Reading # L*a*b* Color (≤) L*a*b* Color (≤) L*a*b* Color (≤) 199.41-0.131.90B698.07-0.225.12B597.53-0.588.33B4 299.33-0.131.93B698.05-0.225.12B597.54-0.578.32B4 399.34-0.131.91B698.07-0.215.14B597.31-0.588.29B4 499.34-0.131.91B697.92-0.235.08B597.57-0.588.31B4 599.34-0.131.91B698.05-0.225.14B597.61-0.588.31B4 699.37-0.141.89B698.08-0.235.16B597.54-0.558.38B4 Max ΔE*2000 0.150.090.16 Instrument ID#1#2Max ΔE*2000 Sample IDL*a*b*L*a*b* Mab 399.36-0.131.9199.40-0.151.90 0.08 Mab 598.04-0.225.1398.10-0.245.16 0.11 Mab 1497.52-0.578.3297.54-0.618.36 0.17 Analyst ID#1#2Max ΔE*2000 Sample IDL*a*b*L*a*b* Mab 399.21-0.141.8799.36-0.131.91 0.23 Mab 597.96-0.225.1698.04-0.225.13 0.14 Mab 1497.34-0.578.3297.52-0.578.32 0.30 Cuvette3.5 mL standard cuvette1.4 mL semi-micro cuvette Reading #L*a*b*color (≤)L*a*b*color (≤) 198.75-0.344.91 B5 98.49-0.295.01 B5 298.76-0.344.90 B5 98.18-0.314.91 B5 398.48-0.334.91 B5 98.45-0.284.98 B5 Average98.66-0.344.91 98.37-0.294.97 SD0.160.01 0.170.020.05 The HunterLab UltraScan Vis spectrophotometer is suitable for quantitatively measuring the color of a protein solution. The spectral measurement of color solutions independent of protein concentration and/or turbidity. A didymium standard and a 0.2 OD neutral density filter are suitable for the system suitability check. The quantitative spectral method is comparable to the current EP visual assessment method, but can produce more precise color values. The precision of the spectral assay is acceptable compared to that of the visual assessment.. The qualitative spectral method is suitable for using in a GMP environment. ACKNOWLEDGMENT Margaret Tang, Travis Horst, Inna Notkin and Samir Sane ASSAY PRECISION : ACCEPTANCE CRITERIAS CONCLUSIONS ASSAY PRECISION : ASSAY REPEATABILITY MINIMIZING SAMPLE VOLUME ASSAY PRECISION : INSTRUMENT REPEATABILITY SYSTEM SUITABILITY INTRODUCTION Max ΔE*2000 ≤0.05. Sample IDConc.TurbidityCOC report color (≤)HunterLab report color (≤) mg/mL Analyst ID 1234512345 Mab120Ref IIB7BY6B7 B6 Mab222Ref IB9 B8B9 N/AB9 Mab 325Ref IIB7BY6B7B8B6 Mab450Ref IBY4Y4BY4Y4 B5 Mab 550Ref 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 Variable result between different analysts by the visual assessment method Consistent result between different analysts by the spectral method Small bias between the two methods when using protein solutions as testing samples which is partially attributable to the light scattering effect on protein solutions Analyst to analyst variability-Max ΔE*2000 is ≤0.30. The 3.5 mL standard clear wall cuvette (10 x 10 mm) and the 1.4 mL (4 X 10 mm) black wall semi-micro cuvette with 1 cm path length can produce comparable and reproducible result thus are recommended to use. The 3.5 mL disposable acrylic cuvette (10 X 10 mm) with 1 cm path length can be used for ADC and potent protein solutions (data not shown). The 0.7 mL black wall cuvette (4 X 5 mm) with 1 cm path length can not produce reproducible result, thus can not be used (data not shown). One analyst measured each sample 6 times on the same instrument without sample refills One analyst measured each sample 6 times on the same instrument with sample refills One analyst measured each sample 6 times on each of the two instruments Two analysts measured each sample 6 times on the same instrument