MABs Stability – Analytical Techniques

Andrew G Watts PhD.
Medicinal Chemistry Group
Department of Pharmacy and Pharmacology
University of Bath

Transcript

1. MABs Stability – Analytical Techniques Andrew G Watts PhD. Medicinal Chemistry Group Department of Pharmacy and Pharmacology September 2012
2. Disclosure Glythera Ltd. Founder and Scientific Advisor Bath ASU Scientific Consultant
3. Key questions which need to be answered Which analytical techniques do we need to use to effectively evaluate the stability of MABs ? What do we need to consider when interpreting the results from these techniques? What amount/type of degradation is significant/important? - Acceptance criteria
4. Source Guidance •  International Conference for Harmonization (ICH) •  Harmonization of British, US, Japanese and European Pharmacopeia's •  ICH Q2 R1 Analytical validation •  ICH Q5C Stability Testing of Biotechnological/Biological products •  ICH Q6B Specifications Test Proceedures and Acceptance criteria for bioltechnological/biological products Guidelines generally aimed at the licensing of new drug products.  
5. Scope of Q5C Q5c  -­‐  characterised  proteins  and  polypeptides   their  derivatives  and  products  of  which  they  are  components  and  which  are   isolated  from  tissues,  body  fluids,  cell  cultures,  or  produced  using  rDNA   technology   Ø  cytokines  (interferons,  interleukins,  colony-­‐stimulating  factors,  tumour  necrosis   factors)   Ø  erythropoietins   Ø  plasminogen  activators   Ø  blood  plasma  factors   Ø  growth  hormones  and  growth  factors   Ø  insulins   Ø  monoclonal  antibodies   Ø  vaccines  consisting  of  well-­‐characterised  proteins  or  polypeptides  
6. ICH Q5C : DOES NOT : Provide us with a ‘recipe’ for how to analyse or interpret MAB stability DOES : Outline criteria for a sound approach to designing protocols for determining stability. •  Primary data to support a requested storage period, long-term, real-time and real-condition stability studies •  Preferable NOT to use accelerated/stressed stability testing
7. Stability-Indicating profile §  no single stability-indicating assay or parameter – profiles the stability characteristics of a biotechnological/biological product §  the stability-indicating profile should provide assurance that changes in the : Identity Purity Potency Other characteristics §  the determination of which tests should be included will be product- specific Chemical analysis Biological activity Cellular response Primary structure Secondary structure Tertiary structure Quaternary structure
8. Degradation Pathways Physical •  Denaturation •  Aggregation •  Surface adsorption Chemical •  Disulfide formation/exchange •  Non-reducible cross linking •  Deamidation •  Isomerization •  Oxidation •  Glycosylation Cellular response Primary structure Secondary structure Tertiary structure Quaternary structure Impact upon:
9. Analytical method Principle Information/use Advantages/ Disadvantages SEC Size Quantify mAb and relative purity + separation of main isoforms - Limited mass resolution SDS-PAGE Size Estimate purity and molecular mass + Cheap, fast - Limited information - Limited mass resolution DLS Size Polydispersity, size distribution, detection of high molecular weight aggregates + Highly sensitive technique - High polydispersity will affect accuracy - Cannot resolve short oligomers CD Shape / 3D structure Estimate ratios of secondary structures, detect changes in tertiary structure/ conformation + speed and ease of use + spectra can be obtained with small volume and concentration of antibody -  Expensive instrument -  Diluents and excipients may show significant absorption in far-UV Microflow Size + morphology Count and characterisation of aggregates + characterisation allows removal of air and oil droplets - Reliable shape information above 4µm diameter pH Hydrogen potential Hydrogen potential of solution + indicative of degradation processes - Limited information LC-MS Polarity + mass Charge to mass ratio, separation, characterisation, quantification of mAb isoforms +Online desalting and fragment separation + highly detailed information -  Significant method optimization -  Expensive and requires trained analyst
10. Aggregation  0µm                    0.5µm    10µm    25µm    50µm    500µm   partial unfolding aggregation Wide dynamic range of aggregate types and sizes means that multiple techniques are need to assess this form of degradation.
11. SEC HPLC (Size-Exclusion) Aggregates Fragments Lower MW Higher MW Monomer Validation (ICH Q2) Selectivity •  Forced degradation studies Linearity •  Calibration Curves •  Limit of detection •  Limit of quantification Accuracy •  Over 3 concentrations levels (min 9 injections) Precision •  Repeatability of 6 injections (%RSD <2%) Robustness
12. SEC HPLC (Size-Exclusion) Day   HMW-4-%   HMW-3-%   HMW-2-%   HMW-1-%   Monomer-%   A   0.00 ± 0.00   0.00 ± 0.00   0.00 ± 0.00   1.76 ± 0.24   98.25 ± 0.24   B   0.00 ± 0.00   0.00 ± 0.00   0.21 ± 0.02   2.18 ± 0.10   97.61 ± 0.12   C   0.00 ± 0.00   0.00 ± 0.00   0.25 ± 0.02   2.40 ± 0.06   97.36 ± 0.09   D   0.00 ± 0.00   0.00 ± 0.00   0.18 ± 0.11   2.14 ± 0.41   97.68 ± 0.52   E   0.00 ± 0.00   0.00 ± 0.00   0.21 ± 0.01   2.25 ± 0.03   97.51 ± 0.05   F   0.00 ± 0.00   0.00 ± 0.00   0.19 ± 0.01   2.27 ± 0.02   97.54 ± 0.02   G   0.00 ± 0.00   0.00 ± 0.00   0.23 ± 0.01   2.34 ± 0.06   97.44 ± 0.07   H   0.00 ± 0.00   0.00 ± 0.00   0.22 ± 0.01   2.30 ± 0.03   97.48 ± 0.04   I   0.18 ± 0.36   0.17 ± 0.01   0.82 ± 0.04   1.39 ± 0.08   97.46 ± 0.34   J   0.00 ± 0.00   0.21 ± 0.01   1.00 ± 0.04   1.54 ± 0.07   97.25 ± 0.08   Linearity of response and LOD, LOQ are crucial. Low molecular weight aggregates can be reversible - constant temperature
13. Dynamic Light Scattering (DLS)   Z-Ave (r.nm)   PdI   %PD   Oligomeric Species mean r.nm   High MW Aggregates mean r.nm   Oligomeric Species (%Volume)   High MW Aggregates (% Volume)   Day A   7.98   0.07   27.33   8.67   0.00   100.00   0.00   Day D   8.27   0.10   31.56   8.75   2584.67   100.00   0.00   Day C   8.03   0.07   25.84   8.69   0.00   100.00   0.00   Day D   8.05   0.08   28.27   8.79   0.00   100.00   0.00   Day E   7.99   0.05   23.15   8.53   0.00   100.00   0.00   Day F   8.02   0.07   26.12   8.67   0.00   100.00   0.00   Day G   8.87   0.17   41.58   9.63   2323.33   99.98   0.02   Day H   7.97   0.05   22.53   8.50   0.00   100.00   0.00   Day I   7.95   0.05   21.93   8.49   0.00   100.00   0.00   Day J   8.09   0.08   27.97   8.79   0.00   100.00   0.00   •  Sample is illuminated with a laser •  Measures time dependant fluctuations in the intensity of scattered light •  Fluctuations occur because particles are constantly undergoing Brownian motion •  Effectively measures velocity of molecules which can be converted on to an equivalent hydrodynamic radius. The size distribution obtained is a plot of the relative intensity of light scattered by particles in various size classes. monomer aggregate
14. Microflow Imaging (Particle counting)   Particles (1000/ml) ± SD   X mg/ml   > 1µm   >2µm   >10µm   >25µm   Day A   275 ± 102   45 ± 29   5 ± 3   0.0 ± 0.1   Day B   441 ± 14   194 ± 10   10 ± 2   0.0 ± 0.2   Day C   166 ± 21   56 ± 14   0.3 ± 3   0.0 ± 0.4   micro-air bubble silicone oil aggregated protein MFI uses high resolution camera to detect insoluble particles in a flow cell •  Captures numerous frames per second •  Software crops particles from each raw image and produces collage •  Results must be corrected using statistical filters to distinguish silicone oil droplets and micro air bubbles
15. SDS – Page (Elecrophoresis) Native Tris Acetate 3-8% polyacrilamide gel Better separation of high molecular weight proteins (300-100kDa) Coomassie Staining Maximum loading of 0.5ug protein Reducing Bis-Tris 10% polyacrilamide gel Superior separation of mid-low molecular weight proteins Silver staining Maximum loading of 1ng protein Qualitative at best. Method of visualisation is very important.
16. Liquid Chromatography – Mass spectroscopy Core heptasaccharide is primarily complex biantennary type structure Licensenced recombinant MAbs generally core fucosylated with low levels of galactosylation Outer arms have variable addition of fucose galactose, bisecting N-acetyleglucosamine sialic acid on the antenna G0 = zero galactose residues G1 = one galactose residue G2 = two galactose residues F denotes fucosylation
17. Circular Dichroism •  Measures difference in absorbance left hand and right hand circularly polarised light •  Molecules must be chiral •  Far-UV range used for analysis of secondary structure (200-260nm) •  Near-UV range used for analysis of tertiary structure (260-350nm) •  Use computer program to estimate ratios of secondary structures β-sheets, α-helices etc •  Compare spectra between time points and also examine thermal stability
18. Variable Temperature Circular Dichroism Can measure changes in secondary structure as a function of changing temperature Can identify the melting temperature of a MAB, which give information as to the integrity of secondary and tertiary structure. Ie. Can identify the IMPACT of chemical changes Temperature   25°C   37.5°C   50°C   62.5°C   75°C   α-helix   5.3   5.4   5.5   5.5   6.6   Antiparallel   40.1   39.9   39.7   39.4   36.4   Parallel   5.4   5.4   5.4   5.4   5.5   Beta-turn   17.7   17.7   17.7   17.7   18.1   Random Coil   34.8   34.8   34.8   34.8   34.9  
19. Other pH Visual appearance Colour Turbidity
20. Potency Receptor Studies Cell Line Studies Clinical Effect
21. ICH Q6B ELISA
22. ELISA -  Receptor binding studies -  Allow one to measure changes in binding ability of Fc and variable region. -  DOES NOT tell us whether this binding leads to a cellular effect. -  Can be carried out quickly at a range of temperatures and concentrations. -  Real system???
23. Cell based activity studies -  Want to ensure that results of the assay are representative. -  Parameters that need to be identified/optimized. -  Cell lines: Need cells which express receptor specific to the MAB -  Cell numbers, Culture time, Passages -  Correct MAB concentration to see a change -  What cellular response are we looking for. Ie. Apoptosis -  Validation is essential. But no instructions on how to do this, but what are some key considerations?
24. Which effect ? Trastuzumab -  As we want the assay to be representative of clinical effect, measurement of ultimate effect of the MAB is preffered. -  Cell death -  Other pathways higher up the cascade could also be monitored. -  pAkt -  Cellular proliferation
25. Which effect? Alemtuzumab - Multiple mechanisms of action contribute to clinical efficacy - More than one mechanism should be tested.
26. Establishing an end point Base line Conc 3 Conc 2 Conc 1 Days Baseline -  Determination of a baseline -  Comparison to baseline over time -  Meaningful comparison requires high level of confidence in the data -  Reducing standard deviation between points is key.
27. Thank you ! Download available from: mabstalk.com