Chemicals and recombinant protein
Recombinant insulin drug substance of analytical purity of >99% pure containing deamidation variant of 0.3% was procured from Biocon Limited, Bangalore, India. Purified insulin drug substance was solubilized in 0.01N HCl targeting concentration of 4-10 mg/ml. Liquid solution was incubated at 45°C for minimum 24.0 hours. The sample was analysed in analytical HPLC to measure the level of enrichment of A21 deamidated variant in forced degradation condition. Processing of deamidated enriched material is pH adjusted to 7±0.1 using 1N NaOH. Conductivity of this solution was maintained at ≤ 3mS/cm and final load was filtered through 1.2 and 0.45µm filters After attaining this level, the material was used for feed for 1st step of purification through Anion Exchange Chromatography. The other regular laboratory chemicals and chemicals required for chromatography were procured from qualified vendors. The raw materials used for characterization studies were Hydrochloric acid (12N 37% Sigma Aldrich), Acetonitrile (J.T Baker (ACN)), Trifluoroacetic acid (Sigma Aldrich (TFA), C18 column (ACE), C8-column (Waters), Dithiothreitol (DTT), Glu-C (Endoproteinase, Roche), tris(hydroxymethyl) aminomethane (Sigma Aldrich (TRIS). Insulin phosphorylation in-vitro assay was performed using engineered CHO-K1 cells overexpressing insulin receptor B (IR-B).
Chromatographic media and instrumentation
Anion exchange chromatography was performed using Polyquat resin (Bakerbond XWP 500 Polyquat-35 from J.T.baker). RP-HPLC was performed with Kromasil C8 100Å 13µresin. Laboratory scale chromatography experiments were performed using a GE Healthcare ÄKTA Explorer 100 system. Anion exchange chromatography was performed using 1.6 cm & 5 cm diameter columns (XK-16 & HS50) obtained from GE healthcare, while RP-HPLC was performed using 2.12 cm & 1 cm Diameter prepacked columns (C8,13µ,100A°).
Anion exchange and Reversed phase chromatography
PolyQuat resin packed in the column was pre-equilibrated with 10 column volumes(CV) with at a linear velocity of ≤200cm/hr, with the buffer containing 20mM Imidazole, 10% Acetonitrile (Buffer pH was adjusted to 7.0±0.1 with HCl and conductivity was targeted to be 1-2mS/cm). Selection of Imidazole was done for its excellent buffering capacity at above mentioned pH range (7.0±0.1). The starting material (enriched deamidated material) was loaded onto the column with binding capacity of ≤6 grams of A21 impurity per litre of resin at a linear velocity of ≤200cm/hr. Once loading was completed, the column was washed with the 4 CV of same buffer at same flow rate. Product was eluted with 30 CV linear gradient from 0% to 100% of the elution buffer containing 20mM Imidazole, 200mM NaCl, 10% Acetonitrile at pH 7.0±0.1 with a target conductivity of 18-21mS/cm. While elution of the product was monitored at UV280 nm at preparative scale, eluted fractions were further analysed for its product quality in analytical RP-HPLC at UV220 nm. The fractions qualified for pre-determined acceptable criteria were pooled. Step recovery was also calculated by RP-HPLC analysis.
Elution pool from PolyQuat resin was diluted with purified water (having 10% acetonitrile) to target conductivity of ≤7.0mS/cm. pH was adjusted to 4.0±0.1 with 3.0 M glacial acetic acid. Load was filtered through 0.45- and 0.2-micron filter. Reverse Phase chromatography was carried out in pre-packed column using 0.05M Citrate buffer at pH 4.0±0.1 (Buffer A) and Acetonitrile (Buffer B) as organic modifier. The reversed phase chromatographic runs were carried out at a flow rate of ≤360 cm/h in loading, equilibration, washing and elution steps and monitored throughout at 280nm. Column was equilibrated with 4 CV of 10% buffer B (90% Buffer A). Column loading was performed with a binding capacity of ≤10 grams of A21 impurity per litre of resin and followed by washing with Column with > 4 CV of same buffer. Product was eluted from the column using a step elution of 32% Buffer B (68% Buffer A). Elution fractions were collected and analysed in analytical RP-HPLC at UV220 nm. The fractions qualified for pre-determined acceptable criteria were pooled. Step recovery was also calculated by RP-HPLC analysis.
Precipitation and lyophilisation
RP HPLC elution pool was diluted with water for injection (WFI) to target the final concentration of acetonitrile in FFC (Feed for precipitation) in the range of 14-16%. pH was adjusted to 5.0±0.15 using 1.0N NaOH & it was mixed for 3-5 min at a tip speed of 0.42 m/s. Post pH adjustment, the material was left out for further settling & slurry was allowed to settle for > 60minutes. Slurry sample was further centrifuged for 30 minutes at 8000RCF maintaining the centrifuge temperature at 4±2°C. Centrifuged supernatant was decanted & slurry was further washed by adding 200L of WFI (24±2°C) per kg of A21 impurity. Slurry was allowed to settle for > 60minutes, post mixing (5 minutes of mixing at a tip speed of 0.42 m/s). Slurry sample was further centrifuged for 30 minutes at 8000RCF maintaining the centrifuge temperature at 4±2°C & final centrifuged slurry was made into a final homogenous suspension with mild mixing while adding the WFI (final slurry % targeted was in the range of 10-40% of A21 product). Final homogenous slurry suspension (also called as feed for lyophilization) was further dried up using the freeze-drying recipe to attain an efficient drying (target LOD was ≤10%).
Analytical methods for detection, concentration and purity measurements
Samples at each process stage were analyzed using a reversed phase high-performance liquid chromatography (RP-HPLC) Agilent 1100 &1200 series system (Agilent Technologies, Santa Clara , USA) fitted with an analytical (4.6 mm × 250 mm) symmetry C18, 5 μm, 300 Å column (ACE technology ). A gradient elution was performed at a flow rate of 1mL/min with solvent A (0.1% Trifluoroacetic acid in water) and solvent B (Acetonitrile). A gradient program was set as follows: 25% - 40% solvent B for 15 min and column was equilibrated with 25% solvent B for 3 min . All the chromatograms were monitored under UV 220 nm. The retention time (RT) of product and each product related impurity peak was assigned as the time of elution from the column of corresponding product under the mentioned gradient program. The percentage purity of the target product as well as product concentration at all the stages of process were calculated using the HPLC peak area percentage as described in earlier work 
a) Intact and reduced Mass analysis:
The A21 desamido Human Insulin variant was characterized and identified by state of art technique i.e. LCESI -MS (Thermo Scientific LTQ Orbitrap XL) with a top down approach. The primary structure was determined by injecting the standard sample and the +1 Da variant in LC-MS analysis. Reverse-phase HPLC followed by electrospray ionization LC-MS (LC-ESI-MS; 5 μ C18-300Å 250 × 4.6 mm) was used to determine the intact and reduced masses of the drug product samples. Chromatographic separation of samples was carried out by using a reverse-phase C8 column (5 μ C8 250 × 4.6 mm), which was simultaneously ionized to give a mass to charge ratio (m/z) for particular species. The intact mass of variant was detected and a +1 Da was observed. Reduced masses of both samples A- and B-chains were obtained via dithiothreitol (DTT)-mediated reduction of disulfide bonds.
The difference in the standard protein chain mass and variant chain masses was observed and the site of +1 Da variant was identified.
b) Peptide mass analysis:
The variant was digested with Glutamyl endopeptidase (commercially available as Glu-C from Roche). The pH of the sample was adjusted to 7.0 with 1M tris(hydroxymethyl) aminomethane (Sigma Aldrich) and endoproteinase Glu-C enzyme is added in the enzyme-protein ratio of 1:25. The mixture of reaction was incubated at 37°C for three hours. Further, the disulphide bonds were reduced by using 1M DTT with 1:10 protein to DTT ratio and incubated for an additional hour. The variant was digested and reduced to individual fragments which were identified using LC-MS.
c) MS/MS analysis:
MS/MS spectrum based sequencing of polypeptides was done (CID) by using the nomenclature that was proposed by Roeptorff-Fohlmann-Biemann . Based on the aforementioned theory, a polypeptide chain is thus sequenced to obtain the complete amino-acid sequence identity for the peptides. Software aided annotation of either b ions or Y ions are indicative of sequence coverage at amino acid level.
Near (260-360 nm) and far (190-260 nm) UV circular dichroism (UV-CD) spectroscopy was used to study tertiary and secondary protein structures, respectively. Circular dichroism experiments were carried out on a Jasco J-815 spectrometer equipped with a Peltier-type cell holder. Spectra were recorded at 25°C with a scanning speed of 200 nm/min and a bandwidth of 0.1 nm in a quartz cell, with a path length of 0.1 cm. Far-UVCD spectra were recorded from 190 to 260 nm at a sample concentration of 0.3 mg/mL, whereas near-UVCD spectra were recorded from 260 to 360 nm at a sample concentration of 3 mg/mL. For each sample, 6 accumulations were performed, and baseline correction was applied.
Intrinsic fluorescence measurements were performed on a Cary Eclipse fluorescence spectrometer (Agilent Technologies), and data were analyzed using Scan software version 1.2 (Agilent Technologies). Intrinsic fluorescence analysis was performed by excitation at 278 nm, and emission was scanned from 300 to 400 nm.
CHO-K1 cells overexpressing insulin receptor B (IR-B) were cultured in DMEM F12 medium without serum. 20,000 cells/well/100 µL in culture medium were seeded in a 96 well flat bottom cell culture plate and incubated overnight at 37°C in 5% CO2 incubator. After overnight starvation, 100µl of drug dilutions were added to each well and incubated at 37°C in 5% CO2 incubator for 5-10 minutes. After incubation, drug dilutions were removed and washed with 100 µL of 1X ice cold DPBS followed by addition of 25 µL of 1X lysis buffer (supplied with Alpha SureFire INS-RB p-Y1150/1151 and Alphascreen Protein A kit (Perkin Elmer)). Samples were processed further by adding both acceptor beads and donor beads followed by measuring the alpha counts as per the kit protocol. The data was analysed using SoftMax Pro software.