Research On The Quality Standards Of APIs In The IND Application Stage Of A Targeted Anti-tumor Innovative Drug

Research background and purpose:The HBW-3-20,developed and synthesized in our laboratory with independent intellectual property rights,has superior efficacy over tirabrutinib(the fist-generation irreversible Bruton’s tyrosine kinase(BTK)inhibitor).That can permeate the blood-brain barrier to a large extent,reversible and drug-resistant.It is the first three generation BTK inhibitor reported in the world.HBW-3-20 can effectively treat primary central nervous system lymphoma(PCNSL).This topic is based on the principles of safety,efficacy,and quality control,to study the quality standards of HBW-3-20 drug substance in the declaration phase of a clinical trial(investigational new drug,IND),to provide a scientific reference for the quality control of the drug’s clinical use,as well as for later drug registration and marketing.Methods:1.Physicochemical properties and discrimination.According to the relevant regulations in the 2020 edition of the Chinese Pharmacopoeia,combined with HBW-3-20 own properties sequentially observe and determine the appearance,solubility,melting point and specific rotation of the drug.In addition,the HPLC method combined with IR method are used to discrimination study.2.Examined items.In accordance with the relevant guidelines for impurity studies issued by the International Council on Harmonisation of technical requirements for pharmaceuticals for human use(ICH),the center for drug evaluation(CDE)and the Chinese Pharmacopoeia version 2020,in combination with the actual synthetic process of HBW-3-20,the inspection items for its drug substance are tentatively established.Then,develop the assay method and study methodological.Test representative batches of samples.Determine the corresponding limits.The items studied are: chloride(general practice 0801 to part IV),residue on ignition(general practice 0841 to part IV),heavy metals(second approach 0821 to part IV),moisture(general practice 0832 to part IV),residual solvents(GC),related substances(HPLC),enantiomers(HPLC),mutagens(LC-MS / MS).3.Content determination.The HPLC external standard method is selected for content determination of HBW-3-20 by the technical guidelines for the normalization process established for chemical drug quality standards.Then,method validation is performed and detect representative batch samples.Result:1 Physicochemical properties and discrimination study experimental results(1)Physicochemical properties.HBW-3-20 is a white like powder,practically insoluble or insoluble in water and slightly soluble in methanol.Melt path is 199.0 ℃ ～200.5 ℃,specific rotation is-7.74 °～-7.33 °.(2)Discrimination experiments.The test article is retained at the same time as the control article,and the IR spectra of the two samples are consistent.2 Experimental results of studies examining items(1)Chloride < 0.25%.(2)Residue on ignition ≤ 0.1%.(3)Heavy metals < 20 ppm.(4)Moisture ≤ 0.37%.(5)Residual solvents:The GC method established in this paper is well exclusive,and the resolution between each solvent peak to be tested is ≥ 4.0.The limit of detection concentration of each solvent to be tested is ≥ 0.0153 μg/m L,and the limit of quantification is ≥ 0.0613 μg/m L.Six residual solvents within their limit of quantification ～ 250% of limit concentrations show good linearity,R > 0.999.The precision RSD is < 9.0%.The accuracy is good,and the recoveries ranged from 95.01%to 118.22%.Finally,based on the related principles of the 2020 edition of the Chinese Pharmacopoeia,ICH and EMEA,it is determined that the limits of each residual solvent are: ethanol,n-butanol,n-heptane,ethyl acetate shall not exceed 0.5%,1,4-dioxane shall not exceed 0.038%,and tetrahydrofuran shall not exceed 0.072%.(6)Related substances:The exclusivity of the HPLC method established in this paper is good,and the resolution between the major peak and each impurity peak is >1.8.The limit of quantitation concentration is approximately 0.2 μg/m L,and the limit of detection concentration is approximately 0.05 μg/m L.The linearity is good in the range of 0.2 μg/m L ～ 0.5 mg/m L,R > 0.999.The precision RSD is < 15.0%.At 15 ℃ for 24 h,stability is good.Finally based on representative batches samples,as well as relevant regulatory requirements,limits have been established for related substances.Individual impuritie is ≤ 0.5%,total impurities are ≤ 2.0%.(7)Enantiomers:The present method is well exclusive,and the separation between the major and enantiomeric peaks is ≥ 6.7.The limit of detection is approximately0.1912 μg/ m L,and the limit of quantification is approximately 0.3823 μg/m L 。 In0.3823 μg/m L ～ 1.8250 mg/m L show good linearity,R > 0.999.The precision RSD is≤ 1.08%.For 25 h at room temperature show good stability.Finally based on multiple representative batches samples,as well as relevant regulatory requirements,a limit is established for enantiomers.An enantiomer is ≤ 2.0%.(8)Mutagenic impurities:Method exclusivity is good and interfering peaks present in the chromatograms of the blank solution at the peak positions out of the test are negligible.The LOQs for mutagenic impurity 1 and mutagenic impurity 2 are 0.1566ng/m L,0.0152 ng/m L,and the LODs are 0.0783 ng/m L,0.0076 ng/m L,respectively.The linearity is good in the range of 10%-200% of the limiting concentration,R > 0.995.The precision RSD is ≤ 7.5%.The accuracy is good,and the recoveries ranged from 94.6% to 103.6%.The sample in control temperature 10 ℃placed within 6 h,the solution stability is good.Finally,the basis of representative batches samples and relevant regulatory requirements,a limit for mutagenic impurities is established.Mutagenic impurity 1 and mutagenic impurity 2 limits should not exceed7.5 ppm.3 Experimental results of the study for content determinationThe exclusivity of the HPLC method established in this paper is good.The linearity is good in the range of 0.0797 mg/m L to 0.1195 mg/m L and R is 0.9999.The precision RSD is ≤ 0.5%.The solution stability is good with no less than 24 h in control temperature 15 ℃ condition.Basis of representative batches samples and relevant regulatory requirements,the limit of the content is formulated,which is between98.0%～102.0%.Conclusion:In this paper,the physicochemical properties of HBW-3-20 were examined,the methods of identification,inspection and content determination were investigated.The quality standard of the drug substance HBW-3-20 is tentatively established,which can meet the requirements of IND declaration.