ASP.NET Impersonate="false" returns 404 not found.

I have an old WebForms application that I'm trying to debug to fix an unrelated issue. When I compile I receive zero errors but when I run the application with Impersonate set to False it returns a 404 local host not found. When it's set to true it returns an exception:

Could not load file or assembly 'AjaxControlToolkit' or one of its dependencies. An API call exited abnormally. (Exception from HRESULT: 0x800300FA (STG_E_ABNORMALAPIEXIT))

Is it a code issue or an IIS server issue?