Twig whitespace controls have no effect on fields
Although the whitespace control modifier is supposed to work for all {{ … }} prints, {% … %} statements and {# … #} comments I'd expect to make your sample work it needs to be put directly into the print like that:

{% for item in items %}
  {{- item.content -}}{{- not loop.last ? ', ' -}}
{% endfor %}


Alternatively you could try to use the {% spaceless %} tag. But again, I'd expect this only works right when placed inside the loop:

{% for item in items %}
  {% spaceless %}
    {{ item.content }}{{ not loop.last ? ', ' }}
  {% endspaceless %}
{% endfor %}



  As of Twig 1.38, use the |spaceless filter instead.